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
  • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
  • A61K51/0485—Porphyrins, texaphyrins wherein the nitrogen atoms forming the central ring system complex the radioactive metal
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/101—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals
  • A61K49/106—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being cyclic, e.g. DOTA
• • 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
  • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
  • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
• • 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
• • 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
  • A61K51/1036—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 hormone receptors
• • 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
• • 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
• • 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 [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/286—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against neuromediator receptors, e.g. serotonin receptor, dopamine receptor
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IGs], 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2121/00—Preparations for use in therapy

Definitions

• the insulin-like growth factor-1 receptor (IGF-1 R) has been evaluated as a potential therapeutic target in the treatment of cancer.
• the present invention describes an alternate method for leveraging IGF-1 R by delivering therapeutic radioisotopes that produce enhanced tumor efficacy with substantially lower doses than that of the antibody alone.
• the present invention is directed to monoclonal antibodies that target the insulin-like growth factor-1 receptor and the radioimmunoconjugates thereof that demonstrate increased potency and enhance the excretion of a chelating moiety, or a metal complex thereof, when conjugated to a therapeutic moiety, a targeting moiety, or a cross-linking group.
• the invention features a compound having the structure:
• A is chelating moiety or a metal complex thereof
• L 1 is optionally substituted C C 6 alkyl, substituted C C 6 heteroalkyl, substituted aryl or heteroaryl;
• B is a therapeutic moiety, a targeting moiety, or cross-linking group
• n 1 -5;
• each L 2 independently, has the structure:
• R 1 and R 1 is H or optionally substituted C C 6 alkyl or optionally substituted C C 6 heteroalkyl, substituted aryl or heteroaryl;
• L 3 is optionally substituted C C 50 alkyl or optionally substituted C C 5 o heteroalkyl or C 5 -C 2 o polyethylene glycol;
• the chelating moiety is DOTA (1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid), DOTMA (1 R,4R,7R, 10R)-a, a', a", cT-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), D03AM-acetic acid (2-(4,7, 10-tris(2-amino-2-oxoethyl)-1 , 4,7, 10-tetraazacyclododecan-1 - yl)acetic acid), D03AM-ace
• HOPO octadentate hydroxypyridinones
• chelating moieties in the practice of the invention are not limited to the specific constructs disclosed herein, but rather may include other known chelating moieties.
• the chelating moiety has the structure:
• Y 1 is H
• Y 2 is L 1 -(L 2 ) n -B
• L 1 has the structure:
• R 2 is optionally substituted hydrogen or -C0 2 H
• the metal can be selected from Bi, Pb, Y, Mri, Cr, Fe, Co, Zn, Ni, Tc,
• radionuclides suitable for complexlng to a compound of formula (I) include 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 86 Y, 87 Y, 90 Y, 97 Ru, 105 Rh, 109 Pd, 11 1 ln, 1 17m Sn, 149 Pm, 149 Tb, 153 Sm, 177 Lu, 186 Re, 188 Re, 199 Au, 201 TI, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 225 Ac, and 227 Th.
• B is a therapeutic moiety or targeting moiety.
• the therapeutic moiety or targeting moiety is an antibody, an antigen- binding fragment thereof or other targeting protein such as nanobodies, affibodies, and consensus sequences from Fibronectin type III domains.
• the antibody, or an antigen-binding fragment thereof specifically binds insulin-like growth factor-1 receptor (IGF-1 R), such as figitumumab, cixutumumab, ganitumab, AVE1642 (also known as humanized EM164 and huEM164), BIIB002, robatumumab, and teprotumumab.
• IGF-1 R insulin-like growth factor-1 receptor
• the antibody, or an antigen binding fragment thereof is AVE1642.
• the antibody, or antibody-binding fragment thereof includes a light chain variable domain including at least one, two, or all three complementarity determining regions (CDRs) selected from:
• the antibody, or antibody-binding fragment thereof includes a heavy chain variable domain including at least one, two, or all three CDRs selected from:
• the antibody, or antibody-binding fragment thereof includes a heavy chain variable domain and a light chain variable domain including at least one, two, three, four, five, or all six CDRs selected from:
• CDR-H2 including the amino acid sequence of SEQ ID NO: 6;
• the heavy chain variable domain includes the amino acid sequence of SEQ ID NO: 4.
• the light chain variable domain includes the amino acid sequence of SEQ ID NO: 8.
• the cross-linking group is an amino-reactive cross-linking group, a methionine-reactive cross-linking group, a thiol-reactive cross-linking group or a sortase-mediated coupling sequence.
• the amino-reactive, methionine-reactive, or thiol-reactive cross-linking group comprises an activated ester such as a hydroxysuccinimide ester, N-hydroxysulfosuccinimide, 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 an oxaziridine.
• an activated ester such as a hydroxysuccinimide ester, N-hydroxysulfosuccinimide, 2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or an imidate
• 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.
• cross linking groups in the practice of the invention are not limited to the specific constructs disclosed herein, but rather may include other known cross linking groups.
• the cross-linking group is selected from the group consisting of:
• Y 1 is H.
• Z 1 is -CH 2 .
• L 2 has n value of 1 .
• the compound is selected from the group consisting of:
• the metal is a radionuclide.
• the radionuclide is 111 In.
• the radionuclide is 68 Ga.
• the radionuclide is 86 Y.
• the metal is a beta-emitting radionuclide.
• the radionuclide are 67 Cu, 177 Lu' or 90 Y
• the metal is an alpha-emitting radionuclide.
• the radionuclide is 225 Ac, 212 Pb, 227 Th or the progeny (daughter isotopes) thereof.
• the invention features a pharmaceutical composition including any of the foregoing compounds and a pharmaceutically acceptable excipient.
• the invention features a method of radiation treatment planning and/or radiation treatment, the method comprising administering to a subject in need thereof any of the foregoing compounds or pharmaceutical compositions.
• the invention features a method of detecting and/or treating cancer, the method including administering to a subject in need thereof a first dose of any of the foregoing compounds or pharmaceutical compositions in an amount effective for radiation treatment planning, followed by administering subsequent doses of any of the foregoing compounds or pharmaceutical compositions in a therapeutically effective amount.
• the compound or composition administered in the first dose and the compound or composition administered in the second dose, or subsequent doses are the same.
• the compound or composition administered in the first dose and the compound or composition administered in the second dose, or subsequent doses are different.
• the cancer is a solid tumor or hematologic (liquid) cancer.
• the solid tumor cancer is breast cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, colorectal cancer, sarcoma, adrenocortical carcinoma, neuroendocrine cancer, Ewing's Sarcoma, multiple myeloma, or acute myeloid leukemia.
• the foregoing methods further include administering an
• antiproliferative agent radiation sensitizer, or an immunoregulatory or immunomodulatory agent.
• any of the foregoing compounds or compositions thereof and an antiproliferative agent or radiation sensitizer are administered within 28 days (e.g., within 14, 7, 6, 5, 4, 3, 2, or 1 day(s)) of each other.
• any of the above-described compounds or compositions thereof and an immunoregulatory or immunomodulatory agent are administered within 90 days (e.g., within 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 day(s)) of each other.
• the invention features a method of making a radioconjugate (e.g., any of the radioconjugates described herein).
• the method includes the steps of (a) conjugating a bifunctional chelate to a biological molecule, (b) purifying the conjugate produced by step (a), and (c) chelating one or more radionuclides (e.g., one or more Ac-225 radionuclides) with the purified conjugate of step (b) at a temperature of less than 35 °C (e.g., 20-25 °C) to produce a radioconjugate (e.g. an actinium radioconjugate).
• a radioconjugate e.g., any of the radioconjugates described herein.
• the radioconjugate is a radioimmunoconjugate (e.g., any of the radioimmunoconjugates described herein).
• the pH of the reaction mixture of conjugation step (a) is less than 6.4 (e.g., 6.3, 6.2, 6.1 , 6.0, 5.9, or 5.8 or less). In some embodiments, the pH of the reaction mixture of conjugation step (c) is less than 5.5 (e.g., 5.4, 5.3, 5.2, 5.1 , or 5.0 or less) or more than 7.0 (e.g., 7.1 , 7.2, 7.3, 7.4, 7.5 or more).
• the temperature of the reaction mixture of conjugation step (c) is 20- 34 °C (e.g., 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27°C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, or 34 °C).
• alkyl is inclusive of both straight chain and branched chain saturated groups from 1 to 20 carbons (e.g., from 1 to 10 or from 1 to 6), unless otherwise specified.
• Alkyl groups are exemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- and tert-butyl, neopentyl, and the like, and may be optionally substituted with one, two, three, or, in the case of alkyl groups of two carbons or more, four substituents independently selected from the group consisting of: (1 ) alkoxy; (2) d- 6 alkylsulfinyl; (3) amino, as defined herein (e.g., unsubstituted amino (i.e.
• alkyl e.g., alkyl
• C 2 . 20 alkenyl e.g., C 2 . 6 alkenyl
• C 6 . 10 aryl C 6 . 10 aryl
• hydrogen hydrogen
• alk-C 6 . 10 aryl amino-C ⁇ o alkyl
• s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 1 0), and R' is H or Ci- 20 alkyl, and (h) amino-polyethylene glycol of - NR N1 (CH 2 ) s2 (CH 2 CH 2 0) s1 (CH 2 ) s3 NR N1 , wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R N1
• R D is selected from the group consisting of (a) Ci_ 6 alkyl, (b) C 6 . 10 aryl, (c) alk-C 6 . 10 aryl, and (d) hydroxy; (17) -S0 2 NR E R F , where each of R E and R F is, independently, selected from the group consisting of (a) hydrogen, (b) Ci_ 6 alkyl, (c) C 6 . 10 aryl and (d) alk-C 6 . 10 aryl; (18) -C(0)R G , where R G is selected from the group consisting of (a) Ci.
• alkyl e.g., alkyl
• C 2 . 20 alkenyl e.g., C 2 -6 alkenyl
• C 6 . 10 aryl e.g., hydrogen
• hydrogen e.g., hydrogen, hydrogen, alk-C 6 .
• each R N1 is, independently, hydrogen or optionally substituted d-6 alkyl; (19) -NR H C(0)R' , wherein R H is selected from the group consisting of (a1 ) hydrogen and (b1 ) d-6 alkyl, and R 1 is selected from the group consisting of (a2) d- 20 alkyl (e.g., d-6 alkyl), (b2) C 2 . 20 alkenyl (e.g., C 2 . 6 alkenyl), (c2) C 6 . 10 aryl, (d2) hydrogen, (e2) d-6 alk-C 6 .
• R H is selected from the group consisting of (a1 ) hydrogen and (b1 ) d-6 alkyl
• R 1 is selected from the group consisting of (a2) d- 20 alkyl (e.g., d-6 alkyl), (b2) C 2 . 20 alkenyl (e.g., C 2 . 6 alkenyl), (c2) C 6
• NR N1 (CH 2 ) s2 (CH 2 CH 2 0) s1 (CH 2 ) s3 NR N1 , wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R N1 is, independently, hydrogen or optionally substituted d-e alkyl; (20) -NR J C(0)OR K , wherein R J is selected from the group consisting of (a1 ) hydrogen and (b1 ) d-6 alkyl, and R K is selected from the group consisting of (a2) Ci- 20 alkyl (e.g., d-6 alkyl), (b2) C 2 .
• alkenyl e.g., C 2 . 6 alkenyl
• C 6 . 10 aryl e.g., C 2 . 6 alkenyl
• c2 C 6 . 10 aryl
• d2 hydrogen
• e2 d-e alk-C 6 . 10 aryl
• f2 amino-d-20 alkyl
• polyethylene glycol of -(CH 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 OR' wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or d-20 alkyl, and (h2) amino-polyethylene glycol of -N R N1 (CH 2 ) s2 (CH 2 CH 2
• alkylene and the prefix "alk-,” as used herein, represent a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, and is exemplified by methylene, ethylene, isopropylene, and the like.
• C x . y alkylene and the prefix "C x . y alk-” represent alkylene groups having between x and y carbons. Exemplary values for x are 1 , 2, 3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (e.g., d-e, d-10, C 2 . 20 , C 2 . 6 , C 2 . 10 , or C 2 . 20 alkylene).
• the alkylene can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for an alkyl group.
• alkenyl represents monovalent straight or branched chain groups of, unless otherwise specified, from 2 to 20 carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one or more carbon-carbon double bonds and is exemplified by ethenyl, 1 -propenyl, 2- propenyl, 2-methyl-1 -propenyl, 1 -butenyl, 2-butenyl, and the like. Alkenyls include both cis and trans isomers.
• Alkenyl groups may be optionally substituted with 1 , 2, 3, or 4 substituent groups that are selected, independently, from amino, aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituent groups described herein.
• alkynyl represents monovalent straight or branched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from 2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bond and is exemplified by ethynyl, 1 -propynyl, and the like.
• Alkynyl groups may be optionally substituted with 1 , 2, 3, or 4 substituent groups that are selected, independently, from aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituent groups described herein.
• amino represents -N(R N1 ) 2 , wherein each R N1 is, independently, H, OH, N0 2 , N(R N2 ) 2 , S0 2 OR N2 , S0 2 R N2 , SOR N2 , an W-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl (e.g., optionally substituted with an O-protecting group, such as optionally substituted arylalkoxycarbonyl groups or any described herein), sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., optionally substituted with an O-protecting group, such as optionally substituted arylalkoxycarbonyl groups or any
• each of these recited R N1 groups can be optionally substituted, as defined herein for each group; or two R N1 combine to form a heterocyclyl or an /V-protecting group, and wherein each R N2 is, independently, H, alkyl, or aryl.
• the amino groups of the invention can be an unsubstituted amino (i.e., -NH 2 ) or a substituted amino (i.e., -N(R N1 ) 2 ).
• amino is -NH 2 or - NHR N1 , wherein R N1 is, independently, OH, N0 2 , NH 2 , NR N2 2 , S0 2 OR N2 , S0 2 R N2 , SOR N2 , alkyl, carboxyalkyl, sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein),
• alkoxycarbonylalkyl e.g., t-butoxycarbonylalkyl
• each R N2 can be H, Ci- 20 alkyl (e.g., alkyl), or C 6 . 10 aryl.
• amino acid refers to a molecule having a side chain, an amino group, and an acid group (e.g., a carboxy group of -C0 2 H or a sulfo group of -S0 3 H), wherein the amino acid is attached to the parent molecular group by the side chain, amino group, or acid group (e.g., the side chain).
• the amino acid is attached to the parent molecular group by a carbonyl group, where the side chain or amino group is attached to the carbonyl group.
• Exemplary side chains include an optionally substituted alkyl, aryl, heterocyclyl, alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl.
• Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, and valine.
• Amino acid groups may be optionally substituted with one, two, three, or, in the case of amino acid groups of two carbons or more, four substituents independently selected from the group consisting of: (1 ) alkoxy; (2) d- 6 alkylsulfinyl; (3) amino, as defined herein (e.g., unsubstituted amino (i.e. , -NH 2 ) or a substituted amino (i.e., -N(R N1 ) 2 , where R N1 is as defined for amino); (4) C 6 . 10 aryl-C ⁇ alkoxy; (5) azido; (6) halo; (7) (C 2 .
• substituents independently selected from the group consisting of: (1 ) alkoxy; (2) d- 6 alkylsulfinyl; (3) amino, as defined herein (e.g., unsubstituted amino (i.e. , -NH 2 ) or a substituted amino (i.e.,
• R D is selected from the group consisting of (a) d-6 alkyl, (b) C 6 . 10 aryl, (c) d-6 alk-C 6 . 10 aryl, and (d) hydroxy; (17) -S0 2 NR E R F , where each of R E and R F is, independently, selected from the group consisting of (a) hydrogen, (b) d-6 alkyl, (c) C 6 . 10 aryl and (d) d-6 alk-C 6 .
• R G is selected from the group consisting of (a) d-20 alkyl (e.g., d-6 alkyl), (b) C2-20 alkenyl (e.g., C 2 -6 alkenyl), (c) C 6 . 10 aryl, (d) hydrogen, (e) d-6 alk-C 6 .
• each R N1 is, independently, hydrogen or optionally substituted d-6 alkyl; (19) -NR H C(0)R' , wherein R H is selected from the group consisting of (a1 ) hydrogen and (b1 ) d-6 alkyl, and R 1 is selected from the group consisting of (a2) d- 20 alkyl (e.g., d-6 alkyl), (b2) C2-20 alkenyl (e.g., C 2 -6 alkenyl), (c2) C 6 . 10 aryl, (d2) hydrogen, (e2) d-6 alk-C 6 . 10 aryl, (f2) amino-d.
• R H is selected from the group consisting of (a1 ) hydrogen and (b1 ) d-6 alkyl
• R 1 is selected from the group consisting of (a2) d- 20 alkyl (e.g., d-6 alkyl), (b2) C2-20 alkenyl (e.g., C 2 -6 alkenyl
• aryl represents a mono-, bicyclic, or multicyclic carbocyclic ring system having one or two aromatic rings and is exemplified by phenyl, naphthyl, 1 ,2-dihydronaphthyl, 1 ,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl, indanyl, indenyl, and the like, and may be optionally substituted with 1 , 2, 3, 4, or 5 substituents independently selected from the group consisting of: (1 ) d- 7 acyl (e.g., carboxyaldehyde); (2) Ci- 20 alkyl (e.g., d-6 alkyl, d-6 alkoxy-d-6 alkyl, d-6 alkylsulfinyl-C-i_ 6 alkyl, amino-d-6 alkyl, azido-d-6 alkyl, (carboxyal
• R E and R F is, independently, selected from the group consisting of (a) hydrogen, (b) d-6 alkyl, (c) C 6 -io aryl, and (d) d- 6 alk-d- ! o aryl; (21 ) thiol; (22) C 6 . 10 aryloxy; (23) d- 8 cycloalkoxy; (24) C 6 .
• each of these groups can be further substituted as described herein.
• the alkylene group of a d-alkaryl or a d-alkheterocyclyl can be further substituted with an oxo group to afford the respective aryloyl and (heterocyclyl)oyl substituent group.
• arylalkyl represents an aryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein.
• exemplary unsubstituted arylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as d-6 alk- d- 1 0 aryl, d- 1 0 alk-C 6 . 10 aryl, or d- 2 0 alk-C 6 . 10 aryl).
• the alkylene and the aryl each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective groups.
• Other groups preceded by the prefix "alk-" are defined in the same manner, where “alk” refers to a d_ 6 alkylene, unless otherwise noted, and the attached chemical structure is as defined herein.
• cyano represents an -CN group.
• cycloalkyi represents a monovalent saturated or unsaturated non- aromatic cyclic hydrocarbon group from three to eight carbons, unless otherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicycle heptyl, and the like.
• the cycloalkyi group includes one carbon-carbon double bond or one carbon-carbon triple bond, the cycloalkyi group can be referred to as a "cycloalkenyl" or "cycloalkynyl” group respectively.
• Exemplary cycloalkenyl and cycloalkynyl groups include cyclopentenyl, cyclohexenyl, cyclohexynyl, and the like.
• the cycloalkyi groups of this invention can be optionally substituted with: (1 ) d- 7 acyl (e.g., carboxyaldehyde); (2) d- 2 0 alkyl (e.g., d-6 alkyl, d-6 alkoxy-d-6 alkyl, d-6 alkylsulfinyl-d-6 alkyl, amino-d-6 alkyl, azido-d-6 alkyl, (carboxyaldehyde)-d_ 6 alkyl, halo-d-6 alkyl (e.g., perfluoroalkyl), hydroxy-d-6 alkyl, nitro-d-6 alkyl, or d -6 thioalkoxy-d-6 alkyl); (3) d- 2 0 alk
• (CH 2 ) q CON R B R c where q is an integer from zero to four and where R B and R c are independently selected from the group consisting of (a) hydrogen, (b) C 6 . 10 alkyl, (c) C 6 . 10 aryl, and (d) d-6 alk-C 6 . 10 aryl; (19) -(CH 2 ) q S0 2 R D , where q is an integer from zero to four and where R D is selected from the group consisting of (a) C 6 . 10 alkyl, (b) C 6 . 10 aryl, and (c) d-e alk-C 6 .
• R E and R F is, independently, selected from the group consisting of (a) hydrogen, (b) C 6 . 10 alkyl, (c) C 6 . 10 aryl, and (d) d-6 alk-C 6 . 10 aryl; (21 ) thiol; (22) C 6 -io aryloxy; (23) C 3 . 8 cycloalkoxy; (24) C 6 .
• each of these groups can be further substituted as described herein.
• the alkylene group of a d-alkaryl or a d-alkheterocyclyl can be further substituted with an oxo group to afford the respective aryloyl and (heterocyclyl)oyl substituent group.
• diastereomer as used herein means stereoisomers that are not mirror images of one another and are non-superimposable on one another.
• enantiomer means each individual optically active form of a compound of the invention, having an optical purity or enantiomeric excess (as determined by methods standard in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% and more preferably at least 98%.
• halogen represents a halogen selected from bromine, chlorine, iodine, or fluorine.
• heteroalkyl refers to an alkyl group, as defined herein, in which one or two of the constituent carbon atoms have each been replaced by nitrogen, oxygen, or sulfur.
• the heteroalkyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for alkyl groups.
• the terms "heteroalkenyl” and heteroalkynyl,” as used herein refer to alkenyl and alkynyl groups, as defined herein, respectively, in which one or two of the constituent carbon atoms have each been replaced by nitrogen, oxygen, or sulfur.
• the heteroalkenyl and heteroalkynyl groups can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for alkyl groups.
• heteroaryl represents that subset of heterocyclyls, as defined herein, which are aromatic: i.e., they contain 4n+2 pi electrons within the mono- or multicyclic ring system.
• exemplary unsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 1 1 , 1 to 10, 1 to 9, 2 to
• heteroaryl is substituted with 1 , 2, 3, or 4 substituents groups as defined for a heterocyclyl group.
• heteroarylalkyi refers to a heteroaryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein.
• exemplary unsubstituted heteroarylalkyi groups are from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to 17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to 12 carbons, such as d-6 alk-d-12 heteroaryl, Ci -10 alk-C 1-12 heteroaryl, or Ci- 20 alk-C 1-12 heteroaryl).
• the alkylene and the heteroaryl each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective group.
• Heteroarylalkyl groups are a subset of heterocyclylalkyl groups.
• heterocyclyl represents a 5-, 6- or 7-membered ring, unless otherwise specified, containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
• the 5-membered ring has zero to two double bonds, and the 6- and 7-membered rings have zero to three double bonds.
• Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to 1 1 , 1 to 10, 1 to 9, 2 to 12, 2 to 1 1 , 2 to 10, or 2 to 9) carbons.
• heterocyclyl also represents a heterocyclic compound having a bridged multicyclic structure in which one or more carbons and/or heteroatoms bridges two non-adjacent members of a monocyclic ring, e.g., a quinuclidinyl group.
• heterocyclyl includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused to one, two, or three carbocyclic rings, e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another monocyclic heterocyclic ring, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like.
• fused heterocyclyls include tropanes and 1 ,2,3,5,8,8a-hexahydroindolizine.
• Heterocyclics include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinoly
• heterocyclyls include: 2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1 H-imidazolyl; 2,3,4,5-tetrahydro-5-oxo-1 H-pyrazolyl (e.g., 2,3,4,5-tetrahydro-2-phenyl-5-oxo-1 H-pyrazolyl); 2,3,4,5- tetrahydro-2,4-dioxo-1 H-imidazolyl (e.g., 2,3,4, 5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1 H- imidazolyl); 2,3-dihydro-2-thioxo-1 ,3,4-oxadiazolyl (e.g., 2,3-dihydro-2-thioxo-5-phenyl-1 ,3,4- oxadiazolyl); 4, 5-dihydro-5-oxo-1 H-triazoly
• heterocyclics include 3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and 2,5- diazabicyclo[2.2.1 ]heptan-2-yl, homopiperazinyl (or diazepanyl), tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl, thiepanyl, azocanyl, oxecanyl, and thiocanyl.
• Heterocyclic groups also include groups of the formula , where
• E' is selected from the group consisting of -N- and -CH-;
• G' is selected from the group consisting of -CH- and -N-.
• any of the heterocyclyl groups mentioned herein may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1 ) d-7 acyl (e.g., carboxyaldehyde ); (2) Ci- 20 alkyl (e.g., d-6 alkyl, d-6 alkoxy-d-6 alkyl, d-6 alkylsulfinyl-Ci_ 6 alkyl, amino-d-6 alkyl, azido-d-6 alkyl, (carboxyaldehyde)-d-6 alkyl, halo-d-6 alkyl (e.g., perfluoroalkyl), hydroxy-d-6 alkyl, nitro-d-6 alkyl, or d-6 thioalkoxy-d-6 alkyl) ; (3) d-20 alkoxy (e.g., d-6 alkoxy, such as perfluoroalkoxy); (4) d-6 alkylsulfin
• each of these groups can be further substituted as described herein.
• the alkylene group of a d-alkaryl or a d-alkheterocyclyl can be further substituted with an oxo group to afford the respective aryloyl and (heterocyclyl)oyl substituent group.
• hydrocarbon represents a group consisting only of carbon and hydrogen atoms.
• hydroxyl represents an -OH group. In some embodiments, the hydroxyl group can be substituted with 1 , 2, 3, or 4 substituent groups (e.g., O-protecting groups) as defined herein for an alkyl.
• isomer means any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the invention. It is recognized that the compounds of the invention can have one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis/trans isomers). According to the invention, the chemical structures depicted herein, and therefore the compounds of the invention, encompass all of the corresponding stereoisomers, that is, both the stereomerically pure form (e.g., geometrically pure, enantiomerically pure, or
• Enantiomeric and stereoisomeric mixtures of compounds of the invention can typically be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent.
• stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.
• V-protected amino refers to an amino group, as defined herein, to which is attached one or two /V-protecting groups, as defined herein.
• V-protecting group represents those groups intended to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N- protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3 rd Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference.
• /V-protecting groups include acyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2- chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, a- chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine, and the like; sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl, and the like; carbamate forming groups such as benzyloxycarbon
• allyloxycarbonyl 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fluorenyl- 9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl, and the like and silyl groups, such as trimethylsilyl, and the like.
• Preferred /V-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).
• O-protecting group represents those groups intended to protect an oxygen containing (e.g., phenol, hydroxyl, or carbonyl) group against undesirable reactions during synthetic procedures.
• O-protecting groups include acyl, aryloyl, or carbamyl groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, a-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4- bromobenzoyl, i-butyldimethylsilyl, tri-/so-propylsilyloxymethyl, 4,4'-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4-is
• arylalkoxycarbonyl groups such as benzyloxycarbonyl, p-methylbenzyloxycarbonyl, p- methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl, 3,5- dimethylbenzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-bromobenzyloxy-carbonyl,
• aryloxycarbonyl groups such as phenoxycarbonyl, p-nitrophenoxycarbonyl, o-nitrophenoxycarbonyl, 2,4-dinitrophenoxycarbonyl, p- methyl-phenoxycarbonyl, m-methylphenoxycarbonyl, o-bromophenoxycarbonyl, 3,5- dimethylphenoxycarbonyl, p-chlorophenoxycarbonyl, 2-chloro-4-nitrophenoxy-carbonyl, and the like); substituted alkyl, aryl, and alkaryl ethers (e.g., trityl; methylthiomethyl; methoxymethyl;
• benzyloxymethyl siloxymethyl; 2,2,2,-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl; 1 -[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; t-butyl ether; p-chlorophenyl, p- methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and nitrobenzyl) ; silyl ethers (e.g., trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl; t- butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and diphenymethylsilyl); carbonates (e.g.,
• polyethylene glycol represents an alkoxy chain comprised of one or more momomer units, each monomer unit consisting of -OCH 2 CH 2 -.
• PEG polyethyelene glycol
• PEO polyethylene oxide
• POE polyoxyethylene
• a polyethylene glycol may have the structure, -(CH 2 ) S2 (OCH 2 CH 2 ) s i (CH 2 ) s3 0-, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), and each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10).
• Polyethylene glycol may also be considered to include an amino-polyethylene glycol of -N R N1 (CH 2 ) S2 (CH 2 CH 2 0) s1 (CH 2 ) s3 NR N1 .
• s1 is an integer from 1 to 10 (e.g. , from 1 to 6 or from 1 to 4)
• each of s2 and s3, independently is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10)
• each R N1 is, independently, hydrogen or optionally substituted alkyl.
• stereoisomer refers to all possible different isomeric as well as conformational forms which a compound may possess (e.g., a compound of any formula described herein), in particular all possible stereochemical ⁇ and conformationally isomeric forms, all diastereomers, enantiomers and/or conformers of the basic molecular structure. Some compounds of the present invention may exist in different tautomeric forms, all of the latter being included within the scope of the present invention.
• sulfonyl represents an -S(0) 2 - group.
• thiol as used herein represents an -SH group.
• the term “administered in combination” or “combined administration” means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within 90 days (e.g., within 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 day(s)), within 28 days (e.g., with 14, 7, 6, 5, 4, 3, 2, or 1 day(s), within 24 hours (e.g., 12, 6, 5, 4, 3, 2, or 1 hour(s), or within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the
• administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.
• antibody refers to a polypeptide whose amino acid sequence including 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, Ig E, IgG, or IgM) or subtype (e.g., lgA1 , lgA2, lgG1 , lgG2, lgG3, or lgG4).
• 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.
• bifunctional chelate or “bifunctional conjugate” as used interchangeably herein, refer to a compound that contains a chelating group or metal complex thereof, a linker group, and a therapeutic moiety, targeting moiety, or cross linking group.
• 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 therapeutic moiety, targeting moiety, or cross linking group.
• the term "compound,” is meant to include all stereoisomers, geometric isomers, and tautomers of the structures depicted.
• the compounds described herein can be asymmetric (e.g., having one or more
• Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton.
• Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
• Examples prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1 H- and 3H-imidazole, 1 H-, 2H- and 4H- 1 ,2,4-triazole, 1 H- and 2H- isoindole, and 1 H- and 2H-pyrazole.
• Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
• substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges.
• the term "d- 6 alkyl” is specifically intended to individually disclose methyl, ethyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, and C 6 alkyl.
• a phrase of the form "optionally substituted X" e.g., optionally substituted alkyl
• X optionally substituted alkyl
• alkyl wherein said alkyl is optionally substituted
• detection agent refers to a molecule or atom which is useful in diagnosing a disease by locating the cells containing the antigen.
• 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., FITC, rhodamine, lanthanide phosphors, cyanine, and near IR dyes), and magnetic agents, such as gadolinium chelates.
• 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,"Tc, 99m Tc 105 Rh, 109 Pd, 111 In, 123 l, 124 l, 125 l, 131 1, 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, 229Th , 66 Ga, 67 Ga, 68 Ga, 82 Rb, 117m Sn, 201 TI).
• radioactive decay
• radioactive nuclide may also be used to describe a radionuclide.
• Radionuclides may be used as detection agents, as described above.
• the radionuclide may be an alpha-emitting radionuclide.
• an "effective amount” of an agent 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.
• immunoconjugate refers to a conjugate that includes a targeting moiety, such as an antibody, 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).
• radioconjugate 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.
• radioimmunotherapy refers a method of using a
• 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, in some embodiments the cell is a cancer cell in a subject having cancer.
• composition represents a composition containing a compound 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.
• unit dosage form e.g., a tablet, capsule, caplet, gelcap, or syrup
• topical administration e.g., as a cream, gel, lotion, or ointment
• intravenous administration e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use
• a "pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being nontoxic and non-inflammatory in a patient.
• 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,
• pharmaceutically acceptable salt represents 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.
• the 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. Methods for preparation of the appropriate salts are well- established in the art.
• 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, oleate, oxalate, palmitate, pamoate,
• 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.
• therapeutic moiety refers to any molecule or any part of a molecule that confers a therapeutic benefit.
• the therapeutic moiety is a protein or polypeptide, e.g., an antibody, an antigen-binding fragment thereof.
• the therapeutic moiety is a small molecule.
• targeting moiety refers to any molecule or any part of a molecule that binds to a given target.
• the targeting moiety is a protein or polypeptide such as an antibody or antigen binding fragment thereof, a nanobody, an affibody, or a consensus sequence from a Fibronectin type II I domain.
• cross-linking group refers to any reactive group that is able to join two or more molecules by a covalent bond.
• the cross-linking group is an amino-reactive or thiol-reactive cross-linking group.
• 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.
• 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 halo
• the cross linking group may be glycine-glycine-glycine and/or leucine-proline-(any amino acid)-threonine-glycine, which are the recognition sequences for coupling targeting agents with the linker using a sortase-mediated coupling reaction.
• the person having ordinary skill in the art will understand that the use of cross linking groups in the practice of the invention are not limited to the specific constructs disclosed herein, but rather may include other known cross linking groups.
• 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, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 1 00, 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, Wl 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
• 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.
• Figure 1 is a schematic depicting the general structure of a conjugate comprising a chelate, a linker, and a cross-linking group (top) and a conjugate comprising a chelate, a linker, and a targeting moiety (bottom).
• Figure 2 is a schematic depicting the synthesis of the bifunctional chelate, 4- ⁇ [1 1 -oxo-1 1 - (2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl ⁇ -2-[4,7, 10-tris(carboxymethyl)-1 ,4,7, 10- tetraazacyclododecan-1 -yl]butanoic acid (Compound B).
• Compound B The synthesis of Compound B is described in Example 3.
• Figure 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). The synthesis of Compound C is described in Example 4.
• Figure 4 is a graph depicting the percent residualization of three bifunctional chelated antibodies (Compound A, Compound B, and Compound C) determined as CPM (lysate)/CPM (efflux+recycled+lysate). The residualization assay used is described in detail in Example 6.
• Figure 5 is a series of graphs depicting the metabolic excretion profile of non-targeted human IgG antibody conjugates [ 177 Lu]-Compound B-HuMIGF-1 R, and [ 177 Lu]-Compound C-HuMIGF-1 R as compared to [ 177 Lu]-Compound A-HuMIGF-1 R, the methods and results of which are described in detail in Example 9.
• Figure 8 is a series of graphs depicting the metabolic excretion profile of non-targeted human IgG antibody conjugates [ 177 Lu]-Compound B-HuMIgG, and [ 177 Lu]-Compound C-HuMIgG as compared to [ 177 Lu]-Compound A-HuMIgG, the methods and results of which are described in detail in Example 14.
• Radiolabeled targeting moieties 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 specifically binds to IGF-1 R), 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 radiolabeled, the final radioimmunoconjugate is formed.
• a biological targeting moiety e.g, an antibody or antigen binding fragment thereof that specifically binds to IGF-1 R
• a radioisotope e.g., an antibody or antigen binding fragment thereof that specifically binds to IGF-1 R
• a radioisotope e.g., an antibody or antigen binding fragment thereof that specifically binds to IGF-1 R
• a radioisotope e.g., an antibody or antigen binding fragment thereof that specifically binds to IGF-1 R
• a radioisotope e
• Bifunctional chelates structurally contain a chelate, the linker, and cross-linking group ( Figure 1 ). When developing new bifunctional chelates, most efforts focus around the chelating portion of the molecule. Several examples of bifunctional chelates have been described with various cyclic and acyclic structures conjugated to a targeted moiety. [Bioconjugate Chem. 2000, 1 1 , 510-519, Bioconjugate Chem.2012, 23, 1029-1039, Mol Imaging Biol (201 1 ) 13:215-221 , Bioconjugate Chem.2002, 13, 1 10-1 15]
• 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).
• the most obvious mechanism is to increase the rate of clearance of the biological targeting agent.
• This approach also likely requires identifying ways to shorten the half-life of the biological targeting agent, which is a topic not well described for biological targeting agents.
• increasing drug clearance will also negatively impact the pharmacodynamics/efficacy in that the more rapid removal of drug from the body will lower the effective concentration at the site of action, which, in turn, would require a higher total dose and would not achieve the desired results of reducing total radioactive dose to normal tissue.
• 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; 12(1 1 ) November 2013, Methods in Molecular Biology, 2009, 539, 191 -21 1 , Bioconjugate chemistry, Volume 14, Issue 5, p.927-33 (2003)].
• the focus of the embodiments described herein centers on more effectively eliminating radioactivity from the body after catabolism and/or metabolism of the radioimmunoconjugate by making modifications to the linker region of the bifunctional chelate.
• radioimmunoconjugate with that same bifunctional chelate. Based on these data, one would expect that following catabolism/metabolism of the radioimmunoconjugate, the metabolite containing the bifunctional chelate would also be rapidly eliminated.
• the linker region of bifunctional chelates can directly impact the elimination of the radioactivity from the body following catabolism of the radioconjugate while not having a detrimental impact to the overall in vitro properties or the in vivo pharmacokinetics and pharmacodynamics of the radioimmunoconjugate.
• Data are presented below that demonstrates that the certain bifunctional chelates available commercially produce a slower rate and a lower extent of elimination of the total radioactivity from the body when compared to the embodiments described herein.
• bifunctional chelates when attached to biological targeting moieties or therapeutic agents, have been identified that achieve a reduction of total body radioactivity by increasing the extent of excretion of the catabolic/metabolic products while maintaining the pharmacokinetics of the intact molecule when compared to similar bifunctional chelates in the public domain.
• This reduction in total body radioactivity has been determined to be due to the clearance of catabolic/metabolic by-products and does not impact the other in vitro and in vivo properties such as degree of specificity (in vitro binding), cellular retention, and tumor uptake in vivo. When taken in whole, these embodiments achieve the desired properties of
• Radioimmunoconjugates by reducing the body burden of radioactivity while maintaining on-target activity.
• Therapeutic or targeting moieties include any molecule or any part of a molecule that confers a therapeutic benefit.
• the therapeutic moiety is a protein or polypeptide, e.g., an antibody, an antigen-binding fragment thereof.
• the therapeutic moiety is a small molecule.
• Targeting moieties include any molecule or any part of a molecule that binds to a given target.
• the targeting moiety is a protein or polypeptide such as antibodies or antigen binding fragments thereof, nanobodies, affibodies, and consensus sequences from Fibronectin type III domains (e.g., Centyrins or Adnectins).
• 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.
• hematologic agents including, for instance, erythropoietin, blood-clotting factors, etc.
• interferons including, for instance, erythropoietin, blood-clotting factors, etc.
• colony stimulating factors antibodies, enzymes, and hormones.
• any polypeptide of interest can be a polypeptide in the present methods.
• a reference polypeptide described herein can include a target-binding domain that binds to a target of interest (e.g., binds to an antigen).
• a polypeptide such as an antibody, can bind to a transmembrane polypeptide (e.g., receptor) or ligand (e.g., a growth factor).
• Exemplary molecular targets (e.g., antigens) for polypeptides described herein include CD proteins such as CD2, CD3, CD4, CD8, CD1 1 , CD1 9, CD20, CD22, CD25, CD33, CD34, CD40, CD52; members of the ErbB receptor family such as the EGF receptor (EGFR, HER1 , ErbB1 ), HER2 (ErbB2), HER3 (ErbB3) or HER4 (ErbB4) receptor; macrophage receptors such as CRIg; tumor necrosis factors such as TNFa or TRAIL/Apo-2; cell adhesion molecules such as LFA-1 , Mad , p150,95, VLA-4, ICAM-1 , VCAM and ⁇ 3 integrin including either a or ⁇ subunits thereof (e.g., anti- CD1 1 a, anti-CD18 or anti-CD1 1 b antibodies); growth factors and receptors such as EGF, FGFR (e).
• EGF EGF
• molecular targets include Tweak, B7RP-1 , proprotein convertase subtilisin/kexin type 9 (PCSK9), sclerostin, c-kit, Tie-2, c-fms, and anti-M1 .
• PCSK9 proprotein convertase subtilisin/kexin type 9
• sclerostin c-kit
• Tie-2 c-fms
• anti-M1 anti-M1 .
• An IgG antibody consists of 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 found in 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.
• the light chain includes 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).
• the heavy chain includes an additional constant region (CH4).
• Antibodies described herein 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.
• Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass.
• antigen binding fragment of an antibody 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).
• CDR complementarity determining region
• Antibodies or 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 ).
• IGF-1R Insulin-like growth factor 1
• Insulin-like growth factor 1 receptor is a transmembrane protein found on the surface of human cells activated by insulin-like growth factor 1 (IGF-1 ) and 2 (IGF-2).
• Radioimmunoconjugates of the invention may include the insulin-like growth factor-1 receptor (IGF-1 R).
• IGF-1 R insulin-like growth factor-1 receptor
• IGF-1 R promotes initiation and progression of cancer, playing a critical role in mitogenic transformation and maintenance of the transformed phenotype.
• IGF-1 R has been associated with development of multiple common cancers including breast, lung (e.g., non-small lung), liver, prostate, pancreas, ovarian, colon, melanoma, adrenocortical carcinoma, and various types of sarcomas.
• IGF- 1 R signaling stimulates tumour cell proliferation and metabolism, supports angiogenesis, and confers protection from apoptosis. It affects metastatic factors (e.g.HIF-1 dependent hypoxia signaling), anchorage independent growth, as well as growth and survival of tumour metastases after extravasation. IGF-1 R has also been implicated in the development, maintenance and enrichment of therapeutic resistant cancer stem cell populations.
• Radioimmunotherapy may provide a viable mechanism for treating cancers over expressing the IGF-1 receptor by utilizing the ability of IGF-1 R to undergo antibody triggered internalization and lysosomal degradation to deliver targeted radioisotopes inside cancer cells. Internalization and lysosomal degradation of an IGF-1 R targeted radioimmunoconjugate prolongs the residence time of the delivered radioisotope inside cancer cells thereby maximizing the potential for a cell killing emission to occur.
• cell death can be accomplished by as little as 1 atom of radionuclide delivered per cell [Sgouros, et al. J Nucl Med. 2010, 51 :31 1 -2].
• Cell killing due to direct DNA impact and breakage by an alpha particle may occur in the targeted cell or in a radius of 2 or 3 non-targeted cells for a given alpha particle decay.
• IGF-1 R targeted radioimmunoconjugates may not generate mechanistic resistance as they do not rely on blocking ligand binding to the receptor to inhibit the oncologic process, as needed with a therapeutic antibody.
• IGF-1 R antibodies have been developed and investigated for the treatment of various types of cancers including figitumumab, cixutumumab, ganitumab, AVE1642 (also known as humanized EM164 and huEM164), BIIB002, robatumumab, and teprotumumab. After binding to IGF- 1 R, these antibodies are internalized into the cell and degraded by lysosomal enzymes. The combination of overexpression on tumor cells and internalization offers the possibility of delivering detection agents directly to the tumor site while limiting the exposure of normal tissues to toxic agents.
• the CDRs of the light chain variable region of AVE1642 have the sequences:
• the light chain variable region of AVE1642 has the sequence:
• the CDRs of the heavy chain variable region of AVE1642 have the sequences:
• the heavy chain variable region of AVE1642 has the sequence:
• 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 bind 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).
• 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).
• Affibody molecules showing specific binding to a variety of different proteins e.g. insulin, fibrinogen, transferrin, tumor necrosis factor-a, IL-8, gp120, CD28, human serum albumin, IgA, IgE, IgM, H ER2 and EGFR
• proteins e.g. insulin, fibrinogen, transferrin, tumor necrosis factor-a, IL-8, gp120, CD28, human serum albumin, IgA, IgE, IgM, H ER2 and EGFR
• the Fibronectin type III domain is an evolutionarily conserved protein domain found in a wide- variety of extracellular proteins.
• the Fibronectin type III domain has been used as a molecular scaffold to produce molecules capable of selectively binding a specific antigen.
• Variants of the Fibronectin type III domains (FN3) that have been engineered for selective-binding may also be referred to as monobodies.
• FN3 domains may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., CIS-display, phage display, yeast display, bacterial display, mRNA display, ribosome display). Modified polypeptides
• the polypeptides of the invention may have a modified amino acid sequence.
• 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 IGF-1 R).
• 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 processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination.
• 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 any of the polypeptides of the invention 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.
• 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, or (c) the bulk of the side chain.
• a cross-linking group is a reactive group that is able to join two or more molecules by a covalent
• 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.
• 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
• 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.
• a detection agent is a molecule or atom which is administered conjugated to a polypeptide, e.g., an antibody or antigen-binding fragment thereof, and is useful in diagnosing a disease by locating the cells containing the antigen, radiation treatment planning, or treatment of a disease.
• useful detection agents include, but are not limited to, radioisotopes, dyes (such as with the biotin- streptavidin complex), contrast agents, fluorescent compounds or molecules, luminescent agents, and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI).
• MRI magnetic resonance imaging
• Radioisotopes and radionuclides known in the art for their utility as detection agents 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, 67 Cu, 75 Br, 76 Br , 77 Br , 89 Zr, 86 Y, 87 Y, 90 Y, 97 Ru,"Tc, 99m Tc 105 Rh, 109 Pd, 11 1 ln, 123 l, 124 l, 125 l, 131 l, 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, 229Th , 66 Ga, 67 Ga, 68 Ga, 82 Rb, 117m Sn, 201 TI.
• Chelating moieties are known in the art for their utility as detection agents include, but are not limited to, DOTA (1 ,4,7, 10-tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid), DOTMA (1 R,4R,7R, 10R)-a, a', a", a' etramethyl-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), D03AM-acetic acid (2-(4,7, 10-tris(2-amino-2- oxoethyl)-1 ,4,7, 10-tetraazacyclodo
• 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 4,000 keV), such as 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 86 Y, 87 Y, 90 Y, 97 Ru, 99m Tc, 105 Rh, 109 Pd, 111 In, 117m Sn, 149 Tb, 149 Pm, 153 Sm, 177 Lu, 186 Re, 188 Re, 199 Au, 201 TI, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 225 Ac, and 227 Th.
• metals such as manganese, iron, and gadolinium and isotopes (e.g., isotopes in the general energy range of 60 to 4,000
• Linkers of the invention may have the structure of Formula I:
• A is chelating moiety or a metal complex thereof
• L 1 is optionally substituted C C 6 alkyl, substituted C C 6 heteroalkyl, substituted aryl or heteroaryl;
• B is a is a therapeutic moiety, a targeting moiety, or cross-linking group,
• n 1 -5;
• each L 2 independently, has the structure:
• L 3 is optionally substituted C C 5 o alkyl or optionally substituted C C50 heteroalkyl or C 5 -C 2 o
• the conjugates of the invention comprise three distinct modules that together result in their increased effectiveness compared to those known in the art.
• Module A is included for incorporation of a detection agent (e.g., a chelating moiety or metal complex thereof).
• a metal complex may include an imaging radionuclide.
• A is chelating moiety or a metal complex thereof
• L 1 is optionally substituted C C 6 alkyl, substituted C C 6 heteroalkyl, substituted aryl or heteroaryl;
• B is a is a therapeutic moiety, a targeting moiety, or cross-linking group
• n 1 -5;
• each L 2 independently, has the structure:
• NR 1 and R 1 is H or optionally substituted C r C 6 alkyl or optionally substituted C C 6 heteroalkyl, substituted aryl or heteroaryl;
• L 3 is optionally substituted C C 5 o alkyl or optionally substituted C C50 heteroalkyl or C 5 -C 20 polyethylene glycol;
• Module B is a therapeutic moiety (e.g., antibodies, antigen-binding fragments), a targeting moiety (e.g. nanobodies, affibodies, consensus sequences from Fibronectin type III domains), or a cross- linking group (e.g. amino-reactive, thiol-reactive cross-linking group, or a sortase-mediated coupling).
• a therapeutic moiety e.g., antibodies, antigen-binding fragments
• a targeting moiety e.g. nanobodies, affibodies, consensus sequences from Fibronectin type III domains
• a cross- linking group e.g. amino-reactive, thiol-reactive cross-linking group, or a sortase-mediated coupling.
• the present invention also features pharmaceutical compositions that contain a
• compositions can be formulated for use in a variety of drug delivery systems.
• One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).
• the pharmaceutical compositions are intended for parenteral, intranasal, topical, oral, or local administration, such as by a transdermal means, for prophylactic and/or therapeutic treatment.
• the pharmaceutical compositions can be administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application or intraarticular injection at areas affected by the vascular or cancer condition. Additional routes of administration include intravascular, intra-arterial, intratumor, intraperitoneal, intraventricular, intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or aerosol inhalation administration.
• compositions for parenteral administration that include the above mention agents dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, or PBS, among others.
• an acceptable carrier preferably an aqueous carrier, e.g., water, buffered water, saline, or PBS, among others.
• the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, or detergents, among others.
• compositions for oral delivery which may contain inert ingredients such as binders or fillers for the formulation of a unit dosage form, such as a tablet or a capsule.
• this invention provides compositions for local administration, which may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, a gel, a paste, or an eye drop.
• compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered.
• the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
• the pH of the preparations typically will be between 3 and 1 1 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 6 and 7, such as 6 to 6.5.
• the resulting compositions in solid form may be packaged 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.
• the composition 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.
• compositions containing an effective amount can be administered for radiation treatment planning, diagnostic, or therapeutic treatments.
• the conjugate is administered to a subject in a diagnostically effective dose and/or an amount effective to determine the therapeutically effective dose.
• compositions are 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.
• 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 will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the disease or condition symptoms are ameliorated, or the term of the disease or condition is changed or, for example, is less severe or recovery is accelerated in an individual.
• the conjugates of the invention can be used for the treatment of cancer by administering to a subject a first dose of any of the foregoing conjugates or compositions in an amount effective for radiation treatment planning, followed by administering a second dose of any of the foregoing conjugates or compositions in a therapeutically effective amount.
• Amounts effective for these uses may depend on the severity of the disease or condition and the weight and general state of the subject.
• the therapeutically effective amount of the compositions of the invention and used in the methods of this invention applied to mammals can be determined by the ordinarily-skilled artisan with consideration of individual differences in age, weight, and the condition of the mammal. Because certain conjugates of the invention exhibit an enhanced ability to target cancer cells and residualize, the dosage of the compounds of the invention can be lower than (e.g.
• agents of the invention are administered to a subject (e.g., a mammal, such as a human) in an effective amount, which is an amount that produces a desirable result in a treated subject.
• an effective amount can also be determined empirically by those of skill in the art.
• compositions of the invention including an effective amount can be carried out with dose levels and pattern being selected by the treating physician.
• the 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 conjugates of the present invention may be used in combination with either conventional methods of treatment or therapy or may be used separately from conventional methods of treatment or therapy.
• compositions according to the present invention may be comprised of a combination of a compound of the present invention in association with a pharmaceutically acceptable excipient, as described herein, and another therapeutic or prophylactic agent known in the art.
• antiproliferative or “antiproliferative agent,” as used interchangeably herein, is meant any anticancer agent, including those antiproliferative agents listed in Table 2, any of which can be used in combination with a conjugate of the invention to treat the medical conditions recited herein.
• Antiproliferative agents also include organo-platinum derivatives, naphtoquinone and benzoquinone derivatives, chrysophanic acid and anthroquinone derivatives thereof.
• immuno-modulatory agent or “immunomodulatory agent,” as used interchangeably herein, is meant any immuno-modulator, including those listed in Table 2, any of which can be used in combination with a conjugate of the invention to treat the medical conditions recited herein.
• Radiation sensitizer includes any agent that increases the sensitivity of cancer cells to radiation therapy.
• Radiation sensitizers may include, but are not limited to, 5- fluorouracil, analogs of platinum (e.g., cisplatin, carboplatin, oxaliplatin), gemcitabine, EGFR antagonists (e.g., cetuximab, gefitinib), farnesyltransferase inhibitors, COX-2 inhibitors, bFGF antagonists, and VEGF anatagonists.
• Alkylating agents Busulfan Chlorambucil
• picoplatin AP-5280 (Access) oxaliplatin cisplatin
• etoposide gimatecan (Sigma-Tau) teniposide or mitoxantrone diflomotecan (Beaufour-lpsen)
• vindesine IDN 5109 (Bayer) dolastatin 10 (NCI)
• NCI dolastatin 10
• a 105972 Abbott
• rhizoxin (Fujisawa)
• a 204197 Abbott) mivobulin (Warner-Lambert)
• LU 223651 (BASF)
• TXD 258 (Aventis) combretastatin A4 (BMS) epothilone B (Novartis) isohomohalichondrin-B (PharmaMar)
• Fabre vinflunine
• AVLB Prescient NeuroPharma
• auristatin PE Teikoku Hormone
• azaepothilone B BMS
• BMS 188797 BMS 188797 (BMS) dolastatin-10 (NIH) taxoprexin (Protarga) CA-4 (OXiGEN E)
• glufosfamide (Baxter International) mafosfamide (Baxter International) albumin + 32P (Isotope Solutions) apaziquone (Spectrum
• TNF alpha virulizin (Lorus Therapeutics) revimid (Celgene)
• Immuno-modulators interferon dexosome therapy (Anosys) oncophage (Antigenics) pentrix (Australian Cancer GMK (Progenies) Technology) adenocarcinoma vaccine (Biomira) ISF-154 (Tragen)
• SR-27897 CCK A inhibitor, Sanofi-Synthelabo ceflatonin (apoptosis promotor, ChemGenex) tocladesine (cyclic AMP agonist, Ribapharm) BCX-1777 (PN P inhibitor, BioCryst) alvocidib (CDK inhibitor, Aventis) ranpirnase (ribonuclease stimulant, Alfacell) CV-247 (COX-2 inhibitor, Ivy Medical) galarubicin (RNA synthesis inhibitor, Dong-A) P54 (COX-2 inhibitor, Phytopharm) tirapazamine (reducing agent, SRI CapCellTM (CYP450 stimulant, Bavarian Nordic) International)
• GCS-100 gal3 antagonist, GlycoGenesys
• N-acetylcysteine reducing agent, Zambon
• G17DT immunogen Gastrin inhibitor, Aphton
• R-flurbiprofen NF-kappaB inhibitor, Encore
• efaproxiral oxygenator, Alios Therapeutics
• 3CPA N F-kappaB inhibitor, Active Biotech
• PI-88 heparanase inhibitor, Progen
• seocalcitol vitamin D receptor agonist, Leo
• tesmilifene histamine antagonist, YM 131 -I-TM-601 (DNA antagonist,
• histamine histamine H2 receptor agonist, Maxim
• ODC inhibitor eflornithine
• tiazofurin IMPDH inhibitor
• Ribapharm tiazofurin
• minodronic acid osteoclast inhibitor, cilengitide (integrin antagonist, Merck KGaA) Yamanouchi
• SR-31747 (IL-1 antagonist, Sanofi-Synthelabo) indisulam (p53 stimulant, Eisai)
• CCI-779 mTOR kinase inhibitor, Wyeth
• PPT inhibitor PharmaMar
• exisulind PDE V inhibitor, Cell Pathways
• gemtuzumab CD33 antibody, Wyeth Ayerst
• CP-461 PDE V inhibitor, Cell Pathways
• PG2 hematopoiesis enhancer
• WX-UK1 plasmaogen activator inhibitor, Wilex
• ImmunolTM triclosan oral rinse, Endo
• PBI-1402 PMN stimulant, ProMetic LifeSciences
• triacetyluridine uridine prodrug , Wellstat
• bortezomib proteasome inhibitor, Millennium
• SRL-172 T cell stimulant, SR Pharma) Bioscience
• TLK-286 glutthione S transferase inhibitor, TransMID-107TM (immunotoxin, KS Biomedix)
• PT-100 growth factor agonist, Point doranidazole (apoptosis promotor, Pola)
• CHS-828 cytotoxic agent, Leo
• midostaurin PLC inhibitor, Novartis
• trans-retinoic acid differentiator, NIH
• bryostatin-1 PLC stimulant, GPC Biotech
• MX6 apoptosis promotor, MAXIA
• CDA-II apoptosis promotor, Everlife
• apomine apoptosis promotor, ILEX Oncology
• SDX-101 apoptosis promotor, Salmedix
• urocidin apoptosis promotor, Bioniche
• rituximab CD20 antibody, Genentech Ro-31 -7453 (apoptosis promotor, La Roche) carmustine brostallicin (apoptosis promotor, Pharmacia) Mitoxantrone ⁇ -lapachone Bleomycin gelonin Absinthin cafestol Chrysophanic acid kahweol Cesium oxides caffeic acid
• BRAF inhibitors Tyrphostin AG
• PD-L1 inhibitors PD-1 inhibitors
• MEK inhibitors CTLA-4 inhibitors
• the antibodies used were HuMIgG (Aldrich, I4506) and HuMIGF-1 R (AVE1642).
• Lutetium- 177 was received from Perkin Elmer as lutetium chloride in a 0.05 N hydrochloric acid solution.
• Analytical H PLC-MS was performed using a Waters Acquity H PLC-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 x50 (1.7 ⁇ ) column.
• a Waters Acquity H PLC-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
• Preparative H PLC was performed using a Waters HPLC system comprised of a Waters 1525 Binary H PLC pump, a Waters 2489 UV/Visible Detector (monitoring at 254 nm and 214 nm) and a Waters XBridge Prep phenyl or C18 1 9x 1 00 mm (5 pm) column.
• HPLC elution method 1 Waters Acquity BEH C18 2.1 x50 mm (1.7 pm) column; mobile phase
• Analytical Size Exclusion Chromatography was performed using a Waters system comprised of a Waters 1525 Binary H PLC pump, a Waters 2489 UV/Visible Detector (monitoring at 280 nm), a Bioscan Flow Count radiodetector (FC-3300) and TOSOH TSKgel G3000SWxl, 7.8x300 mm column.
• the bifunctional chelating agent 2,2',2"-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1 ,4,7, 10- tetraazacyclododecane-1 ,4,7-triyl)triacetic acid was obtained from CheMatech.
• the Compound A (3.0 ⁇ ) was dissolved in sodium acetate buffer (0.228 mL, pH 6.5). An aliquot of the Compound A solution (8 ⁇ _, 106 nmoles) was added to a solution containing the antibody HuMIGF-1 R (6.7 nmoles, AVE1642) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate (Compound A)-HuMIGF-1 R was eluted from the column with acetate buffer (pH 6.5). SEC retention time: 8.2 min; MALDI-MS (positive ion): (Compound A)-HuMIGF-1 R found m/z 151759; HuMIGF-1 R found m/z 149835.
• the Lu-177 (1 .1 mCi, 14 ⁇ _) was added to a solution of (Compound A)- HuMIGF-1 R (100 ⁇ g in acetate buffer (pH 6.5) and ascorbic acid (1 ⁇ _, 0.1 M in acetate buffer (pH 6.5)).
• the radiolabeling reaction was incubated at 37 S C for 30 minutes.
• the crude product, [ 177 Lu]- Compound A-HuMIGF-1 R was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). SEC retention time: 8.1 min; radioTLC radiochemical purity: 99%; radiochemical yield: 74%; specific activity: 8.2 mCi/mg.
• Compound B (0.7 ⁇ ) was dissolved in sodium acetate buffer (69 ⁇ _, pH 6.5). An aliquot of Compound B solution (4 ⁇ _, 40 nmoles) was added to a solution containing the antibody HuMIGF- 1 R (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound B-HuMIGF-1 R was eluted from the column with acetate buffer (pH 6.5).
• MALDI-TOF-MS positive ion: Compound B-HuMIGF-1 R: found m/z 152988 [M+H] + ; HuMIGF-1 R: found m/z 149835 [M+H] + .
• the Lu-177 (1 .15 mCi, 14 ⁇ _) was added to a solution of Compound B- HuMIGF-1 R (75 ⁇ g in acetate buffer (pH 6.5) and ascorbic acid (1 ⁇ _, 0.1 M in acetate buffer (pH 6.5)).
• the radiolabeling reaction was incubated at 37 S C for 30 minutes.
• the crude product, [ 177 Lu]- Compound C-HuMIGF-1 R was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). RadioTLC radiochemical purity: 99%; radiochemical yield: 75%; specific activity: 1 1 .9 mCi/mg.
• the Compound C (17.5 ⁇ ) was dissolved in sodium acetate buffer (1.32 ml_, pH 6.5). An aliquot of Compound C solution (8 ⁇ _, 91 nmoles) was added to a solution containing the antibody HuMIGF-1 R (13.4 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column.
• the Lu-177 (1 .6 mCi, 16 ⁇ _) was added to a solution of Compound C- HuMIGF-1 R (150 ⁇ g in acetate buffer (pH 6.5) and ascorbic acid (1 ⁇ _, 0.1 M in acetate buffer (pH 6.5)).
• the radiolabeling reaction was incubated at ambient temperature for 20 minutes.
• [ 177 Lu]- Compound C-HuMIGF-1 R was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). RadioTLC radiochemical purity: 99%; radiochemical yield: 91 %; specific activity: 15.6 mCi/mg.
• K d ligand concentration that binds to half the receptor sites at equilibrium
• Bmax maximum number of binding sites
• both total and nonspecific binding are measured, where specific binding to the receptor is calculated by subtracting the difference.
• Nonspecific binding is typically assessed by measuring radioconjugate binding in the presence of a fixed concentration of HumlGF-1 R that binds to essentially all the receptors. Since all the receptors are occupied by the HumlGF-1 R, the radioconjugate only binds nonspecifically.
• K d and B max values are calculated by nonlinear regression analysis and computerized curve fitting.
• This assay was to ensure that these new radioconjugates maintained binding characteristics consistent with the native antibody in an IGF-1 R expressing A431 cell line. Twenty- four hours prior to the start of the experiment, 1 .5x10 5 A431 cells were seeded in 48-well microplates in 500 ⁇ supplemented medium. The radioconjugate was diluted with binding buffer (PBS+0.5% BSA) to a range of concentrations from 0.08nM to 40nM; final assay concentration 0.04 to 20 nM. At the start of the assay, the media is aspirated, discarded and 500 ⁇ of serum-free DMEM was added to each well. The plates were incubated at 37°C for 1 hour.
• binding buffer PBS+0.5% BSA
• the lysates were transferred to counting tubes and run with radioconjugate standards on the Wizard 1470 gamma counter to determine the radioactivity content (in counts per minute (CPM)) for each lysate.
• the remaining lysate from each well (25 ⁇ ) was transferred to a 96- well plate, and the protein content of each lysate determined using a standard protein quantification assay.
• Total, non-specific and specific ligand binding determinations, mass of bound conjugate in each lysate were calculated by converting lysate CPM to fmol bound using the specific activity of the conjugate standards and then normalizing the fmol bound to the protein content of each lysate(in milligrams). Specific binding was determined by subtracting the non-specific binding from total binding.
• the residualization assay was designed to determine the degree of cell retention of radiolabeled-linker-antibody derivatives.
• the assay relies on the inherent ability of the IGF-1 receptor to internalize when bound to ligand and the ability to track radiolabeled compounds.
• a constant amount of radioconjugate is incubated with an IGF-1 R expressing cell line for a fixed period of time. Following incubation, the cells are stripped with a mild acid buffer to remove any external or membrane-bound radioconjugate. Fresh medium is re-applied and the cells are again incubated for a pre-determined amount of time.
• Residualization is determined by calculating the amount of internalized radioactivity as a percentage of the total cell-associated activity following acid wash.
• A431 cells were plated in 24-well plates at a concentration of 2.5x10 5 cells/well in full medium (DMEM). Following overnight incubation, the cells were changed to serum-free DMEM and incubated for 1 hour at 37 °C. Media was decanted and plates were washed once with sterile PBS. The radioconjugate was diluted in serum-free DMEM to a concentration of 2nM. 500uL of radioconjugate was loaded into each well and incubated for 4 hours at 37 °C. After incubation, plates were immediately placed on ice and medium was discarded into pre-labeled (non-bound) gamma counting tubes.
• DMEM full medium
• mice normal CD-1 or athymic CD-1 nude
• Immunoconjugates with various linkers were synthesized and radiolabelled with lutetium-177.
• animals were sacrificed at specific timepoints, and blood and tumor (when applicable) were analyzed for total radioactivity.
• metabolism studies animals were placed in metabolic cages (4-5 per cage) for urine and feces collection every 24 hours for up to 7 days. The radioactive content of urine and feces samples was quantified and converted to total urine or feces output based on weight.
• Excretion profiles for urine, feces, or total excretion (urine + feces) were generated by plotting cumulative % injected dose (%ID) over time.
• the route of synthesis of the actinium-225 (Ac-225) radiolabeled compounds were similar to that for the corresponding Lu-177 analogs.
• Therapeutic efficacy studies were carried out using the IGF-1 R overexpressing colon cancer cell line Colo-205 (ATCC #CCL-222). Tumor xenografts are established in 5-7 week old female Balb/c athymic nude mice (Charles River Laboratories).
• non-radiolabelled, non-conjugated antibody (HuMIGF-1 R) was administered at a protein mass equivalent corresponding to the highest radioactivity dose of the actinium-225 radioimmunoconjugates tested in a study.
• Tumor growth was expressed as relative tumor volume (RTV) which is tumor volume measured on day X divided by the tumor volume measured on the day of dosing.
• the compound Compound A (1 .34 ⁇ ) was dissolved in sodium acetate buffer (20 ⁇ _, pH 6.5) and added to a solution containing the antibody Human-lgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 45 minutes at ambient temperature, the resulting immunoconjugate was purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). The antibody conjugate Compound A-Human-lgG.
• MALDI-TOF-MS positive ion: Compound A-Human-lgG: found m/z 150360 [M+H] + ; Human-lgG: found m/z 148339 [M+H] + .
• the Lu-177 (1 .1 mCi, 5 ⁇ _) was added to a solution of Compound A- Human-lgG (90 ⁇ g in acetate buffer (pH 6.5) and ascorbic acid (1 ⁇ _, 0.1 M in acetate buffer (pH 6.5)).
• the radiolabeling reaction was incubated at 37 S C for 90 minutes.
• the crude product, [ 177 Luj- Compound A-Human-lgG, was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid. RadioTLC radiochemical purity: 98%; radiochemical yield: 45%; specific activity: 15.1 mCi/mg.
• Compound B (1 .17 ⁇ ) was dissolved in sodium acetate buffer (0.1 1 7 ml_, pH 6.5). An aliquot of the Compound B solution (2 ⁇ _, 10 nmoles) was added to a solution containing the antibody Human-lgG (6.7 nmoles) in a bicarbonate buffer (pH 8.5).
• the human IgG preparation used consisted of a purified mixture of all IgG isotypes (lgG1 -4). After 1 hour at ambient temperature the antibody conjugate product was purified via a Sephadex G-50 resin packed column. Compound A-Human-lgG was eluted from the column with acetate buffer (pH 6.5).
• MALDI-TOF-MS positive ion: Compound B- Human-lgG found m/z 149949 [M+H] + ; Human-lgG found m/z 148540 [M+H] + .
• the Lu-177 (1 .1 mCi, 5 ⁇ -) was added to a solution of Compound B- Human-lgG (100 ⁇ g in acetate buffer (pH 6.5) and ascorbic acid (1 ⁇ , 0.1 M in acetate buffer (pH 6.5)).
• the radiolabeling reaction was incubated at 37 °C for 30 minutes.
• the crude product, 177 Lu- Compound B-Human-lgG was purified via a H PLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa,). RadioTLC radiochemical purity: 98%; radiochemical yield: 51 %; specific activity: 9.68 mCi/mg.
• the compound Compound C (0.96 ⁇ ) was dissolved in sodium acetate buffer (95 ⁇ , pH 6.5). An aliquot of the Compound C solution (2 ⁇ , 20 nmoles) was added to a solution containing the antibody Human-lgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate product was purified via a Sephadex G-50 resin packed column. Compound C-Human-lgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound C-Human-lgG: found m/z 150095 [M+H] + ; Human-lgG: found m/z 148540 [M+H] + .
• the Lu-177 (1 .1 mCi, 5 ⁇ -) was added to a solution of Compound C- Human-lgG (100 ⁇ g in acetate buffer (pH 6.5) and ascorbic acid (1 ⁇ , 0.1 M in acetate buffer (pH 6.5)).
• the radiolabeling reaction was incubated at 37 S C for 30 minutes.
• the crude product, [ 177 Luj- Compound C-Human-lgG was purified via a H PLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa,). RadioTLC radiochemical purity: 98%; radiochemical yield: 37%; specific activity: 9.99 mCi/mg.
• Example 14 Pharmacokinetic and Metabolism Study Results for HuMlgG based compounds
• Non-targeted human IgG antibodies were used for metabolic excretion studies in order to demonstrate that the alterations in radioactivity excretion profiles directed by conjugation with linker Compound B and Compound C is a general process demonstrating that these finding are not-limited to HuMIGF-1 R antibody.
• Pharmacokinetic and metabolism studies were carried out using [ 177 Luj- Compound A-HuMIgG, [ 177 Lu]-Compound B-HuMIgG, and [ 177 Lu]-Compound C-HuMIgG as described for the HuMIGF-1 R antibody based compounds described previously.
• the compound Compound A (1 .34 ⁇ ) was dissolved in sodium acetate buffer (20 ⁇ , pH 6.5) and added to a solution containing the antibody Human-lgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 45 minutes at ambient temperature the antibody conjugate product was purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).
• MALDI-TOF-MS positive ion: Compound A-Human-lgG: found m/z 150360 [M+H] + ; Human- IgG: found m/z 148339 [M+H] + .
• the compound Compound B (1 .17 ⁇ ) was dissolved in sodium acetate buffer (0.1 17 ml_, pH 6.5). An aliquot of the Compound B solution (2 ⁇ _, 10 nmoles) was added to a solution containing the antibody Human-lgG (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature the antibody conjugate product was purified via a Sephadex G-50 resin packed column. The antibody conjugate Compound A-Human-lgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound B-Human-lgG found m/z 149949 [M+H] + ; Human-lgG found m/z 148540 [M+H] + .
• the Ac-225 (1 .1 mCi, 5 ⁇ _) is added to a solution of Compound B- Human-lgG (100 ⁇ g in acetate buffer (pH 6.5) and ascorbic acid (1 ⁇ _, 0.1 M in acetate buffer (pH 6.5)).
• the radiolabeling reaction is incubated at ambient temperature (e.g., 20-25 s C) for 30 minutes.
• the crude product, [ 225 Ac]-Compound B-Human-lgG is purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa).
• the compound Compound C (0.96 ⁇ ) was dissolved in sodium acetate buffer (95 ⁇ _, pH 6.5). An aliquot of the Compound C solution (2 ⁇ _, 20 nmoles) was added to a solution containing the antibody Human-lgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature the antibody conjugate product was purified via a Sephadex G-50 resin packed column. The antibody conjugate Compound C-Human-lgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound C-Human-lgG: found m/z 150095 [M+H] + ; Human-lgG: found m/z 148540 [M+H] + .
• the Ac-225 (1 .1 mCi, 5 ⁇ _) is added to a solution of Compound C- Human-lgG (100 ⁇ g in acetate buffer (pH 6.5) and ascorbic acid (1 ⁇ _, 0.1 M in acetate buffer (pH 6.5)).
• the radiolabeling reaction is incubated at ambient temperature (e.g., 20-25 s C) for 30 minutes.
• the crude product, [ 225 Ac]-Compound C-Human-lgG is purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa,).
• the Compound A (3.0 ⁇ ) was dissolved in sodium acetate buffer (0.228 mL, pH 6.5). An aliquot of the Compound A solution (8 ⁇ _, 106 nmoles) was added to a solution containing the antibody HuMIGF-1 R (6.7 nmoles, AVE1642) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate (Compound A)-HuMIGF-1 R was eluted from the column with acetate buffer (pH 6.5). SEC retention time: 8.2 min; MALDI-MS (positive ion): (Compound A)-HuMIGF-1 R found m/z 151759; HuMIGF-1 R found m/z 149835.
• the Ac-225 (1 .1 mCi, 14 ⁇ _) is added to a solution of (Compound A)- HuMIGF-1 R (100 ⁇ g in acetate buffer (pH 6.5) and ascorbic acid (1 ⁇ _, 0.1 M in acetate buffer (pH 6.5)).
• the radiolabeling reaction is incubated at ambient temperature (e.g., 20-25 s C) for 30 minutes.
• the crude product, [ Ac]-Compound A-HuMIGF-1 R is purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).
• Compound B (0.7 ⁇ ) was dissolved in sodium acetate buffer (69 ⁇ _, pH 6.5). An aliquot of Compound B solution (4 ⁇ _, 40 nmoles) was added to a solution containing the antibody HuMIGF- 1 R (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound B-HuMIGF-1 R was eluted from the column with acetate buffer (pH 6.5).
• MALDI-TOF-MS positive ion: Compound B-HuMIGF-1 R: found m/z 152988 [M+H] + ; HuMIGF-1 R: found m/z 149835 [M+H] + .
• the Ac-225 (1 .15 mCi, 14 ⁇ _) iss added to a solution of Compound B- HuMIGF-1 R (75 ⁇ g in acetate buffer (pH 6.5) and ascorbic acid (1 ⁇ _, 0.1 M in acetate buffer (pH 6.5)).
• the radiolabeling reaction is incubated at ambient temperature (e.g., 20-25 s C) for 30 minutes.
• the crude product, [ 225 Ac]-Compound C-HuMIGF-1 R is purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).
• the Compound C (17.5 ⁇ ) was dissolved in sodium acetate buffer (1.32 ml_, pH 6.5). An aliquot of Compound C solution (8 ⁇ _, 91 nmoles) was added to a solution containing the antibody HuMIGF-1 R (13.4 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column.
• the Ac-225 (1 .6 mCi, 16 ⁇ _) is added to a solution of Compound C- HuMIGF-1 R (150 ⁇ g in acetate buffer (pH 6.5) and ascorbic acid (1 ⁇ _, 0.1 M in acetate buffer (pH 6.5)).
• the radiolabeling reaction is incubated at ambient temperature (e.g., 20-25 s C) for 30 minutes.

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