WO2013063001A1 - Associations thérapeutiques et méthodes de traitement du mélanome - Google Patents
Associations thérapeutiques et méthodes de traitement du mélanome Download PDFInfo
- Publication number
- WO2013063001A1 WO2013063001A1 PCT/US2012/061533 US2012061533W WO2013063001A1 WO 2013063001 A1 WO2013063001 A1 WO 2013063001A1 US 2012061533 W US2012061533 W US 2012061533W WO 2013063001 A1 WO2013063001 A1 WO 2013063001A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- etbr
- mpk
- seq
- inhibitor
- Prior art date
Links
- 0 CCC(CC)(C(C)C1IC)*1(C1)*(CC)(CC)C2[C@]1C(C)C2 Chemical compound CCC(CC)(C(C)C1IC)*1(C1)*(CC)(CC)C2[C@]1C(C)C2 0.000 description 4
- NJIVQNBISDYANJ-DTWKUNHWSA-N CCN[C@H](C)[C@H](C)C(C)(C)C Chemical compound CCN[C@H](C)[C@H](C)C(C)(C)C NJIVQNBISDYANJ-DTWKUNHWSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
- A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
-
- 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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
-
- 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
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3053—Skin, nerves, brain
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
Definitions
- This invention concerns in general, a treatment of melanoma by using certain antibody and small molecule drug combinations.
- melanomas can demonstrate molecular variations, for example in certain signal transduction pathways necessary for cell responsiveness to growth factors. Therefore, rather than treating melanoma as a single disease, at have been made to stratify it into molecular subtypes in order to treat each subtype with the most appropriate therapies.
- the MAPK pathway is a phosphorylation-driven signal transduction cascade that couples intracellular responses to the binding of growth factors to cell surface receptors. This pathway regulates several processes including cell proliferation and differentiation, and is often dysregulated in a variety of cancers. (Sebolt-Leopold JS, Herrera R. Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat Rev Cancer. 2004;4:937-947).
- the classical MAPK pathway consists of RAS, RAF, MEK and ERK, where RAS triggers the formation of a RAF/MEK/ERK kinase complex which then drives transcription of key regulators through protein phosphorylation.
- RAS triggers the formation of a RAF/MEK/ERK kinase complex which then drives transcription of key regulators through protein phosphorylation.
- the inhibition of MAPK signaling with agents targeted toward critical proteins in the pathway has the potential to inhibit growth in a variety of tumor types (Wong K-K et al., Recent developments in anti-cancer agents targeting the Ras/Raf/MEK/ERK pathway. Recent Pat Anticancer Drug Discov. 2009;4:28-35.
- GDC-0973 (a.k.a. XL518) is a potent and highly selective small molecule inhibitor of MEK1/2, a MAPK kinase that activates ERK1/2 (Johnston S. XL518, a potent, selective, orally bioavailable MEK1 inhibitor, downregulates the Ras/Raf/MEK/ERK pathway in vivo, resulting in tumor growth inhibition and regression in preclinical models.
- AACR-NCI-EORTC Symposium on Molecular Targets and Cancer Therapeutics October 22, 2007; San Francisco, CA. Abstract C209.
- vemurafenib also known as Zelboraf®, which is a B-Raf enzyme inhibitor
- Zelboraf® which is a B-Raf enzyme inhibitor
- Vemurafenib has been shown to cause programmed cell death in melanoma cell lines (Sala E, et al., BRAF silencing by short hairpin RNA or chemical blockade by PLX4032 leads to different responses in melanoma and thyroid carcinoma cells. Mol. Cancer Res. 6 (5): 751-9 (May 2008).
- Vemurafenib interrupts the B-Raf/MEK step on the B-Raf/MEK/ERK pathway if the B-Raf has the common V600E mutation.
- Vemurafenib is effective in melanoma patients whose cancer has a V600E BRAF mutation (that is, at amino acid position number 600 on the B-RAF protein, the normal valine is replaced by glutamic acid). About 60% of melanomas have the V600E BRAF mutation.
- the present invention contemplates a method of tumor growth inhibition (TGI) in a subject suffering from melanoma comprising administering to the subject an effective amount of an anti- endothelin B receptor (ETBR) antibody drug conjugate in combination with an effective amount of a MAP kinase inhibitor.
- TGI tumor growth inhibition
- the combination of an anti-ETBR antibody drug conjugate and a MAP kinase inhibitor is synergistic.
- the TGI is greater than the TGI seen using an anti-ETBR antibody drug conjugate alone or greater than the TGI seen using a MAP kinase inhibitor alone.
- the TGI is about 10% greater, or about 15%> greater, or about 20% greater, or about 25% greater, or about 30% greater, or about 35% greater, or about 40% greater, or about 45% greater, or about 50% greater, or about 55%o greater, or about 60% greater, or about 65% greater, or about 70% greater than use of an anti-ETBR antibody drug conjugate alone or the TGI is about 10% greater, or about 15% greater, or about 20% greater, or about 25% greater, or about 30% greater, or about 35% greater, or about 40% greater, or about 45%o greater, or about 50% greater, or about 55%> greater, or about 60% greater, or about 65%> greater, or about 70%) greater than use of a MAP kinase inhibitor alone.
- the anti-ETBR antibody specifically binds an ETBR epitope consisting of amino acids number 64 to 101 of SEQ ID NO: 10.
- the anti-ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs wherein VH CDR1 is SEQ ID NO: l, VH CDR2 is SEQ ID NO:2, VH CDR3 is SEQ ID NO:3 and wherein VL CDR1 is SEQ ID NO:4, VL CDR2 is SEQ ID NO:5, VL CDR3 is SEQ ID NO:6.
- the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO:7 or 9.
- a further aspect of this method also includes an anti-ETBR antibody also having a VL which is SEQ ID NO: 8.
- the anti-ETBR antibody is conjugated to a cytotoxin, wherein said cytotoxin is a cytotoxic agent that is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes, and wherein said cytotoxin is a toxin.
- the toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin.
- the toxin is a maytansinoid.
- the MAP kinase inhibitor is a BRAF inhibitor.
- the BRAF inhibitor is propane- 1- sulfonic acid ⁇ 3-[5-(4-chlorophenyl)-lH-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl ⁇ -amide.
- the BRAF inhibitor has the following chemical structure:
- the MAP kinase inhibitor is a MEK inhibitor.
- the MEK inhibitor is (S)-(3,4- difuoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)(3-hydoxy-3-(piperidin-2yl)azetidin-l-yl)methanone.
- the MEK inhibitor has the following chemical structure:
- a method of treating melanoma comprising administering to a subject in need thereof a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody is described.
- said melanoma is ETBR positive.
- said melanoma is metastatic.
- said subject has not had prior therapy with a MAP kinase inhibitor.
- said subject has a V600E BRAF gene mutation or said subject is BRAF wildtype, having no V600E BRAF mutation.
- the subject has not had prior therapy with a MAP kinase inhibitor.
- the combination of an anti-ETBR antibody and a MAP kinase inhibitor is synergistic.
- the TGI is greater than the TGI seen using an anti-ETBR antibody alone or greater than the TGI seen using a MAP kinase inhibitor alone.
- the TGI is about 10% greater, or about 15%> greater, or about 20% greater, or about 25% greater, or about 30% greater, or about 35% greater, or about 40% greater, or about 45% greater, or about 50%) greater, or about 55% greater, or about 60% greater, or about 65% greater, or about 70% greater than use of an anti-ETBR antibody alone or the TGI is about 10% greater, or about 15% greater, or about 20% greater, or about 25% greater, or about 30% greater, or about 35% greater, or about 40% greater, or about 45%) greater, or about 50% greater, or about 55%) greater, or about 60%) greater, or about 65% greater, or about 70%) greater than use of a MAP kinase inhibitor alone.
- the anti-ETBR antibody specifically binds an ETBR epitope consisting of amino acids number 64 to 101 of SEQ ID NO: 10.
- the anti-ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs wherein VH CDR1 is SEQ ID NO: l, VH CDR2 is SEQ ID NO:2, VH CDR3 is SEQ ID NO:3 and wherein VL CDR1 is SEQ ID NO:4, VL CDR2 is SEQ ID NO:5, VL CDR3 is SEQ ID NO:6.
- the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO:7 or 9.
- a further aspect of this method also includes an anti-ETBR antibody also having a VL which is SEQ ID NO: 8.
- the anti-ETBR antibody is conjugated to a cytotoxin, wherein said cytotoxin is cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes, and wherein said cytotoxin is a toxin.
- the toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin.
- the toxin is a maytansinoid.
- the MAP kinase inhibitor is a BRAF inhibitor.
- the BRAF inhibitor is propane- 1 -sulfonic acid ⁇ 3-[5-(4-chlorophenyl)-lH-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl ⁇ -amide.
- the BRAF inhibitor has the following chemical structure:
- the MAP kinase inhibitor is a MEK inhibitor.
- the MEK inhibitor is (S)-(3,4- difuoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)(3-hydoxy-3-(piperidin-2yl)azetidin-l-yl)methanone.
- the MEK inhibitor has the following chemical structure:
- a method of treating melanoma comprising administering to a subject in need thereof a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody drug conjugate is described, wherein the MAP kinase inhibitor is administered first to said subject in need thereof.
- said anti-ETBR antibody drug conjugate is administered after administration of said MAP kinase inhibitor.
- contemplated methods of the invention include where the anti-ETBR antibody and the MAP kinase inhibitor are administered simultaneously.
- a method of treating melanoma comprising administering to a subject in need thereof a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody drug conjugate is described, wherein the anti-ETBR antibody drug conjugate and the MAP kinase inhibitor are administered sequentially, wherein the anti-ETBR antibody drug conjugate is administered to the subject first and the MAP kinase inhibitor is administered to the subject after administration of the anti-ETBR antibody drug conjugate.
- a method of treating melanoma comprising administering to a subject in need thereof a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody drug conjugate is described, wherein the MAP kinase inhibitor is administered to the subject first and the anti-ETBR antibody drug conjugate is administered to the subject after administration of the MAP kinase inhibitor.
- a method of treating melanoma comprising administering to a subject in need thereof a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody drug conjugate is described, wherein said anti-ETBR antibody drug conjugate is administered intraveneously.
- the anti-ETBR antibody drug conjugate is dosed at about 0.1 mpk, or about 0.2 mpk, or about 0.3 mpk, or about 0.5 mpk, or about 1 mpk, or about 5 mpk, or about 10 mpk, or about 15 mpk, or about 20 mpk, or about 25 mpk, or about 30 mpk in the claimed methods of the invention.
- a method of treating melanoma comprising administering to a subject in need thereof a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody drug conjugate is described, wherein the MAP kinase inhibitor is administered orally.
- the BRAF inhibitor is dosed at about 1 mpk, or about 2 mpk, or about 3 mpk, or about 4 mpk, or about 5 mpk, or about 6 mpk, or about 7 mpk, or about 8 mpk, or about 9 mpk, or about 10 mpk, or about 11 mpk, or about 12 mpk, or about 15 mpk, or about 20 mpk or about 30 mpk in the claimed methods of the invention.
- an article of manufacture is used for TGI in a subject suffering from melanoma comprising a package comprising an anti-ETBR antibody drug conjugate composition and a MAP kinase inhibitor composition.
- said anti- ETBR antibody drug conjugate specifically binds an ETBR epitope consisting of amino acids number 64 to 101 of SEQ ID NO: 10.
- said anti-ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs wherein VH CDRl is SEQ ID NO: l, VH CDR2 is SEQ ID NO:2, VH CDR3 is SEQ ID NO:3 and wherein VL CDRl is SEQ ID NO:4, VL CDR2 is SEQ ID NO:5, VL CDR3 is SEQ ID NO:6.
- an anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO:7 or 9.
- an anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or 9 and the VL is SEQ ID NO: 8.
- the anti-ETBR antibody is conjugated to a cytotoxin, wherein said cytotoxin is cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
- the cytotoxin is a toxin wherein said toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin. In one aspect, the toxin is a maytansinoid.
- the MAP kinase inhibitor is a BRAF inhibitor.
- the BRAF inhibitor is propane- 1 -sulfonic acid ⁇ 3-[5-(4-chlorophenyl)-lH-pyrrolo[2,3-b]pyridine-3- carbonyl]-2,4-difluoro-phenyl ⁇ -amide.
- the BRAF inhibitor has the following chemical structure:
- the MAP kinase inhibitor is a MEK inhibitor.
- the MEK inhibitor is (S)-(3,4- difuoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)(3-hydoxy-3-(piperidin-2yl)azetidin-l-yl)methanone.
- the MEK inhibitor has the following chemical structure:
- an article of manufacture for treating melanoma in a subject comprising a package comprising an anti-ETBR antibody drug conjugate composition and a MAP kinase inhibitor composition. It is further contemplated that said anti-ETBR antibody specifically binds an ETBR epitope consisting of amino acids number 64 to 101 of SEQ ID NO: 10.
- said anti-ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs wherein VH CDRl is SEQ ID NO: l, VH CDR2 is SEQ ID NO:2, VH CDR3 is SEQ ID NO:3 and wherein VL CDRl is SEQ ID NO:4, VL CDR2 is SEQ ID NO:5, VL CDR3 is SEQ ID NO:6.
- VH CDRl is SEQ ID NO:1
- VH CDR2 is SEQ ID NO:3
- VL CDR2 is SEQ ID NO:5
- VL CDR3 is SEQ ID NO:6.
- an anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO:7 or 9.
- an anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or 9 and the VL is SEQ ID NO: 8.
- the anti-ETBR antibody is conjugated to a cytotoxin, wherein said cytotoxin is cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
- the cytotoxin is a toxin wherein said toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin.
- the toxin is a maytansinoid.
- the MAP kinase inhibitor is a BRAF inhibitor.
- the BRAF inhibitor is propane- 1 -sulfonic acid ⁇ 3-[5-(4-chlorophenyl)-lH-pyrrolo[2,3-b]pyridine-3- carbonyl]-2,4-difluoro-phenyl ⁇ -amide.
- the BRAF inhibitor has the following chemical structure:
- the MAP kinase inhibitor is a MEK inhibitor.
- the MEK inhibitor is (S)-(3,4-dimoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)(3-hydoxy-3-(piperidin-2yl)azetidin-l - yl)methanone.
- the MEK inhibitor has the following chemical structure:
- one aspect of the invention is use of an anti-ETBR antibody drug conjugate and a MAP kinase inhibitor in the preparation of a medicament for TGI of a melanoma. It is further contemplated that said anti-ETBR antibody specifically binds an ETBR epitope consisting of amino acids number 64 to 101 of SEQ ID NO: 10.
- said anti- ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs wherein VH CDR1 is SEQ ID NO: l, VH CDR2 is SEQ ID NO:2, VH CDR3 is SEQ ID NO:3 and wherein VL CDR1 is SEQ ID NO:4, VL CDR2 is SEQ ID NO:5, VL CDR3 is SEQ ID NO:6.
- VH CDR1 is SEQ ID NO: l
- VH CDR2 is SEQ ID NO:2
- VH CDR3 is SEQ ID NO:3
- VL CDR1 is SEQ ID NO:4
- VL CDR2 is SEQ ID NO:5
- VL CDR3 is SEQ ID NO:6.
- an anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO:7 or 9.
- an anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or 9 and the VL is SEQ ID NO: 8.
- the anti-ETBR antibody is conjugated to a cytotoxin, wherein said cytotoxin is cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
- the cytotoxin is a toxin wherein said toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin.
- the toxin is a maytansinoid.
- the MAP kinase inhibitor is a BRAF inhibitor.
- the BRAF inhibitor is propane- 1 -sulfonic acid ⁇ 3-[5-(4-chlorophenyl)-lH-pyrrolo[2,3-b]pyridine-3- carbonyl]-2,4-difluoro-phenyl ⁇ -amide.
- the BRAF inhibitor has the following chemical structure:
- the MAP kinase inhibitor is a MEK inhibitor.
- the MEK inhibitor is (S)-(3,4-dimoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)(3-hydoxy-3-(piperidin-2yl)azetidin-l - yl)methanone.
- the MEK inhibitor has the following chemical structure:
- one aspect of the invention is use of an article of manufacture comprising an anti-ETBR antibody drug conjugate composition and a MAP kinase inhibitor composition in the preparation of a medicament for TGI of a melanoma. It is further contemplated that said anti-ETBR antibody specifically binds an ETBR epitope consisting of amino acids number 64 to 101 of SEQ ID NO: 10.
- said anti-ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs wherein VH CDRl is SEQ ID NO: l, VH CDR2 is SEQ ID NO:2, VH CDR3 is SEQ ID NO:3 and wherein VL CDRl is SEQ ID NO:4, VL CDR2 is SEQ ID NO:5, VL CDR3 is SEQ ID NO:6.
- VH CDRl is SEQ ID NO:1
- VH CDR2 is SEQ ID NO:3
- VL CDR2 is SEQ ID NO:5
- VL CDR3 is SEQ ID NO:6.
- an anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO:7 or 9.
- an anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or 9 and the VL is SEQ ID NO: 8.
- the anti-ETBR antibody is conjugated to a cytotoxin, wherein said cytotoxin is cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
- the cytotoxin is a toxin wherein said toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin.
- the toxin is a maytansinoid.
- the MAP kinase inhibitor is a BRAF inhibitor.
- the BRAF inhibitor is propane- 1 -sulfonic acid ⁇ 3-[5-(4-chlorophenyl)-lH- pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl ⁇ -amide. Further, it is contemplated that the BRAF inhibitor has the following chemical structure:
- the MAP kinase inhibitor is a MEK inhibitor.
- the MEK inhibitor is (S)-(3,4-difuoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)(3-hydoxy-3- (piperidin-2yl)azetidin-l -yl)methanone.
- the MEK inhibitor has the following chemical structure:
- Figure 1 is a schematic of the MAP kinase pathway.
- Figure 2 demonstrates the relationship of receptor level to ADC cell killing in vitro.
- the indicated number of receptor copies/cell was estimated by Scatchard analysis.
- Panel A shows cell killing by anti-ET B R ADC titration for the melanoma cell line UACC-257X2.2 and panel B for melanoma cell line A2058.
- the indicated concentrations of anti-ET B R ADC Hu5E9vl-vc-MMAE
- control IgG-vc- MMAE or equivalent amount of PBS vehicle control were incubated with cells for 5 days and relative cell viability (y-axis) assessed using CellTiter-Glo.
- Figure 3 shows the in vivo efficacy of anti-ETBR ADC in xenografts mouse models.
- Subcutaneous tumors were established in mice inoculated with UACC-257X2.2 (Panel A) or A2058 (Panel B) cells. When tumor volumes reached approximately 200 mm 3 (day 0), animals were given a single IV injection of either control ADC (Control-vc-MMAE) or anti-ETBR ADC (Hu5E9vl -vc- MMAE) at the indicated doses. Average tumor volumes with standard deviations were determined from 10 animals per groups (indicated on graph).
- FIG. 4 shows ET B R expression in UACC-257X2.2 melanoma cells treated for 24h with varying concentrations of BRAFi-945.
- Panel A shows ETBR transcript normalized to RPL19 transcript.
- Panel B shows the expression of total ETBR and GAPDH (Control) protein in 50 ⁇ g whole cell lysates.
- Panel C shows surface ETBR protein expression in live cells as seen by flow cytometry, where the first peak indicates cells treated to secondary detection reagent alone, the middle peak indicates cells untreated with BRAF inhibitor, and the last peak indicates BRAF inhibitor treated cells.
- Figure 5 shows in vivo combination efficacy of anti-ET B R ADC (Hu5E9vl-vc-MMAE) and BRAFi-945 against UACC-257X2.2 melanoma xenograft mouse models at varying doses.
- Subcutaneous tumors were established in mice inoculated with UACC -257X2.2 cell lines. When tumor volumes reached approximately 200 mm 3 (day 0), animals were dosed orally once a day for 21 days with BRAFi- 945 or vehicle control. On day 1 (after two doses of BRAFi-945), animals were given a single IV injection of either vehicle or anti-ET B R ADC at the indicated doses. Average tumor volumes with standard deviations were determined from 10 animals per group.
- Panel A shows a 1 mpk BRAFi-945 and 1 mpk anti-ET B R-ADC (Hu5E9vl -vc-MMAE) combination
- panel B shows a 1 mpk BRAFi-945 and 3 mpk anti-ET B R-ADC (Hu5E9vl -vc-MMAE) combination
- panel C shows a 6 mpk BRAFi-945 and 1 mpk anti-ET B R-ADC (Hu5E9vl -vc-MMAE) combination
- panel D shows a 6 mpk BRAFi-945 and 3 mpk anti-ET B R-ADC (Hu5E9vl -vc-MMAE) combination
- panel E shows a 20 mpk BRAFi-945 and 3 mpk anti-ET B R-ADC (Hu5E9vl -vc- MMAE) combination.
- Figure 6 shows ETBR expression in COLO 829 melanoma cells treated for 24h with varying concentrations of BRAF inhibitor RG7204.
- Panel A shows ETBR transcript normalized to RPL19 transcript.
- Panel B shows expression of total ETBR and GAPDH (Control) protein in 50 ⁇ g whole cell lysates.
- Panel C shows surface ETBR protein expression in live cells as seen by flow cytometry, where the first peak indicates cells treated to secondary detection reagent alone, the second peak indicates cells untreated with BRAF inhibitor and the third peak indicates BRAF inhibitor treated cells.
- Figure 7 demonstrates the in vivo combination efficacy of anti-ET B R ADC and BRAF inhibitor RG7204 against COLO 829 melanoma xenografts mouse model.
- Subcutaneous tumors were established in mice inoculated with COLO 829 melanoma cell lines. When tumor volumes reached approximately 200 mm 3 (day 0), animals were dosed orally twice a day for 21 days with RG7204. On day 1 (after three doses of RG7204), animals were given a single IV injection of either vehicle or anti-ET B R ADC (Hu5E9vl -vc-MMAE) at the indicated doses. Average tumor volumes with standard deviations were determined from 9 animals per group.
- panel A shows 3 mpk of anti-ET B R-ADC (Hu5E9vl -vc-MMAE) in combination with 30 mpk of RG7204;
- panel B shows 1 mpk of anti-ET B R-ADC (Hu5E9vl -vc-MMAE) in combination with 30 mpk of RG7204;
- panel C shows 1 mpk of anti-ET B R-ADC (Hu5E9vl -vc-MMAE) in combination with 10 mpk of RG7204;
- panel D shows 3 mpk of anti-ET B R-ADC (Hu5E9vl -vc-MMAE) in combination with 10 mpk of RG7204.
- Figure 8 shows ETBR expression in A2058 melanoma cells treated for 24h with varying concentrations of BRAF inhibitor RG7204.
- Panel A shows ETBR transcript normalized to RPL19 transcript;
- panel B shows expression of total ETBR and GAPDH (Control) protein in 100 ⁇ g whole cell lysates;
- panel C shows surface ETBR protein expression in live cells as seen by flow cytometry.
- the first peak indicates cells treated to secondary detection reagent alone, the second peak indicates cells untreated with BRAF inhibitor, and the third peak indicates BRAF inhibitor treated cells.
- Figure 9 demonstrates in vivo combination efficacy of anti-ET B R ADC (Hu5E9vl -vc-MMAE) and BRAF inhibitor RG7204 against A2058 melanoma xenograft mouse models.
- Subcutaneous tumors were established in mice inoculated with A2058 melanoma cell lines. When tumor volumes reached approximately 200 mm 3 (day 0), animals were dosed orally twice a day for 21 days with RG7204. On day 1 (after three doses of RG7204), animals were given a single IV injection of either vehicle or anti- ET B R ADC (Hu5E9vl -vc-MMAE) at the indicated doses. Average tumor volumes with standard deviations were determined from 10 animals per group.
- Panel A shows 6 mpk of anti-ET B R-ADC (Hu5E9vl -vc-MMAE) in combination with 10 mpk of RG7204;
- panel B shows 6 mpk of anti-ET B R-ADC (Hu5E9vl -vc-MMAE) in combination with 30 mpk of RG7204;
- panel C shows 3 mpk of anti-ET B R-ADC (Hu5E9vl -vc-MMAE) in combination with 10 mpk of RG7204;
- panel D shows 3 mpk of anti-ET B R-ADC (Hu5E9vl -vc- MMAE) in combination with 30 mpk of RG7204.
- Figure 10 shows Western blot experiments performed with BRAFi RG7204 showing expression of total ETBR, Perk and erk proteins and control proteins GAPDH and ⁇ -tubulin in 25 to 100 ⁇ g whole cell lysates from IPC-298 melanoma cells.
- Figure 11 shows surface ETBR protein expression in IPC-298 live cells as seen by flow cytometry after incubation with 0.1 ⁇ , 1 ⁇ and 10 ⁇ of BRAFi RG7204 (panels A, B and C respectively).
- the first peak indicates cells treated to secondary detection reagent alone, the second peak indicates BRAF inhibitor treated cells and the third peak indicates cells untreated with BRAF inhibitor.
- Figure 12 shows Western blot experiments performed with MEKi-623 (panel A) and MEKi-973 (panel B) at concentrations of 0 ⁇ , 0.01 ⁇ , 0.1 ⁇ and 1 ⁇ showing expression of total ETBR, Perk and erk proteins and control proteins GAPDH and ⁇ -tubulin in 50 ⁇ g whole cell lysates from COL0829 melanoma cells.
- Figure 13 shows surface ETBR protein expression in COLO 829 live cells as seen by flow cytometry after incubation with 0.01 ⁇ (panels A and D), 0.1 ⁇ (panels B and E) and 1 ⁇ (panel C and F) of MEKi-623 (panels A, B and C respectively) or MEKi-973 (panels D, E and F respectively).
- the first peak indicates cells treated to secondary detection reagent alone, the second peak indicates cells untreated with MEK inhibitor and the third peak indicates MEK inhibitor treated cells.
- Figure 14 shows ETBR mRNA expression in A2058 melanoma cells treated for 24h with varying concentrations of MEKi-623 (panel A) or MEKi-973 (panel B), normalized to RPL19 transcript.
- Figure 15 shows Western blot experiments performed with MEKi-623 (panel A) and MEKi-973 (panel B) at concentrations of 0 ⁇ , 0.01 ⁇ , 0.1 ⁇ and 1 ⁇ showing expression of total ETBR, Perk and erk proteins and control proteins GAPDH and ⁇ -tubulin in 50-100 ⁇ g whole cell lysates from A2058 melanoma cells.
- Figure 16 shows surface ETBR protein expression in A2058 live cells as seen by flow cytometry after incubation with 0.01 ⁇ (panels A and D), 0.1 ⁇ (panels B and E) and 1 ⁇ (panel C and F) of MEKi-623 (panels A, B and C respectively) or MEKi-973 (panels D, E and F respectively).
- the first peak indicates cells treated to secondary detection reagent alone, the second peak indicates cells untreated with MEK inhibitor and the third peak indicates MEK inhibitor treated cells.
- Figure 17 demonstrates in vivo combination efficacy of anti-ET B R ADC (Hu5E9vl -vc-MMAE) and MEKi-973 against A2058 melanoma xenograft mouse models.
- Subcutaneous tumors were established in mice inoculated with A2058 melanoma cell lines. When tumor volumes reached approximately 200 mm 3 (day 0), animals were dosed orally once a day for 21 days with MEKi-973. On day 1 (after two doses of MEKi-973), animals were given a single IV injection of either vehicle or anti- ET B R ADC (Hu5E9vl -vc-MMAE) at the indicated doses. Average tumor volumes with standard deviations were determined from 9 animals per group.
- Panel A shows 7.5 mpk of anti-gD ADC (control) in combination with 7.5 mpk of MEKi-973 as compared to a vehicle control and anti-gD ADC alone
- panel B shows 6 mpk of anti-ET B R- ADC (Hu5E9vl -vc-MMAE) in combination with 7.5 mpk of MEKi-973 as compared to a vehicle control and 7.5 mpk MEKi-973 alone (GDC-0973) or 6 mpk of anti-ET B R-ADC alone.
- Figure 18 shows ET B R transcript expression in SK23-MEL melanoma cells treated for 24h with varying concentrations of MEKi-623 (panel A) or MEKi-973 (panel B), which were normalized to RPL19 transcript.
- Figure 19 shows Western blot experiments performed with MEKi-623 (panel A) and MEKi-973 (panel B) at concentrations of 0 ⁇ , 0.01 ⁇ , 0.1 ⁇ and 1 ⁇ showing expression of total ETBR, Perk and erk proteins and control proteins GAPDH and ⁇ -tubulin in 50 ⁇ g whole cell lysates from SK23- MEL melanoma cells.
- Figure 20 shows surface ETBR protein expression in live SK23-MEL cells as seen by flow cytometry after incubation with 0.01 ⁇ (panels A and D), 0.1 ⁇ (panels B and E) and 1 ⁇ (panel C and F) of MEKi-623 (panels A, B and C respectively) or MEKi-973 (panels D, E and F respectively).
- the first peak indicates cells treated to secondary detection reagent alone, the second peak indicates cells untreated with MEK inhibitor and the third peak indicates MEK inhibitor treated cells.
- Figure 21 demonstrates in vivo combination efficacy of anti-ET B R ADC (Hu5E9vl-vc-MMAE) and MEKi-973 against SK23-MEL melanoma xenograft mouse models.
- Panel A shows 6 mpk of anti-gD ADC (control) in combination with 7.5 mpk of MEKi-973 as compared to a vehicle control, 7.5 mpk MEKi-973 and 6 mpk anti-gD ADC alone
- panel B shows 6 mpk of anti-ET B R-ADC (Hu5E9vl -vc-MMAE) in combination with 7.5 mpk of MEKi-973 ("Combination") as compared to a vehicle control and 7.5 mpk MEKi-973 alone or 6 mpk of anti-ET B R-ADC alone.
- Panel C shows 3 mpk of anti-ET B R-ADC (Hu5E9vl -vc-MMAE) in combination with 3 mpk of MEKi-973 ("Combination"), as compared to a vehicle control, 3 mpk of anti-ET B R-ADC (Hu5E9vl -vc-MMAE) or 3 mpk of MEKi-973.
- Panel D shows 3 mpk of anti-ET B R-ADC (Hu5E9vl -vc- MMAE) in combination with 7.5 mpk of MEKi-973 ("Combination") as compared to a vehicle control and 7.5 mpk MEKi-973 alone or 3 mpk of anti-ET B R-ADC alone.
- Panel E shows 6 mpk of anti-ET B R- ADC (Hu5E9vl -vc-MMAE) in combination with 3 mpk of MEKi-973 ("Combination") as compared to a vehicle control and 3 mpk MEKi-973 alone or 6 mpk of anti-ET B R-ADC alone.
- Figure 22 shows Western blot experiments performed with MEKi-623 (panel A) and MEKi-973 (panel B) at concentrations of 0 ⁇ , 0.01 ⁇ , 0.1 ⁇ and 1 ⁇ showing expression of total ETBR, Perk and erk proteins and control proteins GAPDH and ⁇ -tubulin in 25-100 ⁇ g whole cell lysates from IPC-298 melanoma cells.
- Figure 23 shows surface ETBR protein expression in live IPC-298 cells as seen by flow cytometry after incubation with 0.01 ⁇ (panels A and D), 0.1 ⁇ (panels B and E) and 1 ⁇ (panel C and F) of MEKi-623 (panels A, B and C respectively) or MEKi-973 (panels D, E and F respectively).
- the first peak indicates cells treated to secondary detection reagent alone, the second peak indicates cells untreated with MEK inhibitor and the third peak indicates MEK inhibitor treated cells.
- Figure 24 demonstrates in vivo combination efficacy of anti-ET B R ADC (Hu5E9vl -vc-MMAE) and MEKi-623 against IPC-298 melanoma xenograft mouse models.
- Panel A shows 6 mpk of anti-gD ADC (control) in combination with 1 mpk of MEKi-623 as compared to a vehicle control, 1 mpk MEKi-623 and 6 mpk anti-gD ADC alone
- Panel B shows 6 mpk of anti-ET B R-ADC (Hu5E9vl -vc-MMAE) in combination with 1 mpk of MEKi- 623 ("Combination") as compared to a vehicle control and 1 mpk MEKi-623 alone or 6 mpk of anti- ET B R-ADC alone.
- FIG. 25 in vivo combination efficacy of anti-ET B R ADC (Hu5E9vl -vc-MMAE) and MEKi- 973 against IPC-298 melanoma xenograft mouse models.
- Panel A shows 6 mpk of anti-gD ADC (control) in combination with 7.5 mpk of MEKi-973
- Figure 26 depicts expression of phosphorylated erk and total erk protein in COLO 829 tumors treated with either vehicle or 30mpk BRAFi RG7204.
- Figure 27 depicts ETBR transcript expression in COLO 829 tumors treated with BRAFi RG7204 (panel A) and in A2058 tumors treated with MEKi-973 for 3 days (panel B).
- Panel A shows ETBR transcript normalized to control GAPDH in COLO 829 cell line, in COLO 892 tumors treated with either vehicle control or 10 mpk or 30 mpk RG7204.
- Panel B shows ETBR transcript normalized to control Hprtl in A2058 tumors treated with either vehicle control or 5 or 10 mpk of MEKi-973.
- an "acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
- An acceptor human framework "derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
- the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
- Binding affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
- binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
- the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
- An "affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
- HVRs hypervariable regions
- anti-ETBR antibody and "an antibody that binds to ETBR” refer to an antibody that is capable of binding the endothelin B receptor (ETBR) with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting ETBR.
- the extent of binding of an anti-ETBR antibody to an unrelated, non-ETBR protein is less than about 10% of the binding of the antibody to ETBR as measured, e.g., by a radioimmunoassay (RIA).
- RIA radioimmunoassay
- an antibody that binds to ETBR has a dissociation constant (Kd) of ⁇ 1 ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 "8 M or less, e.g. from 10 "8 M to 10 "13 M, e.g., from 10 "9 M to 10 "13 M).
- Kd dissociation constant
- an anti-ETBR antibody binds to an epitope of ETBR that is conserved among ETBR from different species.
- antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
- an "antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
- antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
- an "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.
- An exemplary competition assay is provided herein.
- BRAF refers to a serine/threonine-protein kinase B-Raf, also known as proto-oncogene B-Raf or v-Raf murine sarcoma viral oncogene homolog Bl, which is a protein that in humans is encoded by the BRAF gene.
- B-Raf protein is involved in sending signals in cells and in cell growth.
- BRAF inhibitor or "BRAFi” as used herein refers to any number of known small molecule drug compounds which can inhibit or interrupt the B-Raf/MEK step on the B-Raf/MEK/ERK pathway.
- suitable BRAFi may include, but are not limited to, those described in
- Another example may be, but is not limited to, GSK 2118436, having a CAS registry number 405554-55- 4, which is also known as 5-[2-[4-[2-(Dimethylamino)ethoxy]phenyl]-5-(4-pyridinyl)-lH-imidazol-4-yl]- 2,3-dihydro-lH-inden-l-one oxime.
- chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
- the "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
- the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
- cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
- Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , Zr 89 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatic
- Antibody effector functions refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
- an "effective amount" of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
- ETBR refers to any native endothelin B receptor (ETBR) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
- the term encompasses "full-length,” unprocessed ETBR as well as any form of ETBR that results from processing in the cell.
- the term also encompasses naturally occurring variants of ETBR, e.g., splice variants or allelic variants.
- amino acid sequence of an exemplary human ETBR is shown in SEQ ID NO: 10 (see Nakamuta M et al., Cloning and Sequence Analysis of a cDNA encoding Human non-selective type of endothelin receptor, Biochem Biophys Res Commun. 1991 May 31 : 177(l):34-9).
- anti-ETBR antibody - ADC refers to any anti-ETBR antibody described herein that is conjugated to a toxin.
- toxins include, but are not limited to, maytansinoids or specifically monomethylauristatin (MMAE).
- MMAE monomethylauristatin
- An anti-ETBR antibody- ADC is contemplated as a species of "anti-ETBR antibodies of the invention”.
- Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
- the term includes native sequence Fc regions and variant Fc regions.
- a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
- the C-terminal lysine (Lys447) of the Fc region may or may not be present.
- numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
- FR Framework or "FR” refers to variable domain residues other than hypervariable region (HVR) residues.
- the FR of a variable domain generally consists of four FR domains: FRl, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FRl -H 1 (L 1 )-FR2-H2(L2)-FR3 -H3 (L3)-FR4.
- VH or VL
- BRAFi-945" refers to a B-Raf enzyme inhibitor that is 4- amino-N-(6-chloro-2-fluoro-3-(3-fluoro propyl sulfonamido) phenyl)thieno[3,2-d]pyrimidine-7- carboxamide and has a structure having the following formula as disclosed in Example 15 of
- host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
- Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
- a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
- a "human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
- the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
- the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.
- the subgroup is subgroup kappa I as in Kabat et al., supra.
- the subgroup III is subgroup III as in Kabat et al., supra.
- a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non- human HVRs and amino acid residues from human FRs.
- a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
- a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
- a "humanized form" of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
- hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops").
- native four-chain antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
- HVRs generally comprise amino acid residues from the hypervariable loops and/or from the "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition.
- CDRs complementarity determining regions
- Exemplary hypervariable loops occur at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3).
- Exemplary CDRs CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 occur at amino acid residues 24-34 of LI, 50-56 of L2, 89- 97 of L3, 31-35B of HI, 50-65 of H2, and 95-102 of H3.
- CDRs generally comprise the amino acid residues that form the hypervariable loops.
- CDRs also comprise "specificity determining residues,” or "SDRs,” which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated- CDRs, or a-CDRs.
- Exemplary a-CDRs (a-CDR-Ll, a-CDR-L2, a-CDR-L3, a-CDR-Hl, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3.
- HVR residues and other residues in the variable domain are numbered herein according to Kabat et al., supra.
- an “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
- mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
- domesticated animals e.g., cows, sheep, cats, dogs, and horses
- primates e.g., humans and non-human primates such as monkeys
- rabbits e.g., mice and rats
- rodents e.g., mice and rats.
- the individual or subject is a human.
- an "isolated" antibody is one which has been separated from a component of its natural environment.
- an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).
- electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
- chromatographic e.g., ion exchange or reverse phase HPLC
- An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
- An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
- isolated nucleic acid encoding an anti-ETBR antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
- MAP kinase mitogen-activated protein kinase
- CMGC CDK/MAPK/GSK3/CLK
- ERK1/2 pathway of mammals is probably the best characterized MAPK system.
- the most important upstream activators of this pathway are the Raf proteins (A-Raf, B-Raf or c-Raf), the key mediators of response to growth factors (EGF, FGF, PDGF, etc.).
- MEK MAPK/ERK kinase
- MEK inhibitor refers to any number of known small molecule drug compounds which can inhibit or interrupt the MEK step on the MAP kinase pathway.
- suitable MEKi may include, but are not limited to, those described as MEKi-623, MEKi-973, or GSK1120212.
- MEKi-973 refers to a MEK inhibitor (S)-(3,4-difuoro-2-((2-fluoro-4- iodo henyl)amino)phenyl)(3-hydoxy-3-(piperidin-2yl)azetidin-l-yl)methanone, having the structure:
- the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
- polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
- each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
- the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
- the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
- a “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.
- Native antibodies refer to naturally occurring immunoglobulin molecules with varying structures.
- native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3).
- VH variable region
- VL variable region
- the light chain of an antibody may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
- package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
- Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
- % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
- the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
- the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code.
- the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
- the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y
- composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
- a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
- a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
- RG7204 refers to a B-Raf enzyme inhibitor that has a molecular formula of C23H 18 CIF 2 N 3 C>3S and the following structure:
- treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
- antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
- variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
- the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs).
- FRs conserved framework regions
- HVRs hypervariable regions
- antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
- vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
- the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
- Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors.”
- the invention is based, in part, on antibodies that bind to ETBR.
- Antibodies of the invention are useful, e.g., for the treatment of melanoma.
- an anti-ETBR antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) CDR-L1 (KS S Q SLLD SDGKTYLN, SEQ ID NO:7), (b) CDR-L2 (LVSKLDS, SEQ ID NO:8), (c) CDR-L3 (WQGTHFPYT; SEQ ID NO:9), (d) CDR-H1 (GYTFTSYWMQ; SEQ ID NO: l), (e) CDR- H2 (TIYPGDGDTSYAQKFKG; SEQ ID NO:2), and (f) CDR-H3 (WGYAYDIDN; SEQ ID NO:3).
- an anti-ETBR antibody is humanized.
- an anti-ETBR antibody comprises CDRs as in any of the above embodiments, and further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.
- the invention provides an isolated anti-ETBR antibody having the VL amino acid sequence of SEQ ID NO:8, and the VH amino acid sequence of SEQ ID NO:7.
- the invention provides an anti-ETBR antibody having a VL sequence of SEQ ID NO: 8 and a VH amino acid sequence of SEQ ID NO:9.
- an anti-ETBR antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:7 or 9.
- VH heavy chain variable domain
- a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti- ETBR antibody comprising that sequence retains the ability to bind to ETBR.
- an anti-ETBR antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 8.
- VL light chain variable domain
- a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-ETBR antibody comprising that sequence retains the ability to bind to ETBR.
- substitutions e.g., conservative substitutions
- insertions, or deletions relative to the reference sequence
- an anti-ETBR antibody comprising that sequence retains the ability to bind to ETBR.
- a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 8.
- the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).
- an anti-ETBR antibody comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
- the antibody comprises the VH and VL sequences in SEQ ID NO: 7 or 9 and SEQ ID NO:8, respectively, including post-translational modifications of those sequences.
- the invention provides an antibody that binds to the same epitope as an anti- ETBR antibody provided herein.
- an antibody is provided that binds to the same epitope as an anti-ETBR antibody comprising a VH sequence of SEQ ID NO: 7 or 9 and a VL sequence of SEQ ID NO: 8.
- an anti-ETBR antibody is provided that binds to an epitope within an N-terminal extracellular domain #1 fragment of ETBR consisting of amino acids number 64 to 101 of SEQ ID NO: 10.
- an anti-ETBR antibody is a monoclonal antibody, including a chimeric, humanized or human antibody.
- an anti-ETBR antibody is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.
- the antibody is a full length antibody, e.g., an intact IgGl antibody or other antibody class or isotype as defined herein.
- an anti-ETBR antibody according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 1-7 below:
- an antibody provided herein has a dissociation constant (Kd) of ⁇ 1 ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 ⁇ 8 M or less, e.g. from 10 ⁇ 8 M to 10 "13 M, e.g., from 10 "9 M to 10 "13 M).
- Kd dissociation constant
- Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay.
- Solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of ( 125 I) -labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)).
- MICROTITER ® multi-well plates (Thermo Scientific) are coated overnight with 5 ⁇ g/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2%> (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C).
- a non-adsorbent plate (Nunc #269620)
- 100 pM or 26 pM [ 125 I]-anti gen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res.
- the Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20 ® ) in PBS. When the plates have dried, 150 ⁇ /well of scintillant (MICROSCINT-20TM; Packard) is added, and the plates are counted on a TOPCOUNTTM gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.
- Kd is measured using surface plasmon resonance assays using a BIACORE ® -2000 or a BIACORE ® -3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at -10 response units (RU).
- CM5 carboxymethylated dextran biosensor chips
- EDC N-ethyl-N'- (3-dimethylaminopropyl)- carbodiimide hydrochloride
- NHS N-hydroxysuccinimide
- Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 ⁇ g/ml (-0.2 ⁇ ) before injection at a flow rate of 5 ⁇ /minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 tiM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20TM) surfactant (PBST) at 25°C at a flow rate of approximately 25 ⁇ /min. Association rates (k on ) and dissociation rates (k 0 ff) are calculated using a simple one-to-one
- the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity
- a spectrometer such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM- AMINCOTM spectrophotometer (ThermoSpectronic) with a stirred cuvette.
- an antibody provided herein is an antibody fragment.
- Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') 2 , Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of
- Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161 ; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).
- Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
- a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).
- Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.
- recombinant host cells e.g. E. coli or phage
- an antibody provided herein is a chimeric antibody.
- Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
- a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
- a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
- a chimeric antibody is a humanized antibody.
- a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
- a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
- HVRs e.g., CDRs, (or portions thereof) are derived from a non-human antibody
- FRs or portions thereof
- a humanized antibody optionally will also comprise at least a portion of a human constant region.
- some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
- a non-human antibody e.g., the antibody from which the HVR residues are derived
- Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit” method (see, e.g., Sims et al. J. Immunol.
- framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271 :22611-22618 (1996)).
- an antibody provided herein is a human antibody.
- Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.
- Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human
- Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse- human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol, 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al.. Proc. Natl Acad. Sci.
- Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.
- Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. m Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol.
- phage display methods repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol, 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
- PCR polymerase chain reaction
- the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993).
- naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol, 227: 381-388 (1992).
- Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
- Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.
- an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody.
- Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites.
- one of the binding specificities is for ETBR and the other is for any other antigen.
- bispecific antibodies may bind to two different epitopes of ETBR.
- Bispecific antibodies may also be used to localize cytotoxic agents to cells which express ETBR.
- Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
- Multispecific antibodies include, but are not limited to, recombinant co- expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., US Patent No.
- Engineered antibodies with three or more functional antigen binding sites are also included herein (see, e.g. US 2006/0025576A1).
- the antibody or fragment herein also includes a "Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to ETBR as well as another, different antigen (see, US 2008/0069820, for example).
- amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
- Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
- antibody variants having one or more amino acid substitutions are provided.
- Sites of interest for substitutional mutagenesis include the HVRs and FRs.
- Conservative substitutions are shown in Table 1 under the heading of "conservative substitutions.” More substantial changes are provided in Table 1 under the heading of "exemplary substitutions,” and as further described below in reference to amino acid side chain classes.
- Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. TABLE 1
- Amino acids may be grouped according to common side -chain properties:hydrophobic:
- Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
- substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
- a parent antibody e.g. a humanized or human antibody
- the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
- An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display- based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
- Alterations may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol.
- Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. m Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).)
- affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
- a secondary library is then created.
- the library is then screened to identify any antibody variants with the desired affinity.
- Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
- substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
- conservative alterations e.g., conservative substitutions as provided herein
- Such alterations may be outside of HVR "hotspots" or SDRs.
- each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
- a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
- a residue or group of target residues e.g., charged residues such as arg, asp, his, lys, and glu
- a neutral or negatively charged amino acid e.g., alanine or polyalanine
- Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
- a crystal structure of an antigen- antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
- Variants may be screened to determine whether they contain the desired properties.
- Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
- terminal insertions include an antibody with an N-terminal methionyl residue.
- Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
- an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated.
- Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
- the carbohydrate attached thereto may be altered.
- Native antibodies produced by mammalian cells typically comprise a branched, biantennary
- oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997).
- the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure.
- modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
- antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
- the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
- the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
- Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
- WO2002/031140 Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).
- Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys.
- knockout cell lines such as alpha-l,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).
- Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided.
- Such antibody variants may have improved CDC function.
- Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
- one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
- the Fc region variant may comprise a human Fc region sequence ⁇ e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification ⁇ e.g. a substitution) at one or more amino acid positions.
- the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
- In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
- Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
- NK cells express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII.
- FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
- Non- limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Natl Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
- non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry
- PBMC peripheral blood mononuclear cells
- NK Natural Killer
- ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Natl Acad. Sci. USA 95:652-656 (1998).
- Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity.
- a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738- 2743 (2004)).
- FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).
- Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056).
- Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
- an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
- alterations are made in the Fc region that result in altered ⁇ i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
- CDC Complement Dependent Cytotoxicity
- Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826).
- cysteine engineered antibodies e.g., "thioMAbs”
- one or more residues of an antibody are substituted with cysteine residues.
- the substituted residues occur at accessible sites of the antibody.
- reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker- drug moieties, to create an immunoconjugate, as described further herein.
- any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.
- Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No.
- an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
- the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
- water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n- vinyl
- Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
- the polymer may be of any molecular weight, and may be branched or unbranched.
- the number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
- conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided.
- the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)).
- the radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.
- Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567.
- isolated nucleic acid encoding an anti-ETBR antibody described herein is provided.
- Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody).
- one or more vectors e.g., expression vectors
- a host cell comprising such nucleic acid is provided.
- a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
- the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
- a method of making an anti-ETBR antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
- nucleic acid encoding an antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
- nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
- Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
- antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
- U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.
- the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
- eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
- Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
- Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).
- Vertebrate cells may also be used as hosts.
- mammalian cell lines that are adapted to grow in suspension may be useful.
- Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.
- monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells.
- Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR " CHO cells (Urlaub et al., Proc. Natl. Acad. Sci.
- Anti-ETBR antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.
- an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc.
- competition assays may be used to identify an antibody that competes with, for example, Hu5E9v. l or Hu5E9v.2 for binding to ETBR.
- a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by Hu5E9v.l or Hu5E9v.2.
- epitope e.g., a linear or a conformational epitope
- Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).
- anti-ETBR antibodies described herein specifically bind an ETBR epitope consisting of amino acids number 64 to 101 of SEQ ID NO: 10.
- immobilized ETBR is incubated in a solution comprising a first labeled antibody that binds to ETBR (e.g., Hu5E9v.l or Hu5E9v.2) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to ETBR.
- the second antibody may be present in a hybridoma supernatant.
- immobilized ETBR is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to ETBR, excess unbound antibody is removed, and the amount of label associated with immobilized ETBR is measured.
- assays are provided for identifying whether anti-ETBR antibodies and/or BRAFi compounds have biological activity.
- Biological activity may include those described in the Examples, e.g., in vitro melanoma cell survival assays or in vivo xenograft models in which melanoma cell lines are transplanted into nude mice and tumor growth inhibition (TGI) is assessed.
- TGI tumor growth inhibition
- the invention also provides immunoconjugates comprising an anti-ETBR antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
- cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
- the invention also provides immunoconjugates (interchangeably referred to as "antibody-drug conjugates,” or "ADCs”) comprising an antibody conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
- cytotoxic agents such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
- Immunoconjugates have been used for the local delivery of cytotoxic agents, i.e., drugs that kill or inhibit the growth or proliferation of cells, in the treatment of cancer (Xie et al (2006) Expert. Opin. Biol. Ther. 6(3):281-291 ; Kovtun et al (2006) Cancer Res. 66(6):3214-3121 ; Law et al (2006) Cancer Res. 66(4):2328-2337; Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al (2005) Nature Biotechnology 23(9): 1137-1146; Payne, G.
- cytotoxic agents i.e., drugs that kill or inhibit the growth or proliferation of cells
- Immunoconjugates allow for the targeted delivery of a drug moiety to a tumor, and intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., Lancet (Mar. 15, 1986) pp. 603-05; Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical
- Efforts to design and refine ADC have focused on the selectivity of monoclonal antibodies (mAbs) as well as drug mechanism of action, drug-linking, drug/antibody ratio (loading), and drug-releasing properties (McDonagh (2006) Protein Eng. Design & Sel.; Doronina et al (2006) Bioconj. Chem. 17: 114-124; Erickson et al (2006) Cancer Res. 66(8): l-8; Sanderson et al (2005) Clin. Cancer Res. 11 :843-852; Jeffrey et al (2005) J. Med. Chem. 48: 1344-1358; Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070).
- the toxins may exert their cytotoxic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
- ZEVALIN® is an antibody-radioisotope conjugate composed of a murine IgGl kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and 11 lln or 90Y radioisotope bound by a thiourea linker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol.
- ZEVALIN has activity against B-cell non-Hodgkin's Lymphoma (NHL), administration results in severe and prolonged cytopenias in most patients.
- MYLOTARGTM (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody-drug conjugate composed of a huCD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25(7):686; US Patent Nos.
- ADCs Antibody-drug conjugates (ADCs) composed of the maytansinoid, DM1, linked to trastuzumab show potent anti-tumor activity in HER2-overexpressing trastuzumab-sensitive and -resistant tumor cell lines and xenograft models of human cancer.
- T-DM1 Trastuzumab-MCC-DMl
- ADC HER2 antibody-drug conjugate
- BC metastatic breast cancer
- auristatin peptides auristatin E (AE) and monomethylauristatin (MMAE), synthetic analogs of dolastatin, were conjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas) and cACIO (specific to CD30 on hematological malignancies) (Doronina et al (2003) Nature Biotechnol. 21(7):778-784) and are under therapeutic development.
- an immunoconjugate comprises an antibody and a chemotherapeutic agent or other toxin.
- Chemotherapeutic agents useful in the generation of immunoconjugates are described herein (e.g., above). Enzymatically active toxins and fragments thereof can also be used and are described herein.
- an immunoconjugate comprises an antibody and one or more small molecule drug moieties (toxins), including, but not limited to, small molecule drugs such as a calicheamicin, maytansinoid, dolastatin, auristatin, anthracycline, taxane, trichothecene, and CC1065, and the derivatives of these drugs that have cytotoxic activity. Examples of such immunoconjugates are discussed in further detail below.
- An immunoconjugate (or "antibody-drug conjugate” (“ADC”)) of the invention may be of Formula I, below, wherein an antibody is conjugated (i.e., covalently attached) to one or more drug moieties (D) through an optional linker (L).
- the antibody may be conjugated to the drug either directly or via a linker.
- p is the average number of drug moieties per antibody, which can range, e.g., from about 1 to about 20 drug moieties per antibody, and in certain embodiments, from 1 to about 8 drug moieties per antibody.
- a linker may comprise one or more linker components.
- exemplary linker components include 6- maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine- phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (a "PAB”), N-Succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“SMCC”), and N-Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”).
- MC 6- maleimidocaproyl
- MP maleimidopropanoyl
- val-cit valine-citrulline
- alanine- phenylalanine ala-p
- a linker may be a "cleavable linker," facilitating release of a drug in the cell.
- an acid-labile linker e.g., hydrazone
- protease-sensitive linker e.g., peptidase-sensitive
- photolabile linker e.g., dimethyl linker or disulfide -containing linker
- dimethyl linker or disulfide -containing linker e.g., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020
- a linker is as shown in the following Formula II:
- A is a stretcher unit, and a is an integer from 0 to 1 ; W is an amino acid unit, and w is an integer from 0 to 12; Y is a spacer unit, and y is 0, 1, or 2; and Ab, D, and p are defined as above for Formula I.
- linkers are described in US 2005-0238649 Al, which is expressly incorporated herein by reference.
- a linker component may comprise a "stretcher unit” that links an antibody to another linker component or to a drug moiety.
- stretcher units are shown below (wherein the wavy line indicates sites of covalent attachment to an antibody):
- a linker component may comprise an amino acid unit.
- the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes. See, e.g., Doronina et al. (2003) Nat. Biotechnol. 21 :778-784.
- Exemplary amino acid units include, but are not limited to, a dipeptide, a tripeptide, a tetrapeptide, and a pentapeptide.
- Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); or N-methyl-valine-citrulline (Me-val-cit).
- Exemplary tripeptides include: glycine -valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).
- An amino acid unit may comprise amino acid residues that occur naturally, as well as minor amino acids and non- naturally occurring amino acid analogs, such as citrulline.
- Amino acid units can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzyme, for example, a tumor- associated protease, cathepsin B, C and D, or a plasmin protease.
- a linker component may comprise a "spacer" unit that links the antibody to a drug moiety, either directly or by way of a stretcher unit and/or an amino acid unit.
- a spacer unit may be "self-immolative” or a "non-self-immolative.”
- a "non-self-immolative" spacer unit is one in which part or all of the spacer unit remains bound to the drug moiety upon enzymatic (e.g., proteolytic) cleavage of the ADC.
- non-self-immolative spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit.
- peptidic spacers susceptible to sequence-specific enzymatic cleavage are also contemplated.
- enzymatic cleavage of an ADC containing a glycine -glycine spacer unit by a tumor-cell associated protease would result in release of a glycine-glycine-drug moiety from the remainder of the ADC.
- the glycine- glycine-drug moiety is then subjected to a separate hydrolysis step in the tumor cell, thus cleaving the glycine-glycine spacer unit from the drug moiety.
- a "self-immolative" spacer unit allows for release of the drug moiety without a separate hydrolysis step.
- a spacer unit of a linker comprises a p-aminobenzyl unit.
- a p-aminobenzyl alcohol is attached to an amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is made between the benzyl alcohol and a cytotoxic agent. See, e.g., Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15: 1087-1 103.
- the spacer unit is p-aminobenzyloxycarbonyl (PAB).
- the phenylene portion of a p-amino benzyl unit is substituted with Qm, wherein Q is -Ci-Cg alkyl, -0-(Ci-Cg alkyl), -halogen,- nitro or -cyano; and m is an integer ranging from 0-4.
- Q is -Ci-Cg alkyl, -0-(Ci-Cg alkyl), -halogen,- nitro or -cyano
- m is an integer ranging from 0-4.
- self-immolative spacer units further include, but are not limited to, aromatic compounds that are electronically similar to p-aminobenzyl alcohol ⁇ see, e.g., US 2005/0256030 Al), such as 2-aminoimidazol-5 -methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett.
- Spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4- aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223); appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J. Amer. Chem. Soc , 1972, 94, 5815); and 2-aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem., 1990, 55, 5867).
- Elimination of amine -containing drugs that are substituted at the a-position of glycine are also examples of self-immolative spacers useful in ADCs.
- a spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit as depicted below, which can be used to incorporate and release multiple drugs.
- BHMS branched bis(hydroxymethyl)styrene
- Q is -Ci-Cg alkyl, -0-(Ci-Cg alkyl), -halogen, -nitro or -cyano;
- m is an integer ranging from 0-4;
- n is 0 or 1 ; and
- p ranges raging from 1 to about 20.
- linker L may be a dendritic type linker for covalent attachment of more than one drug moiety through a branching, multifunctional linker moiety to an antibody (Sun et al (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003) Bioorganic & Medicinal Chemistry 1 1 : 1761 -1768).
- Dendritic linkers can increase the molar ratio of drug to antibody, i.e. loading, which is related to the potency of the ADC.
- a cysteine engineered antibody bears only one reactive cysteine thiol group, a multitude of drug moieties may be attached through a dendritic linker.
- Linkers components including stretcher, spacer, and amino acid units, may be synthesized by methods known in the art, such as those described in US 2005-0238649 Al .
- an immunoconjugate comprises an antibody conjugated to one or more maytansinoid molecules.
- Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Patent No. 3896111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Patent Nos.
- Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because they are: (i) relatively accessible to prepare by fermentation or chemical modification or derivatization of fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation through non-disulfide linkers to antibodies, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.
- Maytansine compounds suitable for use as maytansinoid drug moieties are well known in the art and can be isolated from natural sources according to known methods or produced using genetic engineering techniques (see Yu et al (2002) PNAS 99:7968-7973). Maytansinol and maytansinol analogues may also be prepared synthetically according to known methods.
- Exemplary maytansinoid drug moieties include those having a modified aromatic ring, such as: C-19-dechloro (US Pat. No. 4256746) (prepared by lithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy (or C-20-demethyl) +/-C-19-dechloro (US Pat. Nos. 4361650 and 4307016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20- demethoxy, C-20-acyloxy (-OCOR), +/-dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using acyl chlorides), and those having modifications at other positions.
- C-19-dechloro (US Pat. No. 4256746) (prepared by lithium aluminum hydride reduction of ansamytocin P2)
- C-20-hydroxy (or C-20-demethyl) +/-C-19-dechloro US Pat
- Exemplary maytansinoid drug moieties also include those having modifications such as: C-9-SH (US Pat. No. 4424219) (prepared by the reaction of maytansinol with H 2 S or P 2 S 5 ); C-14- alkoxymethyl(demethoxy/CH 2 OR)(US 4331598); C-14-hydroxymethyl or acyloxymethyl (CH 2 OH or CH 2 OAc) (US Pat. No. 4450254) (prepared from Nocardia); C-15-hydroxy/acyloxy (US 4364866) (prepared by the conversion of maytansinol by Streptomyces); C-15-methoxy (US Pat. Nos.
- R may independently be H or a Ci-C 6 alkyl.
- the alkylene chain attaching the amide group to the sulfur atom may be methanyl, ethanyl, or propyl, i.e., m is 1, 2, or 3 (US 633410; US 5208020; Chari et al (1992) Cancer Res. 52: 127-131 ; Liu et al (1996) Proc. Natl. Acad. Sci USA 93:8618-8623).
- the maytansinoid drug moiety will have the following stereochemistry:
- Exemplary embodiments of maytansinoid drug moieities include: DM1 ; DM3; and DM4, having the structures:
- Exemplary antibody-drug conjugates where DM1 is linked through a BMPEO linker to group of the antibody have the structure and abbreviation:
- Immunoconjugates containing maytansinoids, methods of making the same, and their therapeutic use are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, US 2005/0276812 Al, and European Patent EP 0 425 235 Bl, the disclosures of which are hereby expressly incorporated by reference. Liu et al. Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describe immunoconjugates comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer.
- the conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay.
- Chari et al. Cancer Research 52: 127-131 (1992) describe immunoconjugates in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA. l that binds the HER-2/neu oncogene.
- the cytotoxicity of the TA.l- maytansonoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3 x 10 5 HER-2 surface antigens per cell.
- the drug conjugate achieved a degree of cytotoxicity similar to the free maytansinoid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule.
- the A7 -maytansinoid conjugate showed low systemic cytotoxicity in mice.
- Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. See, e.g., U.S. Patent No. 5,208,020 (the disclosure of which is hereby expressly incorporated by reference). An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources.
- Suitable maytansinoids are disclosed, for example, in U.S. Patent No. 5,208,020 and in the other patents and nonpatent publications referred to hereinabove.
- Preferred maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.
- Antibody-maytansinoid conjugates comprising the linker component SMCC may be prepared as disclosed in US 2005/0276812 Al, "Antibody-drug conjugates and Methods.”
- the linkers comprise disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents. Additional linkers are described and exemplified herein.
- Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HQ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluor
- the coupling agent is N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) or N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.
- SPDP N-succinimidyl-3-(2-pyridyldithio) propionate
- SPP N-succinimidyl-4-(2-pyridylthio)pentanoate
- the linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link.
- an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group.
- the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.
- an immunoconjugate comprises an antibody conjugated to dolastatin or a dolastatin peptidic analog or derivative, e.g., an auristatin (US Pat. Nos. 5635483; 5780588).
- Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (US Pat. No.5663149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965).
- the dolastatin or auristatin drug moiety may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO
- Exemplary auristatin embodiments include the N-terminus linked monomethylauristatin drug moieties DE and DF (US 7498298).
- R 2 is selected from H and C r C 8 alkyl
- R 3 is selected from H, C C 8 alkyl, C 3 -C 8 carbocycle, aryl, C C 8 alkyl-aryl, C C 8 alkyl-(C 3 -C 8 carbocycle), C3-C 8 heterocycle and Ci-C 8 alkyl-(C3-C 8 heterocycle);
- R 4 is selected from H, C C 8 alkyl, C 3 -C 8 carbocycle, aryl, C C 8 alkyl-aryl, C C 8 alkyl-(C 3 -C 8 carbocycle), C 3 -C 8 heterocycle and Ci-C 8 alkyl-(C3-C 8 heterocycle);
- R 5 is selected from H and methyl; or R 4 and R 5 jointly form a carbocyclic ring and have the formula -(CR a R b ) n - wherein R a and R b are independently selected from H, Q-C 8 alkyl and C 3 -C 8 carbocycle and n is selected from 2, 3, 4, 5 and 6;
- R 6 is selected from H and C r C 8 alkyl
- R 7 is selected from H, C r C 8 alkyl, C 3 -C 8 carbocycle, aryl, C r C 8 alkyl-aryl, C r C 8 alkyl-(C 3 -C 8 carbocycle), C 3 -C 8 heterocycle and C r C 8 alkyl-(C 3 -C 8 heterocycle);
- each R 8 is independently selected from H, OH, C r C 8 alkyl, C 3 -C 8 carbocycle and 0-(C r C 8 alkyl);
- R 9 is selected from H and C r C 8 alkyl
- R 10 is selected from aryl or C 3 -C 8 heterocycle
- Z is O, S, NH, or NR 12 , wherein R 12 is C C 8 alkyl;
- R 11 is selected from H, C C 2 o alkyl, aryl, C 3 -C 8 heterocycle, -(R 13 0) m -R 14 , or -(R 13 0) m - CH(R 15 ) 2 ;
- m is an integer ranging from 1 -1000;
- R 13 is C 2 -C 8 alkyl
- R 14 is H or C r C 8 alkyl
- each occurrence of R 15 is independently H, COOH, -(CH 2 ) n -N(R 16 ) 2 , -(CH 2 ) n -S0 3 H, or -(CH 2 ) n - S0 3 -C r C 8 alkyl;
- each occurrence of R 16 is independently H, C C 8 alkyl, or -(CH 2 ) n -COOH;
- R 18 is selected from -C(R 8 ) 2 -C(R 8 ) 2 -aryl, -C(R 8 ) 2 -C(R 8 ) 2 -(C 3 -C 8 heterocycle), and
- n is an integer ranging from 0 to 6.
- R 3 , R 4 and R 7 are independently isopropyl or sec-butyl and R 5 is -H or methyl.
- R 3 and R 4 are each isopropyl, R 5 is -H, and R 7 is sec-butyl.
- R 2 and R 6 are each methyl, and R 9 is -H.
- each occurrence of R 8 is -OCH 3 .
- R 3 and R 4 are each isopropyl, R 2 and R 6 are each methyl, R 5 is -H, R 7 is sec-butyl, each occurrence of R 8 is -OCH 3 , and R 9 is -H.
- Z is -O- or -NH-.
- R 10 is aryl.
- R 10 is -phenyl.
- when Z is -0-, R 11 is -H, methyl or t-butyl.
- R 11 is -CH(R 15 ) 2 , wherein R 15 is -(CH 2 ) n -N(R 16 ) 2 , and R 16 is -C r C 8 alkyl or -(CH 2 ) n -COOH.
- Z is -NH
- R 11 is -CH(R 15 ) 2 , wherein R 15 is -(CH 2 ) n -S0 3 H.
- An exemplary auristatin embodiment of formula D E is MMAE, wherein the wavy line indicates the covalent attachment to a linker (L) of an antibody-drug conjugate:
- An exemplary auristatin embodiment of formula D F is MMAF, wherein the wavy line indicates the covalent attachment to a linker (L) of an antibody-drug conjugate (see US 7498298 and Doronina et al. (2006
- Other exemplary embodiments include monomethylvaline compounds having phenylalanine carboxy modifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008848) and monomethylvaline compounds having phenylalanine sidechain modifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008603).
- hydrophilic groups including but not limited to, triethylene glycol esters (TEG), as shown above, can be attached to the drug moiety at R 11 .
- TEG triethylene glycol esters
- ADCs of Formula I comprising an auristatin/dolastatin or derivative thereof are described in US 7498298 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124, which is expressly incorporated herein by reference.
- Exemplary embodiments of ADCs of Formula I comprising MMAE or MMAF and various linker components have the following structures and abbreviations (wherein "Ab” is an antibody; p is 1 to about 8, "Val-Cit” is a valine -citrulline dipeptide; and "S” is a sulfur atom:
- Exemplary embodiments of ADCs of Formula I comprising MMAF and various linker components further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF.
- immunoconjugates comprising MMAF attached to an antibody by a linker that is not proteolytically cleavable have been shown to possess activity comparable to immunoconjugates comprising MMAF attached to an antibody by a proteolytically cleavable linker. See, Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. In such instances, drug release is believed to be effected by antibody degradation in the cell. Id.
- peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments.
- Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Liibke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.
- Auristatin/dolastatin drug moieties may be prepared according to the methods of: US 2005-0238649 Al ; US Pat. No.5635483; US Pat. No.5780588; Pettit et al (1989) J. Am. Chem. Soc. 111 :5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G.R., et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784.
- auristatin/dolastatin drug moieties of formula D F may be prepared using methods described in US 7498298 and Doronina et al. (2006)
- Auristatin/dolastatin drug moieties of formula D E such as MMAE and derivatives thereof, may be prepared using methods described in Doronina et al. (2003) Nat. Biotech. 21 :778-784.
- Drug-linker moieties MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB- MMAE may be conveniently synthesized by routine methods, e.g., as described in Doronina et al. (2003) Nat. Biotech. 21 :778-784, and US 7498298, and then conjugated to an antibody of interest.
- the immunoconjugate comprises an antibody conjugated to one or more calicheamicin molecules.
- the calicheamicin family of antibiotics are capable of producing double- stranded DNA breaks at sub-picomolar concentrations.
- For the preparation of conjugates of the calicheamicin family see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
- Structural analogues of calicheamicin which may be used include, but are not limited to, ⁇ , ⁇ 2 ', ⁇ 3 ', N-acetyl-y , PSAG and ⁇ ' ⁇ (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the aforementioned U.S. patents to American Cyanamid).
- Another anti-tumor drug to which the antibody can be conjugated is QFA, which is an antifolate.
- QFA Another anti-tumor drug to which the antibody can be conjugated.
- Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody-mediated internalization greatly enhances their cytotoxic effects.
- Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published October 28, 1993.
- the present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
- a compound with nucleolytic activity e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase.
- an immunoconjugate may comprise a highly radioactive atom.
- a variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 Zr 89 and radioactive isotopes of Lu.
- the immunoconjugate When used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc 99m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-I l l, fluorine-19, carbon-13, nitrogen- 15, oxygen- 17, gadolinium, manganese or iron.
- NMR nuclear magnetic resonance
- the radio- or other labels may be incorporated in the immunoconjugate in known ways.
- the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine- 19 in place of hydrogen.
- Labels such as tc 99m or I 123 , Re 186 , Re 188 Zr 89 and In 111 can be attached via a cysteine residue in the peptide.
- Yttrium-90 can be attached via a lysine residue.
- the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.
- an immunoconjugate may comprise an antibody conjugated to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81/01145) to an active drug, such as an anti-cancer drug.
- a prodrug e.g., a peptidyl chemotherapeutic agent, see WO 81/01145
- an active drug such as an anti-cancer drug.
- ADPT antibody- dependent enzyme -mediated prodrug therapy
- Enzymes that may be conjugated to an antibody include, but are not limited to, alkaline phosphatases, which are useful for converting phosphate -containing prodrugs into free drugs; arylsulfatases, which are useful for converting sulfate - containing prodrugs into free drugs; cytosine deaminase, which is useful for converting non-toxic 5- fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), which are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, which are useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as ⁇ - galactosidase and neuraminidase, which are useful for converting
- Drug loading is represented by p, the average number of drug moieties per antibody in a molecule of Formula I. Drug loading may range from 1 to 20 drug moieties (D) per antibody.
- ADCs of Formula I include collections of antibodies conjugated with a range of drug moieties, from 1 to 20. The average number of drug moieties per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of ADC in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
- p may be limited by the number of attachment sites on the antibody.
- an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached.
- higher drug loading e.g. p >5
- the drug loading for an ADC of the invention ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. Indeed, it has been shown that for certain ADCs, the optimal ratio of drug moieties per antibody may be less than 8, and may be about 2 to about 5. See US 2005-0238649 Al .
- an antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent, as discussed below. Generally, antibodies do not contain many free and reactive cysteine thiol groups which may be linked to a drug moiety; indeed most cysteine thiol residues in antibodies exist as disulfide bridges.
- an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphme (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups.
- DTT dithiothreitol
- TCEP tricarbonylethylphosphme
- an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.
- the loading (drug/antibody ratio) of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification.
- the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to an antibody.
- the average number of drugs per antibody may be calculated from the mixture by a dual ELISA antibody assay, which is specific for antibody and specific for the drug.
- Individual ADC molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography (see, e.g., McDonagh et al (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al (2004) Clin. Cancer Res.
- a homogeneous ADC with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.
- An ADC of Formula I may be prepared by several routes employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent to form Ab-L via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with a nucleophilic group of an antibody.
- Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.
- Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges.
- Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP), such that the antibody is fully or partially reduced.
- a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP)
- TCEP tricarbonylethylphosphine
- Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles.
- Additional nucleophilic groups can be introduced into antibodies through modification of lysine residues, e.g., by reacting lysine residues with 2-iminothiolane (Traut's reagent), resulting in conversion of an amine into a thiol.
- Reactive thiol groups may be introduced into an antibody by introducing one, two, three, four, or more cysteine residues (e.
- Antibody-drug conjugates of the invention may also be produced by reaction between an electrophilic group on an antibody, such as an aldehyde or ketone carbonyl group, with a nucleophilic group on a linker reagent or drug.
- an electrophilic group on an antibody such as an aldehyde or ketone carbonyl group
- nucleophilic groups on a linker reagent or drug include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
- an antibody is modified to introduce electrophilic moieties that are capable of reacting with nucleophilic subsituents on the linker reagent or drug.
- the sugars of glycosylated antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or drug moieties.
- the resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g. by borohydride reagents to form stable amine linkages.
- reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the antibody that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques).
- antibodies containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3: 138-146; US
- Such an aldehyde can be reacted with a drug moiety or linker nucleophile.
- Nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.
- the compounds of the invention expressly contemplate, but are not limited to, ADC prepared with the following cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo- SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A.
- Immunoconjugates comprising an antibody and a cytotoxic agent may also be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l -carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate),
- a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987).
- Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026.
- a fusion protein comprising an antibody and a cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis.
- a recombinant DNA molecule may comprise regions encoding the antibody and cytotoxic portions of the conjugate either adjacent to one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.
- an antibody may be conjugated to a "receptor” (such as streptavidin) for utilization in tumor pre -targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a
- the invention further provides pharmaceutical formulations comprising at least one antibody of the invention and/or at least one immunoconjugate thereof.
- a pharmaceutical formulation comprises 1) an antibody of the invention and/or an immunoconjugate thereof, and 2) a pharmaceutically acceptable carrier.
- a pharmaceutical formulation comprises 1) an antibody of the invention and/or an immunoconjugate thereof, and optionally, 2) at least one additional therapeutic agent. Additional therapeutic agents include, but are not limited to, those described below.
- compositions comprising an antibody or immunoconjugate of the invention are prepared for storage by mixing the antibody or immunoconjugate having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers ⁇ Remington 's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of aqueous solutions or lyophilized or other dried formulations.
- Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as
- octadecyldimethylbenzyl ammonium chloride hexamethonium chloride; benzalkonium chloride, benzethonium chloride); phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins;
- hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes ⁇ e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
- Pharmaceutical formulations to be used for in vivo administration are generally sterile. This is readily accomplished by filtration through sterile filtration membranes.
- Active ingredients may also be entrapped in microcapsule prepared, for example, by
- coacervation techniques or by interfacial polymerization for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
- colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
- macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
- Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
- microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3- hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies or immunoconjugates remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved.
- stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
- An antibody may be formulated in any suitable form for delivery to a target cell/tissue.
- antibodies may be formulated as immunoliposomes.
- a "liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
- Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and
- Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
- Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst. 81(19): 1484 (1989).
- an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
- an enzymatically active toxin or fragment thereof including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (
- an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate.
- a variety of radioactive isotopes are available for the production of radioconjugates. Examples include Zr 89 , At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 ,
- the radioconjugate when used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-I l l, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
- NMR nuclear magnetic resonance
- Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HQ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds
- a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987).
- Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See
- the linker may be a "cleavable linker" facilitating release of a cytotoxic drug in the cell.
- a "cleavable linker” facilitating release of a cytotoxic drug in the cell.
- an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020) may be used.
- the immunuoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo- GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc.,
- compositions of an anti-ETBR antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
- Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
- sHASEGP soluble neutral-active hyaluronidase glycoproteins
- rHuPH20 HYLENEX ® , Baxter International, Inc.
- Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968.
- a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
- Aqueous antibody formulations include those described in US Patent No. 6,171,586 and
- the formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
- active ingredients for example, it may be desirable to further provide a BRAF inhibitor, a MEK inhibitor or an anti-CTLA-4 antibody, ipilimumab.
- Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
- Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
- Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
- the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
- any of the anti-ETBR antibodies provided herein may be used in therapeutic methods.
- an anti-ETBR antibody for use as a medicament is provided.
- the methods provide for an anti-ETBR antibody in combination with a BRAF inhibitor as useful as a medicament.
- such a combination is useful in treating melanoma and/or metastatic melanoma.
- an anti-ETBR antibody in combination with a BRAF inhibitor for use in a method of treatment is provided.
- the invention provides an anti-ETBR antibody for use in a method of treating an individual having melanoma and/or metastatic melanoma comprising administering to the individual an effective amount of the anti-ETBR antibody and an effective amount of a BRAF inhibitor.
- the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below, to the combination described.
- the invention provides an anti- ETBR antibody in combination with a BRAF inhibitor for use in tumor growth inhibition (TGI).
- TGI tumor growth inhibition
- the invention provides an anti-ETBR antibody in combination with a BRAF inhibitor for use in a method of inhibiting tumor growth in a subject comprising administering to the subject an effective of the anti-ETBR antibody in combination with a BRAF inhibitor to inhibit tumor growth.
- a "subject" according to any of the above embodiments is preferably a human.
- the invention provides for the use of an anti-ETBR antibody in combination with a BRAF inhibitor in the manufacture or preparation of a medicament.
- the medicament is for treatment of melanoma and/or metastatic melanoma.
- the medicament is for use in a method of treating melanoma and/or metastatic melanoma comprising administering to an individual having melanoma and/or metastatic melanoma an effective amount of the medicament.
- the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.
- the medicament is for tumor growth inhibition.
- the medicament is for use in a method of tumor growth inhibition in an individual comprising administering to the individual an amount effective of the medicament to inhibit tumor growth.
- An "individual" according to any of the above embodiments may be a human.
- the invention provides pharmaceutical formulations comprising any of the anti-ETBR antibodies provided herein, e.g., in combination with a BRAF inhibitor for use in any of the above therapeutic methods.
- a pharmaceutical formulation comprises any of the anti- ETBR antibodies provided herein in combination with a BRAF inhibitor and a pharmaceutically acceptable carrier.
- a pharmaceutical formulation comprises any of the anti- ETBR antibodies provided herein in combination with a BRAF inhibitor and at least one additional therapeutic agent, e.g., as described below.
- Antibodies of the invention can be used either alone or in combination with other agents in a therapy.
- an antibody of the invention may be co-administered with at least one additional therapeutic agent.
- an additional therapeutic agent is a BRAF inhibitor, a MEK inhibitor, or an anti-CTLA-4 antibody, such as, for example, ipilimumab.
- Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
- Antibodies of the invention can also be used in combination with radiation therapy.
- An antibody of the invention (and any additional therapeutic agent, such as, for example, a BRAF inhibitor) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
- Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
- Various dosing schedules including but not limited to single or multiple administrations over various time -points, bolus administration, and pulse infusion are contemplated herein.
- the BRAF inhibitor may be administered orally, in either tablet or capsule or liquid form.
- Antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
- the antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
- an antibody of the invention when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
- the antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 ⁇ g/kg to 15 mg/kg (e.g. O.
- lmg/kg-lOmg/kg can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
- One typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
- the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
- One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg.
- one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
- Such doses may be administered intermittently, e.g.
- Every week or every three weeks e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody.
- An initial higher loading dose, followed by one or more lower doses may be administered.
- other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
- an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
- Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
- the containers may be formed from a variety of materials such as glass or plastic.
- the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
- At least one active agent in the composition is an antibody of the invention.
- the label or package insert indicates that the composition is used for treating the condition of choice.
- the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
- the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
- the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
- Example 1 In vitro evaluations of specific cell killing by an anti-ETBR ADC
- the anti-ETBR antibody- ADC candidate Hu5E9vl -ADC was evaluated in vitro on melanoma cell lines expressing either relatively low ETBR copy number, in the case of cell line A2058 (Obtained from American Type Culture Collection) or high ETBR copy number, in the case of cell line UACC- 257X2.2.
- the UACC-257X2.2 cell line is a derivative of the parental UACC-257 cell line (NCI- Frederick Cancer DCT Tumor Repository) optimized for growth in vivo. Parental UACC-257 cells were injected subcutaneously in the right flank of female NCr nude mice, one tumor was harvested and dissociated grown in vitro resulting in the UACC-257X1.2 cell line.
- the UACC-257X1.2 line was injected again subcutaneously in the right flank of female NCr nude mice in an effort to improve the growth of the cell line.
- a tumor from this study was collected and again adapted for in vitro growth to generate the UACC-257X2.2 cell line.
- This cell line expresses high levels of ET B R as determined by flow cytometry.
- the relationship of receptor levels to Hu5E9vl-vc-MMAE cell killing in these cell lines was evaluated as follows.
- the melanoma cell lines A2058 and UACC-257X2.2 were grown in appropriate media at 37°C and 5% C0 2 .
- Hu5E9vl-ADC Hu5E9vl-ADC concentrations was added to triplicate wells. Five days later, cell survival was determined using CellTiter-Glo Luminescent Cell Viability Reagent (G7572; Promega Corporation) and with an EnVision 2101 Mutilabel Reader (Perkin- Elmer).
- Example 2 In vivo evaluations of specific tumor killing by an anti-ETBR ADC
- melanoma cell lines A2058 and UACC- 257X2.2 were selected as suitable models for in vivo anti-tumor activity studies that represent a wide range of ETBR expression.
- the UACC-257X2.2 melanoma cell line is a derivative of the parental UACC-257 melanoma cell line (National Cancer Institute (NCI)) optimized for growth in vivo.
- NCI National Cancer Institute
- UACC-257 cells were injected subcutaneously in the right flank of female NCr nude mice, one tumor was harvested and grown in vitro resulting in the UACC-257X1.2 cell line.
- the UACC-257X1.2 line was injected again subcutaneously in the right flank of female NCr nude mice in an effort to improve the growth of the cell line.
- a tumor from this study was collected and again adapted for in vitro growth to generate the UACC-257X2.2 cell line.
- This cell line and tumors derived from this line express ETBR comparable to the parental cell line UACC-257 (data not shown).
- the anti-ETBR ADC candidate Hu5E9vl -ADC was administered as a single intravenous (IV) injection on day 0 at 1 mpk, 3 mpk, or 6 mpk (mg/kg).
- a control ADC antibody and vehicle control were also administered. Average tumor volumes with standard deviations were determined from 10 animals per group. Tumor volumes were measured twice per week until study end.
- the primer-probe set for RPL19 is as follows:
- the primer-probe set for ETBR is as follows:
- FACS fluorescence-activated cell sorting
- Example 4 Effect of BRAF inhibitor drugs on in vivo efficacy of anti-ETBR ADC
- Tumor Volume (mm3) (length x width 2 ) x 0.5
- a TGI value of 100% indicates tumor stasis, of > 1% but ⁇ 100% indicates tumor growth delay, and of > 100% indicates tumor regression.
- UIs uncertainty intervals
- the random sample is composed of 1000 simulated realizations of the fitted-mixed model, where the %TGI has been recalculated for each realization.
- UI uncertainty intervals
- the %TGI has been recalculated for each realization.
- the reported UI is the value for which 95% of the time, the recalculated values of %TGI will fall in this region given the fitted model.
- the 2.5 and 97.5 percentiles of the simulated distribution were used as the upper and lower UIs.
- Example 5 Dose Testing Anti-ETBR ADC and BRAFi combinations in vivo in COLO 829 Xenografts
- Tumors were grown to an average size of approximately 200 mm 3 , whereupon animals were randomized into groups of 10 each.
- Table 3 summarizes the three melanoma xenograft models tested at varying doses, as described above, to demonstrate the combination effects, expressed as a percent delta (last column) of the combination use of anti-ETBR ADC with a BRAF inhibitor as compared to either the percent TGI of the anti-ETBR ADC as a single agent or the percent TGI of a BRAF inhibitor as a single agent.
- the percent TGI was calculated using a Linear Mixed Effects (LME) modeling approach, as described above.
- LME Linear Mixed Effects
- ETBR transcript and protein total protein and cell surface protein
- COL0829 BRAF V600E
- A2058 BRAF V600E
- SK23-MEL BRAF WT /RAS WT
- IPC-298 BRAF WT /RAS C61L
- Primer-probe sets were designed with primers flanking a fluorogenic probe dual labeled with Reporter dye FAM and quencher dye TAMRA.
- the primer-probe set for RPL19 is as follows:
- the primer-probe set for ETBR is as follows:
- FACS fluorescence-activated cell sorting
- results for A2058 are shown in Figure 16A-F
- results for COL0829 are shown in Figure 13A-F
- results for SK23-MEL are shown in Figure 20A-F
- results for IPC- 298 are shown in Figure 23A-F.
- Example 8 Effect of MEK inhibitor drugs on in vivo efficacy of anti-ETBR ADC
- Example 7 Given the results demonstrated in Example 7 above, the impact of the MEK inhibitors described herein on the in vivo efficacy of an anti-ET B R ADC was tested. To do this, the in vivo efficacy for various combinations of Hu5E9vl -ADC and MEKi-623 and/or MEKi-973 were evaluated against A2058 and SK-MEL-23 and IPC-298 melanoma in vivo models, performed as described above in Example 4. An appropriate methylcellulose twee vehicle control (0.5% methylcellulose, 0.2% Tween-80 (MCT) or MEK inhibitor at doses of 1 mpk, 3 mpk or 7.5 mpk were administered orally once a day x 21 days beginning on study Day 0. A single 3 mpk or 6 mpk dose of Hu5E9vl-ADC or control, a histidine buffer #8, was administered intraveneously (after two doses of a MEK inhibitor) via tail vein at study Day 1.
- MCT methylcellulose
- Example 9 PD studies of A2058 and COLO 829 melanoma xenografts
- A2058 or COLO 829 tumors were grown to an average size of approximately 200 mm 3 , whereupon animals were randomized into groups of 5-6 each.
- an appropriate vehicle control Kerel LF
- RG7204 at doses of 10 mpk or 30 mpk were administered twice a day for 3 days ( Figure 27A).
- an appropriate vehicle control or MEKi-973 at doses of 5mpk and lOmpk were administered orally once a day for 3 days (Figure 27B). Flash frozen tumors harvested at end of study were homogenized and processed for RNA and/or protein.
- Taqman assays were set up using reagents from Applied Biosystems (ABI, Foster City, CA) and assayed using 7500 Real Time PCR machine and software from ABI. Primer-probe sets were designed with primers flanking a fluorogenic probe dual labeled with Reporter dye FAM and quencher dye TAMRA. ETBR transcript levels in the tumors were normalized against transcript levels of reference genes such as Hprtl
- the primer-probe set for reference gene Hprtl (hypoxanthine phosphoribosyltransferase 1) is as follows: [0380] Forward primer -5' CAC ATC AAA GAC AGC ATC TAA GAA (SEQ ID NO: 17); Reverse primer-5' CAA GTT GGA AAA TAC AGT CAA CAT T (SEQ ID NO: 18) and probe-5' TTT TGT TCT GTC CTG GAA TTA TTT TAG TAG TGT TTC A (SEQ ID NO: 19).
- the primer-probe set for ETBR is as follows:
- the primer-probe set for reference gene GAPDH (Glyceraldehyde 3 phosphate dehydrogenase) is as follows:
- Figure 27A shows that BRAFi induces ETBR niRNA in vivo as compared to control vehicle.
- Figure 27B shows that MEKi-973 induces ETBR mRNA in vivo as compared to control vehicle as well.
- Phosphorylated erk and total erk protein levels were evaluated in the tumors by western blotting using the following reagents: for detection of proteins: anti-Phospho-p44/42 MAPK (Erkl/2)
- BRAFi appears to inhibit Perk in vivo as compared to control.
Abstract
Priority Applications (14)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2012328980A AU2012328980A1 (en) | 2011-10-28 | 2012-10-24 | Therapeutic combinations and methods of treating melanoma |
US14/354,362 US20140341916A1 (en) | 2011-10-28 | 2012-10-24 | Therapeutic combinations and methods of treating melanoma |
EA201490879A EA201490879A1 (ru) | 2011-10-28 | 2012-10-24 | Терапевтические комбинации и способы лечения меланомы |
BR112014009953A BR112014009953A2 (pt) | 2011-10-28 | 2012-10-24 | método de inibição do crescimento de tumores, de tratamento de melanoma, artigo industrializado e uso |
SG11201401815XA SG11201401815XA (en) | 2011-10-28 | 2012-10-24 | Therapeutic combinations and methods of treating melanoma |
IN3062CHN2014 IN2014CN03062A (fr) | 2011-10-28 | 2012-10-24 | |
EP12842985.9A EP2776051A4 (fr) | 2011-10-28 | 2012-10-24 | Associations thérapeutiques et méthodes de traitement du mélanome |
CA2850034A CA2850034A1 (fr) | 2011-10-28 | 2012-10-24 | Associations therapeutiques et methodes de traitement du melanome |
KR1020147013878A KR20140097205A (ko) | 2011-10-28 | 2012-10-24 | 흑색종을 치료하는 치료 조합물 및 방법 |
CN201280065175.XA CN104039340B (zh) | 2011-10-28 | 2012-10-24 | 治疗黑素瘤的方法及治疗剂组合 |
JP2014538892A JP6251682B2 (ja) | 2011-10-28 | 2012-10-24 | メラノーマ治療の治療の組み合わせ及び方法 |
MX2014004991A MX2014004991A (es) | 2011-10-28 | 2012-10-24 | Combinaciones terapeuticas y metodos para tratar el melanoma. |
IL232135A IL232135A0 (en) | 2011-10-28 | 2014-04-22 | Therapeutic combinations and methods for treating melanoma |
MA37028A MA35645B1 (fr) | 2011-10-28 | 2014-05-13 | Associations thérapeutiques et méthodes de traitement du mélanome |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161552893P | 2011-10-28 | 2011-10-28 | |
US61/552,893 | 2011-10-28 | ||
US201261678978P | 2012-08-02 | 2012-08-02 | |
US61/678,978 | 2012-08-02 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2013063001A1 true WO2013063001A1 (fr) | 2013-05-02 |
Family
ID=48168400
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2012/061533 WO2013063001A1 (fr) | 2011-10-28 | 2012-10-24 | Associations thérapeutiques et méthodes de traitement du mélanome |
Country Status (18)
Country | Link |
---|---|
EP (1) | EP2776051A4 (fr) |
JP (2) | JP6251682B2 (fr) |
KR (1) | KR20140097205A (fr) |
CN (1) | CN104039340B (fr) |
AR (1) | AR088509A1 (fr) |
AU (1) | AU2012328980A1 (fr) |
BR (1) | BR112014009953A2 (fr) |
CA (1) | CA2850034A1 (fr) |
CL (1) | CL2014001092A1 (fr) |
CO (1) | CO7151543A2 (fr) |
EA (1) | EA201490879A1 (fr) |
IL (1) | IL232135A0 (fr) |
IN (1) | IN2014CN03062A (fr) |
MX (1) | MX2014004991A (fr) |
PE (1) | PE20142312A1 (fr) |
SG (1) | SG11201401815XA (fr) |
TW (1) | TW201325613A (fr) |
WO (1) | WO2013063001A1 (fr) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9216170B2 (en) | 2012-03-19 | 2015-12-22 | Hoffmann-La Roche Inc. | Combination therapy for proliferative disorders |
WO2016055907A1 (fr) * | 2014-10-10 | 2016-04-14 | Pfizer Inc. | Associations synergiques d'auristatine |
US9724413B2 (en) | 2011-08-01 | 2017-08-08 | Genentech, Inc. | Methods of treating cancer using PD-1 axis binding antagonists and MEK inhibitors |
WO2017220739A1 (fr) | 2016-06-24 | 2017-12-28 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | Anticorps dirigé contre le sous-type bêta du récepteur de l'endothéline |
US10946093B2 (en) | 2014-07-15 | 2021-03-16 | Genentech, Inc. | Methods of treating cancer using PD-1 axis binding antagonists and MEK inhibitors |
US11040027B2 (en) | 2017-01-17 | 2021-06-22 | Heparegenix Gmbh | Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10695400B2 (en) * | 2015-08-03 | 2020-06-30 | Enb Therapeutics, Inc. | Compositions and methods for treating cancers associated with ETBR activation |
Citations (148)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US633410A (en) | 1898-09-22 | 1899-09-19 | George A Ames | Ice-cutter. |
US3773919A (en) | 1969-10-23 | 1973-11-20 | Du Pont | Polylactide-drug mixtures |
US3896111A (en) | 1973-02-20 | 1975-07-22 | Research Corp | Ansa macrolides |
US4137230A (en) | 1977-11-14 | 1979-01-30 | Takeda Chemical Industries, Ltd. | Method for the production of maytansinoids |
US4151042A (en) | 1977-03-31 | 1979-04-24 | Takeda Chemical Industries, Ltd. | Method for producing maytansinol and its derivatives |
US4248870A (en) | 1978-10-27 | 1981-02-03 | Takeda Chemical Industries, Ltd. | Maytansinoids and use |
US4256746A (en) | 1978-11-14 | 1981-03-17 | Takeda Chemical Industries | Dechloromaytansinoids, their pharmaceutical compositions and method of use |
US4260608A (en) | 1978-11-14 | 1981-04-07 | Takeda Chemical Industries, Ltd. | Maytansinoids, pharmaceutical compositions thereof and methods of use thereof |
WO1981001145A1 (fr) | 1979-10-18 | 1981-04-30 | Univ Illinois | Medicaments "pro-drugs" pouvant etre actives par des enzymes hydrolytiques |
US4265814A (en) | 1978-03-24 | 1981-05-05 | Takeda Chemical Industries | Matansinol 3-n-hexadecanoate |
US4294757A (en) | 1979-01-31 | 1981-10-13 | Takeda Chemical Industries, Ltd | 20-O-Acylmaytansinoids |
US4307016A (en) | 1978-03-24 | 1981-12-22 | Takeda Chemical Industries, Ltd. | Demethyl maytansinoids |
US4308268A (en) | 1979-06-11 | 1981-12-29 | Takeda Chemical Industries, Ltd. | Maytansinoids, pharmaceutical compositions thereof and method of use thereof |
US4308269A (en) | 1979-06-11 | 1981-12-29 | Takeda Chemical Industries, Ltd. | Maytansinoids, pharmaceutical compositions thereof and method of use thereof |
US4309428A (en) | 1979-07-30 | 1982-01-05 | Takeda Chemical Industries, Ltd. | Maytansinoids |
US4313946A (en) | 1981-01-27 | 1982-02-02 | The United States Of America As Represented By The Secretary Of Agriculture | Chemotherapeutically active maytansinoids from Trewia nudiflora |
US4315929A (en) | 1981-01-27 | 1982-02-16 | The United States Of America As Represented By The Secretary Of Agriculture | Method of controlling the European corn borer with trewiasine |
US4317821A (en) | 1979-06-08 | 1982-03-02 | Takeda Chemical Industries, Ltd. | Maytansinoids, their use and pharmaceutical compositions thereof |
US4322348A (en) | 1979-06-05 | 1982-03-30 | Takeda Chemical Industries, Ltd. | Maytansinoids |
US4331598A (en) | 1979-09-19 | 1982-05-25 | Takeda Chemical Industries, Ltd. | Maytansinoids |
US4362663A (en) | 1979-09-21 | 1982-12-07 | Takeda Chemical Industries, Ltd. | Maytansinoid compound |
US4364866A (en) | 1979-09-21 | 1982-12-21 | Takeda Chemical Industries, Ltd. | Maytansinoids |
US4371533A (en) | 1980-10-08 | 1983-02-01 | Takeda Chemical Industries, Ltd. | 4,5-Deoxymaytansinoids, their use and pharmaceutical compositions thereof |
US4424219A (en) | 1981-05-20 | 1984-01-03 | Takeda Chemical Industries, Ltd. | 9-Thiomaytansinoids and their pharmaceutical compositions and use |
US4450254A (en) | 1980-11-03 | 1984-05-22 | Standard Oil Company | Impact improvement of high nitrile resins |
US4485045A (en) | 1981-07-06 | 1984-11-27 | Research Corporation | Synthetic phosphatidyl cholines useful in forming liposomes |
US4544545A (en) | 1983-06-20 | 1985-10-01 | Trustees University Of Massachusetts | Liposomes containing modified cholesterol for organ targeting |
US4676980A (en) | 1985-09-23 | 1987-06-30 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Target specific cross-linked heteroantibodies |
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
US4970198A (en) | 1985-10-17 | 1990-11-13 | American Cyanamid Company | Antitumor antibiotics (LL-E33288 complex) |
US4975278A (en) | 1988-02-26 | 1990-12-04 | Bristol-Myers Company | Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells |
EP0404097A2 (fr) | 1989-06-22 | 1990-12-27 | BEHRINGWERKE Aktiengesellschaft | Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application |
US5013556A (en) | 1989-10-20 | 1991-05-07 | Liposome Technology, Inc. | Liposomes with enhanced circulation time |
US5053394A (en) | 1988-09-21 | 1991-10-01 | American Cyanamid Company | Targeted forms of methyltrithio antitumor agents |
US5079233A (en) | 1987-01-30 | 1992-01-07 | American Cyanamid Company | N-acyl derivatives of the LL-E33288 antitumor antibiotics, composition and methods for using the same |
WO1993001161A1 (fr) | 1991-07-11 | 1993-01-21 | Pfizer Limited | Procede de preparation d'intermediaires de sertraline |
US5208020A (en) | 1989-10-25 | 1993-05-04 | Immunogen Inc. | Cytotoxic agents comprising maytansinoids and their therapeutic use |
WO1993008829A1 (fr) | 1991-11-04 | 1993-05-13 | The Regents Of The University Of California | Compositions induisant la destruction de cellules infectees par l'hiv |
WO1993016185A2 (fr) | 1992-02-06 | 1993-08-19 | Creative Biomolecules, Inc. | Proteine de liaison biosynthetique pour marqueur de cancer |
WO1993021232A1 (fr) | 1992-04-10 | 1993-10-28 | Research Development Foundation | IMMUNOTOXINES DIRIGEES CONTRE DES ANTIGENES DE SURFACE APPARENTEES A c-erbB-2(HER-2/neu) |
WO1994011026A2 (fr) | 1992-11-13 | 1994-05-26 | Idec Pharmaceuticals Corporation | Application therapeutique d'anticorps chimeriques et radio-marques contre l'antigene a differentiation restreinte des lymphocytes b humains pour le traitement du lymphome des cellules b |
US5362852A (en) | 1991-09-27 | 1994-11-08 | Pfizer Inc. | Modified peptide derivatives conjugated at 2-hydroxyethylamine moieties |
WO1994029351A2 (fr) | 1993-06-16 | 1994-12-22 | Celltech Limited | Anticorps |
US5500362A (en) | 1987-01-08 | 1996-03-19 | Xoma Corporation | Chimeric antibody with specificity to human B cell surface antigen |
EP0425235B1 (fr) | 1989-10-25 | 1996-09-25 | Immunogen Inc | Agents cytotoxiques contenant des maytansinoides et leur application thérapeutique |
US5571894A (en) | 1991-02-05 | 1996-11-05 | Ciba-Geigy Corporation | Recombinant antibodies specific for a growth factor receptor |
US5585089A (en) | 1988-12-28 | 1996-12-17 | Protein Design Labs, Inc. | Humanized immunoglobulins |
US5587458A (en) | 1991-10-07 | 1996-12-24 | Aronex Pharmaceuticals, Inc. | Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof |
US5606040A (en) | 1987-10-30 | 1997-02-25 | American Cyanamid Company | Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methyl-trithio group |
US5624821A (en) | 1987-03-18 | 1997-04-29 | Scotgen Biopharmaceuticals Incorporated | Antibodies with altered effector functions |
US5635483A (en) | 1992-12-03 | 1997-06-03 | Arizona Board Of Regents Acting On Behalf Of Arizona State University | Tumor inhibiting tetrapeptide bearing modified phenethyl amides |
US5648237A (en) | 1991-09-19 | 1997-07-15 | Genentech, Inc. | Expression of functional antibody fragments |
WO1997030087A1 (fr) | 1996-02-16 | 1997-08-21 | Glaxo Group Limited | Preparation d'anticorps glycosyles |
US5663149A (en) | 1994-12-13 | 1997-09-02 | Arizona Board Of Regents Acting On Behalf Of Arizona State University | Human cancer inhibitory pentapeptide heterocyclic and halophenyl amides |
WO1997038731A1 (fr) | 1996-04-18 | 1997-10-23 | The Regents Of The University Of California | Immunoliposomes optimisant l'internalisation dans des cellules cibles |
US5712374A (en) | 1995-06-07 | 1998-01-27 | American Cyanamid Company | Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates |
US5714586A (en) | 1995-06-07 | 1998-02-03 | American Cyanamid Company | Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates |
US5731168A (en) | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
US5739116A (en) | 1994-06-03 | 1998-04-14 | American Cyanamid Company | Enediyne derivatives useful for the synthesis of conjugates of methyltrithio antitumor agents |
US5750373A (en) | 1990-12-03 | 1998-05-12 | Genentech, Inc. | Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants |
US5770701A (en) | 1987-10-30 | 1998-06-23 | American Cyanamid Company | Process for preparing targeted forms of methyltrithio antitumor agents |
US5770429A (en) | 1990-08-29 | 1998-06-23 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
US5780588A (en) | 1993-01-26 | 1998-07-14 | Arizona Board Of Regents | Elucidation and synthesis of selected pentapeptides |
US5789199A (en) | 1994-11-03 | 1998-08-04 | Genentech, Inc. | Process for bacterial production of polypeptides |
US5821337A (en) | 1991-06-14 | 1998-10-13 | Genentech, Inc. | Immunoglobulin variants |
US5840523A (en) | 1995-03-01 | 1998-11-24 | Genetech, Inc. | Methods and compositions for secretion of heterologous polypeptides |
WO1998058964A1 (fr) | 1997-06-24 | 1998-12-30 | Genentech, Inc. | Procedes et compositions concernant des glycoproteines galactosylees |
US5869046A (en) | 1995-04-14 | 1999-02-09 | Genentech, Inc. | Altered polypeptides with increased half-life |
WO1999022764A1 (fr) | 1997-10-31 | 1999-05-14 | Genentech, Inc. | Compositions renfermant des glycoformes de glycoproteine et methodes afferentes |
US5959177A (en) | 1989-10-27 | 1999-09-28 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
WO1999051642A1 (fr) | 1998-04-02 | 1999-10-14 | Genentech, Inc. | Variants d'anticorps et fragments de ceux-ci |
US6040498A (en) | 1998-08-11 | 2000-03-21 | North Caroline State University | Genetically engineered duckweed |
US6075181A (en) | 1990-01-12 | 2000-06-13 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
WO2000061739A1 (fr) | 1999-04-09 | 2000-10-19 | Kyowa Hakko Kogyo Co., Ltd. | Methode de regulation de l'activite d'une molecule immunologiquement fonctionnelle |
US6150584A (en) | 1990-01-12 | 2000-11-21 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
US6171586B1 (en) | 1997-06-13 | 2001-01-09 | Genentech, Inc. | Antibody formulation |
US6194551B1 (en) | 1998-04-02 | 2001-02-27 | Genentech, Inc. | Polypeptide variants |
WO2001029246A1 (fr) | 1999-10-19 | 2001-04-26 | Kyowa Hakko Kogyo Co., Ltd. | Procede de production d'un polypeptide |
US6248516B1 (en) | 1988-11-11 | 2001-06-19 | Medical Research Council | Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors |
US6267958B1 (en) | 1995-07-27 | 2001-07-31 | Genentech, Inc. | Protein formulation |
US6313276B1 (en) * | 1991-07-12 | 2001-11-06 | Shionogi Sieyaku Kabushiki Kaisha | Human endothelin receptor |
WO2002031140A1 (fr) | 2000-10-06 | 2002-04-18 | Kyowa Hakko Kogyo Co., Ltd. | Cellules produisant des compositions d'anticorps |
US6420548B1 (en) | 1999-10-04 | 2002-07-16 | Medicago Inc. | Method for regulating transcription of foreign genes |
US6441163B1 (en) | 2001-05-31 | 2002-08-27 | Immunogen, Inc. | Methods for preparation of cytotoxic conjugates of maytansinoids and cell binding agents |
US20020164328A1 (en) | 2000-10-06 | 2002-11-07 | Toyohide Shinkawa | Process for purifying antibody |
WO2002088172A2 (fr) | 2001-04-30 | 2002-11-07 | Seattle Genetics, Inc. | Composes pentapeptidiques et leurs utilisations |
WO2003011878A2 (fr) | 2001-08-03 | 2003-02-13 | Glycart Biotechnology Ag | Variants de glycosylation d'anticorps presentant une cytotoxicite cellulaire accrue dependante des anticorps |
US20030115614A1 (en) | 2000-10-06 | 2003-06-19 | Yutaka Kanda | Antibody composition-producing cell |
US6602684B1 (en) | 1998-04-20 | 2003-08-05 | Glycart Biotechnology Ag | Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity |
US20030157108A1 (en) | 2001-10-25 | 2003-08-21 | Genentech, Inc. | Glycoprotein compositions |
US6630579B2 (en) | 1999-12-29 | 2003-10-07 | Immunogen Inc. | Cytotoxic agents comprising modified doxorubicins and daunorubicins and their therapeutic use |
WO2003084570A1 (fr) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Medicament contenant une composition d'anticorps appropriee au patient souffrant de polymorphisme fc$g(g)riiia |
WO2003085107A1 (fr) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Cellules à génome modifié |
WO2003085119A1 (fr) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Procede d'amelioration de l'activite d'une composition d'anticorps de liaison avec le recepteur fc$g(g) iiia |
EP1391213A1 (fr) | 2002-08-21 | 2004-02-25 | Boehringer Ingelheim International GmbH | Compositions et méthodes pour le traitement du cancer en utilisant un conjugué d'un anticorps contre le CD44 avec un maytansinoide et des agents chimiothérapeutiques |
US20040093621A1 (en) | 2001-12-25 | 2004-05-13 | Kyowa Hakko Kogyo Co., Ltd | Antibody composition which specifically binds to CD20 |
US6737056B1 (en) | 1999-01-15 | 2004-05-18 | Genentech, Inc. | Polypeptide variants with altered effector function |
US20040109865A1 (en) | 2002-04-09 | 2004-06-10 | Kyowa Hakko Kogyo Co., Ltd. | Antibody composition-containing medicament |
US20040110282A1 (en) | 2002-04-09 | 2004-06-10 | Kyowa Hakko Kogyo Co., Ltd. | Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost |
US20040132140A1 (en) | 2002-04-09 | 2004-07-08 | Kyowa Hakko Kogyo Co., Ltd. | Production process for antibody composition |
WO2004056312A2 (fr) | 2002-12-16 | 2004-07-08 | Genentech, Inc. | Variants d'immunoglobuline et utilisations |
US20050014934A1 (en) | 2002-10-15 | 2005-01-20 | Hinton Paul R. | Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis |
US20050016993A1 (en) | 2003-04-17 | 2005-01-27 | Koskey James Donald | Heated pet mat |
US20050079574A1 (en) | 2003-01-16 | 2005-04-14 | Genentech, Inc. | Synthetic antibody phage libraries |
WO2005035586A1 (fr) | 2003-10-08 | 2005-04-21 | Kyowa Hakko Kogyo Co., Ltd. | Composition proteique hybride |
WO2005035778A1 (fr) | 2003-10-09 | 2005-04-21 | Kyowa Hakko Kogyo Co., Ltd. | Procede permettant de produire une composition d'anticorps par inhibition par l'arn de la fonction de $g(a)1,6-fucosyltransferase |
WO2005037992A2 (fr) | 2003-10-10 | 2005-04-28 | Immunogen, Inc. | Procede de ciblage de populations cellulaires specifiques a l'aide de conjugues formes d'un agent de liaison cellulaire et de maytansinoides, lies par l'intermediaire d'un lieur non clivable, lesdits conjugues et leurs procedes de preparation |
US20050119455A1 (en) | 2002-06-03 | 2005-06-02 | Genentech, Inc. | Synthetic antibody phage libraries |
US20050123546A1 (en) | 2003-11-05 | 2005-06-09 | Glycart Biotechnology Ag | Antigen binding molecules with increased Fc receptor binding affinity and effector function |
WO2005053742A1 (fr) | 2003-12-04 | 2005-06-16 | Kyowa Hakko Kogyo Co., Ltd. | Medicament contenant une composition a base d'anticorps |
US6913748B2 (en) | 2002-08-16 | 2005-07-05 | Immunogen, Inc. | Cross-linkers with high reactivity and solubility and their use in the preparation of conjugates for targeted delivery of small molecule drugs |
US20050166993A1 (en) | 2004-01-29 | 2005-08-04 | Viken James P. | Automatic fluid exchanger |
US20050238649A1 (en) | 2003-11-06 | 2005-10-27 | Seattle Genetics, Inc. | Monomethylvaline compounds capable of conjugation to ligands |
WO2005100402A1 (fr) | 2004-04-13 | 2005-10-27 | F.Hoffmann-La Roche Ag | Anticorps anti-p-selectine |
US20050256030A1 (en) | 2004-02-23 | 2005-11-17 | Bainian Feng | Heterocyclic self-immolative linkers and conjugates |
US20050260186A1 (en) | 2003-03-05 | 2005-11-24 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases |
US20050266000A1 (en) | 2004-04-09 | 2005-12-01 | Genentech, Inc. | Variable domain library and uses |
US20050276812A1 (en) | 2004-06-01 | 2005-12-15 | Genentech, Inc. | Antibody-drug conjugates and methods |
US6982321B2 (en) | 1986-03-27 | 2006-01-03 | Medical Research Council | Altered antibodies |
US20060025576A1 (en) | 2000-04-11 | 2006-02-02 | Genentech, Inc. | Multivalent antibodies and uses therefor |
WO2006029879A2 (fr) | 2004-09-17 | 2006-03-23 | F.Hoffmann-La Roche Ag | Anticorps anti-ox40l |
WO2006044908A2 (fr) | 2004-10-20 | 2006-04-27 | Genentech, Inc. | Formulations d'anticorps |
US7041870B2 (en) | 2000-11-30 | 2006-05-09 | Medarex, Inc. | Transgenic transchromosomal rodents for making human antibodies |
US20060104968A1 (en) | 2003-03-05 | 2006-05-18 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases |
US7087409B2 (en) | 1997-12-05 | 2006-08-08 | The Scripps Research Institute | Humanization of murine antibody |
US7097840B2 (en) | 2000-03-16 | 2006-08-29 | Genentech, Inc. | Methods of treatment using anti-ErbB antibody-maytansinoid conjugates |
US7125978B1 (en) | 1999-10-04 | 2006-10-24 | Medicago Inc. | Promoter for regulating expression of foreign genes |
WO2007008848A2 (fr) | 2005-07-07 | 2007-01-18 | Seattle Genetics, Inc. | Composes de monomethylvaline presentant des modifications carboxy phenylalanine a la terminaison c |
WO2007008603A1 (fr) | 2005-07-07 | 2007-01-18 | Seattle Genetics, Inc. | Composes de monomethylvaline presentant des modifications de la chaine laterale de phenylalanine au niveau de l'extremite c |
US7189826B2 (en) | 1997-11-24 | 2007-03-13 | Institute For Human Genetics And Biochemistry | Monoclonal human natural antibodies |
US20070061900A1 (en) | 2000-10-31 | 2007-03-15 | Murphy Andrew J | Methods of modifying eukaryotic cells |
US20070117126A1 (en) | 1999-12-15 | 2007-05-24 | Genentech, Inc. | Shotgun scanning |
US20070160598A1 (en) | 2005-11-07 | 2007-07-12 | Dennis Mark S | Binding polypeptides with diversified and consensus vh/vl hypervariable sequences |
US7276497B2 (en) | 2003-05-20 | 2007-10-02 | Immunogen Inc. | Cytotoxic agents comprising new maytansinoids |
US20070237764A1 (en) | 2005-12-02 | 2007-10-11 | Genentech, Inc. | Binding polypeptides with restricted diversity sequences |
US20070292936A1 (en) | 2006-05-09 | 2007-12-20 | Genentech, Inc. | Binding polypeptides with optimized scaffolds |
US20080069820A1 (en) | 2006-08-30 | 2008-03-20 | Genentech, Inc. | Multispecific antibodies |
US7371826B2 (en) | 1999-01-15 | 2008-05-13 | Genentech, Inc. | Polypeptide variants with altered effector function |
WO2008077546A1 (fr) | 2006-12-22 | 2008-07-03 | F. Hoffmann-La Roche Ag | Anticorps contre le récepteur du facteur de croissance i de type insuline et leurs utilisations |
US20090002360A1 (en) | 2007-05-25 | 2009-01-01 | Innolux Display Corp. | Liquid crystal display device and method for driving same |
US7521541B2 (en) | 2004-09-23 | 2009-04-21 | Genetech Inc. | Cysteine engineered antibodies and conjugates |
US7527791B2 (en) | 2004-03-31 | 2009-05-05 | Genentech, Inc. | Humanized anti-TGF-beta antibodies |
WO2009089004A1 (fr) | 2008-01-07 | 2009-07-16 | Amgen Inc. | Méthode de fabrication de molécules hétérodimères fc d'anticorps utilisant les effets de conduite électrostatique |
US20090226925A1 (en) * | 2005-05-20 | 2009-09-10 | Grebe Stefan K G | Methods for Detecting Circulating Tumor Cells |
US20100003240A1 (en) * | 1999-05-04 | 2010-01-07 | New York University | Cancer treatment with endothelin receptor antagonists |
US20100249096A1 (en) * | 2005-10-07 | 2010-09-30 | Exelixis, Inc. | Azetidines as MEK Inhibitors for the Treatment of Proliferative Diseases |
US20110105521A1 (en) * | 2008-07-11 | 2011-05-05 | Novartis Ag | Combination of (a) a phosphoinositide 3-kinase inhibitor and (b) a modulator of ras/raf/mek pathway |
US20110206702A1 (en) * | 2010-02-23 | 2011-08-25 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101489045B1 (ko) * | 2009-10-12 | 2015-02-02 | 에프. 호프만-라 로슈 아게 | Pi3k 억제자 및 mek 억제자의 조합 |
-
2012
- 2012-10-24 MX MX2014004991A patent/MX2014004991A/es unknown
- 2012-10-24 JP JP2014538892A patent/JP6251682B2/ja not_active Expired - Fee Related
- 2012-10-24 AU AU2012328980A patent/AU2012328980A1/en not_active Abandoned
- 2012-10-24 IN IN3062CHN2014 patent/IN2014CN03062A/en unknown
- 2012-10-24 PE PE2014000603A patent/PE20142312A1/es not_active Application Discontinuation
- 2012-10-24 CN CN201280065175.XA patent/CN104039340B/zh not_active Expired - Fee Related
- 2012-10-24 TW TW101139342A patent/TW201325613A/zh unknown
- 2012-10-24 EA EA201490879A patent/EA201490879A1/ru unknown
- 2012-10-24 WO PCT/US2012/061533 patent/WO2013063001A1/fr active Application Filing
- 2012-10-24 SG SG11201401815XA patent/SG11201401815XA/en unknown
- 2012-10-24 BR BR112014009953A patent/BR112014009953A2/pt not_active IP Right Cessation
- 2012-10-24 CA CA2850034A patent/CA2850034A1/fr not_active Abandoned
- 2012-10-24 EP EP12842985.9A patent/EP2776051A4/fr not_active Withdrawn
- 2012-10-24 KR KR1020147013878A patent/KR20140097205A/ko not_active Application Discontinuation
- 2012-10-24 AR ARP120103974A patent/AR088509A1/es unknown
-
2014
- 2014-04-16 CO CO14083118A patent/CO7151543A2/es not_active Application Discontinuation
- 2014-04-22 IL IL232135A patent/IL232135A0/en unknown
- 2014-04-28 CL CL2014001092A patent/CL2014001092A1/es unknown
-
2017
- 2017-08-24 JP JP2017161010A patent/JP2018027948A/ja not_active Withdrawn
Patent Citations (160)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US633410A (en) | 1898-09-22 | 1899-09-19 | George A Ames | Ice-cutter. |
US3773919A (en) | 1969-10-23 | 1973-11-20 | Du Pont | Polylactide-drug mixtures |
US3896111A (en) | 1973-02-20 | 1975-07-22 | Research Corp | Ansa macrolides |
US4151042A (en) | 1977-03-31 | 1979-04-24 | Takeda Chemical Industries, Ltd. | Method for producing maytansinol and its derivatives |
US4137230A (en) | 1977-11-14 | 1979-01-30 | Takeda Chemical Industries, Ltd. | Method for the production of maytansinoids |
US4361650A (en) | 1978-03-24 | 1982-11-30 | Takeda Chemical Industries, Ltd. | Fermentation process of preparing demethyl maytansinoids |
US4265814A (en) | 1978-03-24 | 1981-05-05 | Takeda Chemical Industries | Matansinol 3-n-hexadecanoate |
US4307016A (en) | 1978-03-24 | 1981-12-22 | Takeda Chemical Industries, Ltd. | Demethyl maytansinoids |
US4248870A (en) | 1978-10-27 | 1981-02-03 | Takeda Chemical Industries, Ltd. | Maytansinoids and use |
US4256746A (en) | 1978-11-14 | 1981-03-17 | Takeda Chemical Industries | Dechloromaytansinoids, their pharmaceutical compositions and method of use |
US4260608A (en) | 1978-11-14 | 1981-04-07 | Takeda Chemical Industries, Ltd. | Maytansinoids, pharmaceutical compositions thereof and methods of use thereof |
US4294757A (en) | 1979-01-31 | 1981-10-13 | Takeda Chemical Industries, Ltd | 20-O-Acylmaytansinoids |
US4322348A (en) | 1979-06-05 | 1982-03-30 | Takeda Chemical Industries, Ltd. | Maytansinoids |
US4317821A (en) | 1979-06-08 | 1982-03-02 | Takeda Chemical Industries, Ltd. | Maytansinoids, their use and pharmaceutical compositions thereof |
US4308269A (en) | 1979-06-11 | 1981-12-29 | Takeda Chemical Industries, Ltd. | Maytansinoids, pharmaceutical compositions thereof and method of use thereof |
US4308268A (en) | 1979-06-11 | 1981-12-29 | Takeda Chemical Industries, Ltd. | Maytansinoids, pharmaceutical compositions thereof and method of use thereof |
US4309428A (en) | 1979-07-30 | 1982-01-05 | Takeda Chemical Industries, Ltd. | Maytansinoids |
US4331598A (en) | 1979-09-19 | 1982-05-25 | Takeda Chemical Industries, Ltd. | Maytansinoids |
US4362663A (en) | 1979-09-21 | 1982-12-07 | Takeda Chemical Industries, Ltd. | Maytansinoid compound |
US4364866A (en) | 1979-09-21 | 1982-12-21 | Takeda Chemical Industries, Ltd. | Maytansinoids |
WO1981001145A1 (fr) | 1979-10-18 | 1981-04-30 | Univ Illinois | Medicaments "pro-drugs" pouvant etre actives par des enzymes hydrolytiques |
US4371533A (en) | 1980-10-08 | 1983-02-01 | Takeda Chemical Industries, Ltd. | 4,5-Deoxymaytansinoids, their use and pharmaceutical compositions thereof |
US4450254A (en) | 1980-11-03 | 1984-05-22 | Standard Oil Company | Impact improvement of high nitrile resins |
US4315929A (en) | 1981-01-27 | 1982-02-16 | The United States Of America As Represented By The Secretary Of Agriculture | Method of controlling the European corn borer with trewiasine |
US4313946A (en) | 1981-01-27 | 1982-02-02 | The United States Of America As Represented By The Secretary Of Agriculture | Chemotherapeutically active maytansinoids from Trewia nudiflora |
US4424219A (en) | 1981-05-20 | 1984-01-03 | Takeda Chemical Industries, Ltd. | 9-Thiomaytansinoids and their pharmaceutical compositions and use |
US4485045A (en) | 1981-07-06 | 1984-11-27 | Research Corporation | Synthetic phosphatidyl cholines useful in forming liposomes |
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
US4544545A (en) | 1983-06-20 | 1985-10-01 | Trustees University Of Massachusetts | Liposomes containing modified cholesterol for organ targeting |
US4676980A (en) | 1985-09-23 | 1987-06-30 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Target specific cross-linked heteroantibodies |
US4970198A (en) | 1985-10-17 | 1990-11-13 | American Cyanamid Company | Antitumor antibiotics (LL-E33288 complex) |
US6982321B2 (en) | 1986-03-27 | 2006-01-03 | Medical Research Council | Altered antibodies |
US5500362A (en) | 1987-01-08 | 1996-03-19 | Xoma Corporation | Chimeric antibody with specificity to human B cell surface antigen |
US5079233A (en) | 1987-01-30 | 1992-01-07 | American Cyanamid Company | N-acyl derivatives of the LL-E33288 antitumor antibiotics, composition and methods for using the same |
US5648260A (en) | 1987-03-18 | 1997-07-15 | Scotgen Biopharmaceuticals Incorporated | DNA encoding antibodies with altered effector functions |
US5624821A (en) | 1987-03-18 | 1997-04-29 | Scotgen Biopharmaceuticals Incorporated | Antibodies with altered effector functions |
US5770710A (en) | 1987-10-30 | 1998-06-23 | American Cyanamid Company | Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methlytrithio group |
US5606040A (en) | 1987-10-30 | 1997-02-25 | American Cyanamid Company | Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methyl-trithio group |
US5770701A (en) | 1987-10-30 | 1998-06-23 | American Cyanamid Company | Process for preparing targeted forms of methyltrithio antitumor agents |
US4975278A (en) | 1988-02-26 | 1990-12-04 | Bristol-Myers Company | Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells |
US5053394A (en) | 1988-09-21 | 1991-10-01 | American Cyanamid Company | Targeted forms of methyltrithio antitumor agents |
US6248516B1 (en) | 1988-11-11 | 2001-06-19 | Medical Research Council | Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors |
US5585089A (en) | 1988-12-28 | 1996-12-17 | Protein Design Labs, Inc. | Humanized immunoglobulins |
US5693762A (en) | 1988-12-28 | 1997-12-02 | Protein Design Labs, Inc. | Humanized immunoglobulins |
EP0404097A2 (fr) | 1989-06-22 | 1990-12-27 | BEHRINGWERKE Aktiengesellschaft | Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application |
US5013556A (en) | 1989-10-20 | 1991-05-07 | Liposome Technology, Inc. | Liposomes with enhanced circulation time |
US5416064A (en) | 1989-10-25 | 1995-05-16 | Immunogen, Inc. | Cytotoxic agents comprising maytansinoids and their therapeutic use |
EP0425235B1 (fr) | 1989-10-25 | 1996-09-25 | Immunogen Inc | Agents cytotoxiques contenant des maytansinoides et leur application thérapeutique |
US5208020A (en) | 1989-10-25 | 1993-05-04 | Immunogen Inc. | Cytotoxic agents comprising maytansinoids and their therapeutic use |
US6417429B1 (en) | 1989-10-27 | 2002-07-09 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
US5959177A (en) | 1989-10-27 | 1999-09-28 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
US6075181A (en) | 1990-01-12 | 2000-06-13 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
US6150584A (en) | 1990-01-12 | 2000-11-21 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
US5770429A (en) | 1990-08-29 | 1998-06-23 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
US5750373A (en) | 1990-12-03 | 1998-05-12 | Genentech, Inc. | Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants |
US5571894A (en) | 1991-02-05 | 1996-11-05 | Ciba-Geigy Corporation | Recombinant antibodies specific for a growth factor receptor |
US5821337A (en) | 1991-06-14 | 1998-10-13 | Genentech, Inc. | Immunoglobulin variants |
WO1993001161A1 (fr) | 1991-07-11 | 1993-01-21 | Pfizer Limited | Procede de preparation d'intermediaires de sertraline |
US6313276B1 (en) * | 1991-07-12 | 2001-11-06 | Shionogi Sieyaku Kabushiki Kaisha | Human endothelin receptor |
US5648237A (en) | 1991-09-19 | 1997-07-15 | Genentech, Inc. | Expression of functional antibody fragments |
US5362852A (en) | 1991-09-27 | 1994-11-08 | Pfizer Inc. | Modified peptide derivatives conjugated at 2-hydroxyethylamine moieties |
US5587458A (en) | 1991-10-07 | 1996-12-24 | Aronex Pharmaceuticals, Inc. | Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof |
WO1993008829A1 (fr) | 1991-11-04 | 1993-05-13 | The Regents Of The University Of California | Compositions induisant la destruction de cellules infectees par l'hiv |
WO1993016185A2 (fr) | 1992-02-06 | 1993-08-19 | Creative Biomolecules, Inc. | Proteine de liaison biosynthetique pour marqueur de cancer |
WO1993021232A1 (fr) | 1992-04-10 | 1993-10-28 | Research Development Foundation | IMMUNOTOXINES DIRIGEES CONTRE DES ANTIGENES DE SURFACE APPARENTEES A c-erbB-2(HER-2/neu) |
WO1994011026A2 (fr) | 1992-11-13 | 1994-05-26 | Idec Pharmaceuticals Corporation | Application therapeutique d'anticorps chimeriques et radio-marques contre l'antigene a differentiation restreinte des lymphocytes b humains pour le traitement du lymphome des cellules b |
US5635483A (en) | 1992-12-03 | 1997-06-03 | Arizona Board Of Regents Acting On Behalf Of Arizona State University | Tumor inhibiting tetrapeptide bearing modified phenethyl amides |
US5780588A (en) | 1993-01-26 | 1998-07-14 | Arizona Board Of Regents | Elucidation and synthesis of selected pentapeptides |
WO1994029351A2 (fr) | 1993-06-16 | 1994-12-22 | Celltech Limited | Anticorps |
US5739116A (en) | 1994-06-03 | 1998-04-14 | American Cyanamid Company | Enediyne derivatives useful for the synthesis of conjugates of methyltrithio antitumor agents |
US5877296A (en) | 1994-06-03 | 1999-03-02 | American Cyanamid Company | Process for preparing conjugates of methyltrithio antitumor agents |
US5773001A (en) | 1994-06-03 | 1998-06-30 | American Cyanamid Company | Conjugates of methyltrithio antitumor agents and intermediates for their synthesis |
US5767285A (en) | 1994-06-03 | 1998-06-16 | American Cyanamid Company | Linkers useful for the synthesis of conjugates of methyltrithio antitumor agents |
US5789199A (en) | 1994-11-03 | 1998-08-04 | Genentech, Inc. | Process for bacterial production of polypeptides |
US5663149A (en) | 1994-12-13 | 1997-09-02 | Arizona Board Of Regents Acting On Behalf Of Arizona State University | Human cancer inhibitory pentapeptide heterocyclic and halophenyl amides |
US5731168A (en) | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
US5840523A (en) | 1995-03-01 | 1998-11-24 | Genetech, Inc. | Methods and compositions for secretion of heterologous polypeptides |
US5869046A (en) | 1995-04-14 | 1999-02-09 | Genentech, Inc. | Altered polypeptides with increased half-life |
US5714586A (en) | 1995-06-07 | 1998-02-03 | American Cyanamid Company | Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates |
US5712374A (en) | 1995-06-07 | 1998-01-27 | American Cyanamid Company | Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates |
US6267958B1 (en) | 1995-07-27 | 2001-07-31 | Genentech, Inc. | Protein formulation |
WO1997030087A1 (fr) | 1996-02-16 | 1997-08-21 | Glaxo Group Limited | Preparation d'anticorps glycosyles |
WO1997038731A1 (fr) | 1996-04-18 | 1997-10-23 | The Regents Of The University Of California | Immunoliposomes optimisant l'internalisation dans des cellules cibles |
US6171586B1 (en) | 1997-06-13 | 2001-01-09 | Genentech, Inc. | Antibody formulation |
WO1998058964A1 (fr) | 1997-06-24 | 1998-12-30 | Genentech, Inc. | Procedes et compositions concernant des glycoproteines galactosylees |
WO1999022764A1 (fr) | 1997-10-31 | 1999-05-14 | Genentech, Inc. | Compositions renfermant des glycoformes de glycoproteine et methodes afferentes |
US7189826B2 (en) | 1997-11-24 | 2007-03-13 | Institute For Human Genetics And Biochemistry | Monoclonal human natural antibodies |
US7087409B2 (en) | 1997-12-05 | 2006-08-08 | The Scripps Research Institute | Humanization of murine antibody |
WO1999051642A1 (fr) | 1998-04-02 | 1999-10-14 | Genentech, Inc. | Variants d'anticorps et fragments de ceux-ci |
US6194551B1 (en) | 1998-04-02 | 2001-02-27 | Genentech, Inc. | Polypeptide variants |
US6602684B1 (en) | 1998-04-20 | 2003-08-05 | Glycart Biotechnology Ag | Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity |
US6040498A (en) | 1998-08-11 | 2000-03-21 | North Caroline State University | Genetically engineered duckweed |
US7371826B2 (en) | 1999-01-15 | 2008-05-13 | Genentech, Inc. | Polypeptide variants with altered effector function |
US6737056B1 (en) | 1999-01-15 | 2004-05-18 | Genentech, Inc. | Polypeptide variants with altered effector function |
US7332581B2 (en) | 1999-01-15 | 2008-02-19 | Genentech, Inc. | Polypeptide variants with altered effector function |
WO2000061739A1 (fr) | 1999-04-09 | 2000-10-19 | Kyowa Hakko Kogyo Co., Ltd. | Methode de regulation de l'activite d'une molecule immunologiquement fonctionnelle |
US20100003240A1 (en) * | 1999-05-04 | 2010-01-07 | New York University | Cancer treatment with endothelin receptor antagonists |
US6420548B1 (en) | 1999-10-04 | 2002-07-16 | Medicago Inc. | Method for regulating transcription of foreign genes |
US7125978B1 (en) | 1999-10-04 | 2006-10-24 | Medicago Inc. | Promoter for regulating expression of foreign genes |
WO2001029246A1 (fr) | 1999-10-19 | 2001-04-26 | Kyowa Hakko Kogyo Co., Ltd. | Procede de production d'un polypeptide |
US20070117126A1 (en) | 1999-12-15 | 2007-05-24 | Genentech, Inc. | Shotgun scanning |
US6630579B2 (en) | 1999-12-29 | 2003-10-07 | Immunogen Inc. | Cytotoxic agents comprising modified doxorubicins and daunorubicins and their therapeutic use |
US7097840B2 (en) | 2000-03-16 | 2006-08-29 | Genentech, Inc. | Methods of treatment using anti-ErbB antibody-maytansinoid conjugates |
US20060025576A1 (en) | 2000-04-11 | 2006-02-02 | Genentech, Inc. | Multivalent antibodies and uses therefor |
US20030115614A1 (en) | 2000-10-06 | 2003-06-19 | Yutaka Kanda | Antibody composition-producing cell |
US20020164328A1 (en) | 2000-10-06 | 2002-11-07 | Toyohide Shinkawa | Process for purifying antibody |
WO2002031140A1 (fr) | 2000-10-06 | 2002-04-18 | Kyowa Hakko Kogyo Co., Ltd. | Cellules produisant des compositions d'anticorps |
US20070061900A1 (en) | 2000-10-31 | 2007-03-15 | Murphy Andrew J | Methods of modifying eukaryotic cells |
US7041870B2 (en) | 2000-11-30 | 2006-05-09 | Medarex, Inc. | Transgenic transchromosomal rodents for making human antibodies |
WO2002088172A2 (fr) | 2001-04-30 | 2002-11-07 | Seattle Genetics, Inc. | Composes pentapeptidiques et leurs utilisations |
US6441163B1 (en) | 2001-05-31 | 2002-08-27 | Immunogen, Inc. | Methods for preparation of cytotoxic conjugates of maytansinoids and cell binding agents |
WO2003011878A2 (fr) | 2001-08-03 | 2003-02-13 | Glycart Biotechnology Ag | Variants de glycosylation d'anticorps presentant une cytotoxicite cellulaire accrue dependante des anticorps |
US20030157108A1 (en) | 2001-10-25 | 2003-08-21 | Genentech, Inc. | Glycoprotein compositions |
US20040093621A1 (en) | 2001-12-25 | 2004-05-13 | Kyowa Hakko Kogyo Co., Ltd | Antibody composition which specifically binds to CD20 |
US20040132140A1 (en) | 2002-04-09 | 2004-07-08 | Kyowa Hakko Kogyo Co., Ltd. | Production process for antibody composition |
WO2003084570A1 (fr) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Medicament contenant une composition d'anticorps appropriee au patient souffrant de polymorphisme fc$g(g)riiia |
WO2003085107A1 (fr) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Cellules à génome modifié |
WO2003085119A1 (fr) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Procede d'amelioration de l'activite d'une composition d'anticorps de liaison avec le recepteur fc$g(g) iiia |
US20040109865A1 (en) | 2002-04-09 | 2004-06-10 | Kyowa Hakko Kogyo Co., Ltd. | Antibody composition-containing medicament |
US20040110282A1 (en) | 2002-04-09 | 2004-06-10 | Kyowa Hakko Kogyo Co., Ltd. | Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost |
US20040110704A1 (en) | 2002-04-09 | 2004-06-10 | Kyowa Hakko Kogyo Co., Ltd. | Cells of which genome is modified |
US20050119455A1 (en) | 2002-06-03 | 2005-06-02 | Genentech, Inc. | Synthetic antibody phage libraries |
US6913748B2 (en) | 2002-08-16 | 2005-07-05 | Immunogen, Inc. | Cross-linkers with high reactivity and solubility and their use in the preparation of conjugates for targeted delivery of small molecule drugs |
EP1391213A1 (fr) | 2002-08-21 | 2004-02-25 | Boehringer Ingelheim International GmbH | Compositions et méthodes pour le traitement du cancer en utilisant un conjugué d'un anticorps contre le CD44 avec un maytansinoide et des agents chimiothérapeutiques |
US20050014934A1 (en) | 2002-10-15 | 2005-01-20 | Hinton Paul R. | Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis |
WO2004056312A2 (fr) | 2002-12-16 | 2004-07-08 | Genentech, Inc. | Variants d'immunoglobuline et utilisations |
US20050079574A1 (en) | 2003-01-16 | 2005-04-14 | Genentech, Inc. | Synthetic antibody phage libraries |
US20050260186A1 (en) | 2003-03-05 | 2005-11-24 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases |
US20060104968A1 (en) | 2003-03-05 | 2006-05-18 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases |
US20050016993A1 (en) | 2003-04-17 | 2005-01-27 | Koskey James Donald | Heated pet mat |
US7276497B2 (en) | 2003-05-20 | 2007-10-02 | Immunogen Inc. | Cytotoxic agents comprising new maytansinoids |
WO2005035586A1 (fr) | 2003-10-08 | 2005-04-21 | Kyowa Hakko Kogyo Co., Ltd. | Composition proteique hybride |
WO2005035778A1 (fr) | 2003-10-09 | 2005-04-21 | Kyowa Hakko Kogyo Co., Ltd. | Procede permettant de produire une composition d'anticorps par inhibition par l'arn de la fonction de $g(a)1,6-fucosyltransferase |
WO2005037992A2 (fr) | 2003-10-10 | 2005-04-28 | Immunogen, Inc. | Procede de ciblage de populations cellulaires specifiques a l'aide de conjugues formes d'un agent de liaison cellulaire et de maytansinoides, lies par l'intermediaire d'un lieur non clivable, lesdits conjugues et leurs procedes de preparation |
US20050123546A1 (en) | 2003-11-05 | 2005-06-09 | Glycart Biotechnology Ag | Antigen binding molecules with increased Fc receptor binding affinity and effector function |
US7498298B2 (en) | 2003-11-06 | 2009-03-03 | Seattle Genetics, Inc. | Monomethylvaline compounds capable of conjugation to ligands |
US20050238649A1 (en) | 2003-11-06 | 2005-10-27 | Seattle Genetics, Inc. | Monomethylvaline compounds capable of conjugation to ligands |
WO2005053742A1 (fr) | 2003-12-04 | 2005-06-16 | Kyowa Hakko Kogyo Co., Ltd. | Medicament contenant une composition a base d'anticorps |
US20050166993A1 (en) | 2004-01-29 | 2005-08-04 | Viken James P. | Automatic fluid exchanger |
US20050256030A1 (en) | 2004-02-23 | 2005-11-17 | Bainian Feng | Heterocyclic self-immolative linkers and conjugates |
US7527791B2 (en) | 2004-03-31 | 2009-05-05 | Genentech, Inc. | Humanized anti-TGF-beta antibodies |
US20050266000A1 (en) | 2004-04-09 | 2005-12-01 | Genentech, Inc. | Variable domain library and uses |
WO2005100402A1 (fr) | 2004-04-13 | 2005-10-27 | F.Hoffmann-La Roche Ag | Anticorps anti-p-selectine |
US20050276812A1 (en) | 2004-06-01 | 2005-12-15 | Genentech, Inc. | Antibody-drug conjugates and methods |
WO2006029879A2 (fr) | 2004-09-17 | 2006-03-23 | F.Hoffmann-La Roche Ag | Anticorps anti-ox40l |
US7521541B2 (en) | 2004-09-23 | 2009-04-21 | Genetech Inc. | Cysteine engineered antibodies and conjugates |
WO2006044908A2 (fr) | 2004-10-20 | 2006-04-27 | Genentech, Inc. | Formulations d'anticorps |
US20090226925A1 (en) * | 2005-05-20 | 2009-09-10 | Grebe Stefan K G | Methods for Detecting Circulating Tumor Cells |
WO2007008848A2 (fr) | 2005-07-07 | 2007-01-18 | Seattle Genetics, Inc. | Composes de monomethylvaline presentant des modifications carboxy phenylalanine a la terminaison c |
WO2007008603A1 (fr) | 2005-07-07 | 2007-01-18 | Seattle Genetics, Inc. | Composes de monomethylvaline presentant des modifications de la chaine laterale de phenylalanine au niveau de l'extremite c |
US20100249096A1 (en) * | 2005-10-07 | 2010-09-30 | Exelixis, Inc. | Azetidines as MEK Inhibitors for the Treatment of Proliferative Diseases |
US20070160598A1 (en) | 2005-11-07 | 2007-07-12 | Dennis Mark S | Binding polypeptides with diversified and consensus vh/vl hypervariable sequences |
US20070237764A1 (en) | 2005-12-02 | 2007-10-11 | Genentech, Inc. | Binding polypeptides with restricted diversity sequences |
US20070292936A1 (en) | 2006-05-09 | 2007-12-20 | Genentech, Inc. | Binding polypeptides with optimized scaffolds |
US20080069820A1 (en) | 2006-08-30 | 2008-03-20 | Genentech, Inc. | Multispecific antibodies |
WO2008077546A1 (fr) | 2006-12-22 | 2008-07-03 | F. Hoffmann-La Roche Ag | Anticorps contre le récepteur du facteur de croissance i de type insuline et leurs utilisations |
US20090002360A1 (en) | 2007-05-25 | 2009-01-01 | Innolux Display Corp. | Liquid crystal display device and method for driving same |
WO2009089004A1 (fr) | 2008-01-07 | 2009-07-16 | Amgen Inc. | Méthode de fabrication de molécules hétérodimères fc d'anticorps utilisant les effets de conduite électrostatique |
US20110105521A1 (en) * | 2008-07-11 | 2011-05-05 | Novartis Ag | Combination of (a) a phosphoinositide 3-kinase inhibitor and (b) a modulator of ras/raf/mek pathway |
US20110206702A1 (en) * | 2010-02-23 | 2011-08-25 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
Non-Patent Citations (175)
Title |
---|
ALLEY, S.C. ET AL.: "Controlling the location of drug attachment in antibody-drug conjugates", AMERICAN ASSOCIATION FOR CANCER RESEARCH, 2004 ANNUAL MEETING, MARCH 27-31, 2004, PROCEEDINGS OF THE AACR, vol. 45, 27 March 2004 (2004-03-27) |
ALMAGRO; FRANSSON, FRONT. BIOSCI., vol. 13, 2008, pages 1619 - 1633 |
AMSBERRY ET AL., J. ORG. CHEM., vol. 55, 1990, pages 5867 |
ASCIERTO PA ET AL.: "Melanoma: a model for testing new agents in combination therapies", J TRANSL MED, vol. 8, 2010, pages 38 - 45 |
BACA ET AL., J. BIOL. CHEM., vol. 272, 1997, pages 10678 - 10684 |
BALDWIN ET AL., LANCET, 15 March 1986 (1986-03-15), pages 603 - 05 |
BEERAM ET AL.: "A phase I study of trastuzumab-MCC-DMl (T-DM1), a first-in-class HER2 antibody-drug conjugate (ADC), in patients (pts) with HER2+ metastatic breast cancer (BC", AMERICAN SOCIETY OF CLINICAL ONCOLOGY 43RD, 2 June 2007 (2007-06-02) |
BOERNER ET AL., J. IMMUNOL., vol. 147, 1991, pages 86 |
BRENNAN ET AL., SCIENCE, vol. 229, no. 81, 1985 |
BRODEUR ET AL.: "Monoclonal Antibody Production Techniques and Applications", 1987, MARCEL DEKKER, INC., pages: 51 - 63 |
BRUGGEMANN, M. ET AL., J. EXP. MED., vol. 166, 1987, pages 1351 - 1361 |
CARLSSON ET AL., BIOCHEM. J., vol. 173, 1978, pages 723 - 737 |
CARTER ET AL., PROC. NATL. ACAD. SCI. USA, vol. , 89, 1992, pages 4285 |
CHARI ET AL., CANCER RES., vol. 52, 1992, pages 127 - 131 |
CHARI ET AL., CANCER RESEARCH, vol. 52, 1992, pages 127 - 131 |
CHARI, R.V.J. ET AL.: "Immunoconjugates Containing Novel Maytansinoids: Promising Anticancer Drugs.", CANCER RES., vol. 52, no. 1, 1992, pages 127 - 131, XP000453560 * |
CHARLTON: "Methods in Molecular Biology", vol. 248, 2003, HUMANA PRESS, pages: 245 - 254 |
CHEN ET AL., J. MOL. BIOL., vol. 293, 1999, pages 865 - 881 |
CHOTHIA; LESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917 |
CHOWDHURY, METHODS MOL. BIOL., vol. 207, 2008, pages 179 - 196 |
CLACKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628 |
CLARKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628 |
CLYNES ET AL., PROC. NAT'L ACAD. SCI. USA, vol. 95, 1998, pages 652 - 656 |
CRAGG, M.S. ET AL., BLOOD, vol. 101, 2003, pages 1045 - 1052 |
CRAGG, M.S.; M.J. GLENNIE, BLOOD, vol. 103, 2004, pages 2738 - 2743 |
CUNNINGHAM; WELLS, SCIENCE, vol. 244, 1989, pages 1081 - 1085 |
DALL' ACQUA ET AL., METHODS, vol. 36, 2005, pages 43 - 60 |
DORONINA ET AL., BIOCONJ. CHEM., vol. 17, 2006, pages 114 - 124 |
DORONINA ET AL., BIOCONJUGATE CHEM., vol. 17, 2006, pages 114 - 124 |
DORONINA ET AL., NAT. BIOTECH., vol. 21, 2003, pages 778 - 784 |
DORONINA ET AL., NAT. BIOTECHNOL., vol. 21, 2003, pages 778 - 784 |
DORONINA ET AL., NATURE BIOTECHNOL., vol. 21, no. 7, 2003, pages 778 - 784 |
DORONINA, NAT. BIOTECHNOL., vol. 21, no. 7, 2003, pages 778 - 784 |
DRUGS OF THE FUTURE, vol. 25, no. 7, 2000, pages 686 |
DUBOWCHIK ET AL., BIOORG. & MED. CHEM. LETTERS, vol. 12, 2002, pages 1529 - 1532 |
DUNCAN; WINTER, NATURE, vol. 322, 1988, pages 738 - 40 |
E. SCHRODER; K. LUBKE: "The Peptides", vol. 1, 1965, ACADEMIC PRESS, pages: 76 - 136 |
EPSTEIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 82, 1985, pages 3688 |
ERICKSON ET AL., CANCER RES., vol. 66, no. 8, 2006, pages 1 - 8 |
FELLOUSE, PROC. NATL. ACAD. SCI. USA, vol. 101, no. 34, 2004, pages 12467 - 12472 |
FLATMAN ET AL., J. CHROMATOGR. B, vol. 848, 2007, pages 79 - 87 |
FRAKER ET AL., BIOCHEM. BIOPHYS. RES. COMMUN., vol. 80, 1978, pages 49 - 57 |
GABIZON ET AL., J. NATIONAL CANCER INST., vol. 81, no. 19, 1989, pages 1484 |
GAZZANO-SANTORO ET AL., J. IMMUNOL. METHODS, vol. 202, 1996, pages 163 |
GEOGHEGAN; STROH, BIOCONJUGATE CHEM., vol. 3, 1992, pages 138 - 146 |
GERNGROSS, NAT. BIOTECH., vol. 22, 2004, pages 1409 - 1414 |
GRAHAM ET AL., J. GEN VIROL., vol. 36, 1977, pages 59 |
GRIFFITHS ET AL., EMBO J, vol. 12, 1993, pages 725 - 734 |
GRUBER ET AL., J. IMMUNOL., vol. 152, 1994, pages 5368 |
GUYER ET AL., J. IMMUNOL., vol. 117, 1976, pages 587 |
HALABAN R ET AL.: "PLX4032, a Selective BRAF (V600E) Kinase Inhibitor, Activates the ERK Pathway and Enhances Cell Migration and Proliferation of BRAF(WT) Melanoma Cells", PIGMENT CELL MELANOMA RES, vol. 23, no. 2, February 2010 (2010-02-01), pages 190 - 200 |
HAMANN ET AL., EXPERT OPIN. THER. PATENTS, vol. 15, 2005, pages 1087 - 1103 |
HAMBLETT ET AL., CLIN. CANCER RES., vol. 10, 2004, pages 7063 - 7070 |
HAMBLETT, K.J. ET AL.: "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate", AMERICAN ASSOCIATION FOR CANCER RESEARCH, 2004 ANNUAL MEETING, MARCH 27-31, 2004, PROCEEDINGS OF THE AACR, vol. 45, 27 March 2004 (2004-03-27) |
HARLOW; LANE: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY, article "ch.14" |
HATZIVASSILIOU G ET AL.: "RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.", NATURE, vol. 464, no. 7287, pages 431 - 5, XP055126892, DOI: doi:10.1038/nature08833 |
HAY ET AL., BIOORG. MED. CHEM. LETT., vol. 9, 1999, pages 2237 |
HELLSTROM, I ET AL., PROC. NAT'L ACAD. SCI. USA, vol. 82, 1985, pages 1499 - 1502 |
HELLSTROM, I. ET AL., PROC. NAT'L ACAD. SCI. USA, vol. 83, 1986, pages 7059 - 7063 |
HINMAN ET AL., CANCER RES., vol. 53, 1993, pages 3336 - 3342 |
HINMAN ET AL., CANCER RESEARCH, vol. 53, 1993, pages 3336 - 3342 |
HOLLINGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448 |
HOOGENBOOM ET AL., METHODS IN MOLECULAR BIOLOGY, vol. 178, pages 1 - 37 |
HOOGENBOOM ET AL.: "Methods in Molecular Biology", vol. 178, 2001, HUMAN PRESS, pages: 1 - 37 |
HOOGENBOOM; WINTER, J. MOL. BIOL., vol. 227, 1992, pages 381 - 388 |
HUDSON ET AL., NAT. MED., vol. 9, 2003, pages 129 - 134 |
HWANG ET AL., PROC. NATL ACAD. SCI. USA, vol. 77, 1980, pages 4030 |
IDUSOGIE ET AL., J. IMMUNOL., vol. 164, 2000, pages 4178 - 4184 |
JEFFREY ET AL., BIOORGANIC & MED. CHEM. LETTERS, vol. 16, 2006, pages 358 - 362 |
JEFFREY ET AL., J. MED. CHEM., vol. 48, 2005, pages 1344 - 1358 |
JOHNSTON S.: "XL518, a potent, selective, orally bioavailable MEK1 inhibitor, downregulates the Ras/Raf/MEKJERK pathway in vivo, resulting in tumor growth inhibition and regression in preclinical models", AACR-NCI-EORTC SYMPOSIUM ON MOLECULAR TARGETS AND CANCER THERAPEUTICS, 22 October 2007 (2007-10-22) |
KABAT ET AL.: "Sequences of Proteins of Immunological Interest, 5th Ed.", 1991, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH |
KABAT ET AL.: "Sequences of Proteins of Immunological Interest, Fifth Edition,", vol. 1-3, 1991, NIH PUBLICATION, pages: 91 - 3242 |
KAM ET AL., PROC. NATL. ACAD. SCI. USA, vol. 102, 2005, pages 11600 - 11605 |
KANDA, Y. ET AL., BIOTECHNOL. BIOENG., vol. 94, no. 4, 2006, pages 680 - 688 |
KASHMIRI ET AL., METHODS, vol. 36, 2005, pages 25 - 34 |
KIM ET AL., J. IMMUNOL., vol. 24, 1994, pages 249 |
KINDT ET AL.: "Kuby Immunology", 2007, W.H. FREEMAN AND CO, pages: 91 |
KING ET AL., J. MED. CHEM., vol. 45, 2002, pages 4336 - 4343 |
KINGSBURY ET AL., J. MED. CHEM., vol. 27, 1984, pages 1447 |
KLIMKA ET AL., BR. J. CANCER, vol. 83, 2000, pages 252 - 260 |
KOSTELNY ET AL., J. IMMUNOL., vol. 148, no. 5, 1992, pages 1547 - 1553 |
KOVTUN ET AL., CANCER RES., vol. 66, no. 6, 2006, pages 3214 - 3121 |
KOZBOR, J. IMMUNOL., vol. 133, 1984, pages 3001 |
KRATZ ET AL., CURRENT MED. CHEM., vol. 13, 2006, pages 477 - 523 |
KROP ET AL., EUROPEAN CANCER CONFERENCE ECCO, 23 September 2007 (2007-09-23) |
LAMBERT, J., CURR. OPINION IN PHARMACOLOGY, vol. 5, 2005, pages 543 - 549 |
LAW ET AL., CANCER RES., vol. 66, no. 4, 2006, pages 2328 - 2337 |
LEE ET AL., J. IMMUNOL. METHODS, vol. 284, no. 1-2, 2004, pages 119 - 132 |
LEE ET AL., J. MOL. BIOL., vol. 340, no. 5, 2004, pages 1073 - 1093 |
LI ET AL., NAT. BIOTECH., vol. 24, 2006, pages 210 - 215 |
LI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 103, 2006, pages 3557 - 3562 |
LIU ET AL., PROC. NATL. ACAD. SCI USA, vol. 93, 1996, pages 8618 - 8623 |
LIU ET AL., PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 8618 - 8623 |
LODE ET AL., CANCER RES., vol. 58, 1998, pages 2928 |
LODE ET AL., CANCER RESEARCH, vol. 58, 1998, pages 2925 - 2928 |
LONBERG, CURR. OPIN. IMMUNOL., vol. 20, 2008, pages 450 - 459 |
LONBERG, NAT. BIOTECH., vol. 23, 2005, pages 1117 - 1125 |
MANDLER ET AL., BIOCONJUGATE CHEM., vol. 13, 2002, pages 786 - 791 |
MANDLER ET AL., BIOORGANIC & MED. CHEM. LETTERS, vol. 10, 2000, pages 1025 - 1028 |
MANDLER ET AL., J. NAT. CANCER INST., vol. 92, no. 19, 2000, pages 1573 - 1581 |
MARKS ET AL., J. MOL. BIOL., vol. 222, 1992, pages 581 - 597 |
MARKS; BRADBURY, METHODS IN MOLECULAR BIOLOGY, vol. 248, pages 161 - 175 |
MARTIN ET AL., J. BIOL. CHEM., vol. 257, 1982, pages 286 - 288 |
MATHER ET AL., ANNALS N.Y. ACAD. SCI., vol. 383, 1982, pages 44 - 68 |
MATHER, BIOL. REPROD., vol. 23, 1980, pages 243 - 251 |
MCCAFFERTY ET AL., NATURE, vol. 348, pages 552 - 554 |
MCDONAGH ET AL., PROT. ENGR. DESIGN & SELECTION, vol. 19, no. 7, 2006, pages 299 - 307 |
MCDONAGH, PROTEIN ENG. DESIGN & SEL, 2006 |
MILSTEIN; CUELLO, NATURE, vol. 305, 1983, pages 537 |
MORRIS: "Methods in Molecular Biology", vol. 66, 1996, HUMANA PRESS, article "Epitope Mapping Protocols" |
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855 |
NAGY ET AL., PROC. NATL. ACAD. SCI., vol. 97, 2000, pages 829 - 834 |
NAKAMUTA M ET AL.: "Cloning and Sequence Analysis of a cDNA encoding Human non-selective type of endothelin receptor", BIOCHEM BIOPHYS RES COMMUN., vol. 177, no. l, 31 May 1991 (1991-05-31), pages 34 - 9, XP024771029, DOI: doi:10.1016/0006-291X(91)91944-8 |
NAZARIAN R. ET AL.: "Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation", NATURE, vol. 468, 16 December 2010 (2010-12-16), pages 973 - 977 |
NEUBERGER ET AL., NATURE, vol. 312, 1984, pages 604 - 608 |
NI, XIANDAI MIANYIXUE, vol. 26, no. 4, 2006, pages 265 - 268 |
NICULESCU-DUVAZ; SPRINGER, ADV. DRUG DELIV. REV., vol. 26, 1997, pages 151 - 172 |
OKAZAKI ET AL., J. MOL. BIOL., vol. 336, 2004, pages 1239 - 1249 |
OSBOURN ET AL., METHODS, vol. 36, 2005, pages 61 - 68 |
OSOL, A.: "Remington's Pharmaceutical Sciences 16th edition,", 1980 |
PADLAN, MOL. IMMUNOL., vol. 28, 1991, pages 489 - 498 |
PETKOVA, S.B. ET AL., INT'L. IMMUNOL., vol. 18, no. 12, 2006, pages 1759 - 1769 |
PETTIT ET AL., ANTI-CANCER DRUG DESIGN, vol. 13, 1998, pages 243 - 277 |
PETTIT ET AL., ANTIMICROB. AGENTS CHEMOTHER., vol. 42, 1998, pages 2961 - 2965 |
PETTIT ET AL., J. AM. CHEM. SOC., vol. 111, 1989, pages 5463 - 5465 |
PETTIT ET AL., J. CHEM. SOC. PERKIN TRANS., vol. 1, no. 5, 1996, pages 859 - 863 |
PETTIT, G.R. ET AL., SYNTHESIS, 1996, pages 719 - 725 |
PLUCKTHUN: "The Pharmacology of Monoclonal Antibodies,", vol. 113, 1994, SPRINGER-VERLAG, pages: 269 - 315 |
PORTOLANO ET AL., J. IMMUNOL., vol. 150, 1993, pages 880 - 887 |
PRESTA ET AL., CANCER RES., vol. 57, 1997, pages 4593 - 4599 |
PRESTA ET AL., J. IMMUNOL., vol. 151, 1993, pages 2623 |
QUEEN ET AL., PROC. NAT'L ACAD. SCI. USA, vol. 86, 1989, pages 10029 - 10033 |
RAVETCH; KINET, ANNU. REV. IMMUNOL., vol. 9, 1991, pages 457 - 492 |
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329 |
RIPKA ET AL., ARCH. BIOCHEM. BIOPHYS., vol. 249, 1986, pages 533 - 545 |
RODRIGUES ET AL., CHEMISTRY BIOLOGY, vol. 2, 1995, pages 223 |
ROSOK ET AL., J. BIOL. CHEM., vol. 271, 1996, pages 22611 - 22618 |
ROWLAND ET AL., CANCER IMMUNOL. IMMUNOTHER., vol. 21, 1986, pages 183 - 87 |
SALA E ET AL.: "BRAF silencing by short hairpin RNA or chemical blockade by PLX4032 leads to different responses in melanoma and thyroid carcinoma cells", MOL. CANCER RES., vol. 6, no. 5, May 2008 (2008-05-01), pages 751 - 9, XP055267102, DOI: doi:10.1158/1541-7786.MCR-07-2001 |
SANDERSON ET AL., CLIN. CANCER RES., vol. 11, 2005, pages 843 - 852 |
SEBOLT-LEOPOLD JS; HERRERA R.: "Targeting the mitogen-activated protein kinase cascade to treat cancer", NAT REV CANCER., vol. 4, 2004, pages 937 - 947, XP002446982, DOI: doi:10.1038/nrc1503 |
See also references of EP2776051A4 |
SHIELDS ET AL., J. BIOL. CHEM., vol. 9, no. 2, 2001, pages 6591 - 6604 |
SIDHU ET AL., J. MOL. BIOL., vol. 338, no. 2, 2004, pages 299 - 310 |
SIMS ET AL., J. IMMUNOL., vol. 151, 1993, pages 2296 |
STORM ET AL., J. AMER. CHEM. SOC., vol. 94, 1972, pages 5815 |
SUN ET AL., BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 12, 2002, pages 2213 - 2215 |
SUN ET AL., BIOORGANIC & MEDICINAL CHEMISTRY, vol. 11, 2003, pages 1761 - 1768 |
SYRIGOS; EPENETOS, ANTICANCER RESEARCH, vol. 19, 1999, pages 605 - 614 |
THOMPSON JF ET AL.: "Cutaneous melanoma in the era of molecular profiling", LANCET, vol. 374, 2009, pages 362 - 5, XP026395827, DOI: doi:10.1016/S0140-6736(09)61397-0 |
THOMPSON, LANCET, 2009 |
THORPE ET AL.: "Monoclonal Antibodies '84: Biological And Clinical Applications", 1985, article "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", pages: 475 - 506 |
TORGOV ET AL., BIOCONJ. CHEM., vol. 16, 2005, pages 717 - 721 |
TRAUNECKER ET AL., EMBO J., vol. 10, 1991, pages 3655 |
TUTT ET AL., J. IMMUNOL., vol. 147, 1991, pages 60 |
URLAUB ET AL., PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216 |
VAN DIJK; VAN DE WINKEL, CURR. OPIN. PHARMACOL., vol. 5, 2001, pages 368 - 74 |
VITETTA ET AL., SCIENCE, vol. 238, 1987, pages 1098 |
VOLLMERS; BRANDLEIN, HISTOLOGY AND HISTOPATHOLOGY, vol. 20, no. 3, 2005, pages 927 - 937 |
VOLLMERS; BRANDLEIN, METHODS AND FINDINGS IN EXPERIMENTAL AND CLINICAL PHARMACOLOGY, vol. 27, no. 3, 2005, pages 185 - 91 |
WIDDISON ET AL., J. MED. CHEM., vol. 49, 2006, pages 4392 - 4408 |
WINTER ET AL., ANN. REV. IMMUNOL., vol. 12, 1994, pages 433 - 455 |
WISEMAN ET AL., BLOOD, vol. 99, no. 12, 2002, pages 4336 - 42 |
WISEMAN ET AL., EUR. JOUR. NUCL. MED., vol. 27, no. 7, 2000, pages 766 - 77 |
WITZIG ET AL., J. CLIN. ONCOL., vol. 20, no. 10, 2002, pages 2453 - 63 |
WITZIG ET AL., J. CLIN. ONCOL., vol. 20, no. 15, 2002, pages 3262 - 69 |
WONG K-K ET AL.: "Recent developments in anti-cancer agents targeting the Ras/Raf/MEKJERK pathway", RECENT PAT ANTICANCER DRUG DISCOV., vol. 4, 2009, pages 28 - 35, XP055056888, DOI: doi:10.2174/157489209787002461 |
WOYKE ET AL., ANTIMICROB. AGENTS AND CHEMOTHER., vol. 45, no. 12, 2001, pages 3580 - 3584 |
WRIGHT ET AL., TIBTECH, vol. 15, 1997, pages 26 - 32 |
WU ET AL., NATURE BIOTECHNOLOGY, vol. 23, no. 9, 2005, pages 1137 - 1146 |
XIE ET AL., EXPERT. OPIN. BIOL. THER., vol. 6, no. 3, 2006, pages 281 - 291 |
YAMANE-OHNUKI ET AL., BIOTECH. BIOENG., vol. 87, 2004, pages 614 |
YAZAKI; WU: "Methods in Molecular Biology", vol. 248, 2003, HUMANA PRESS, pages: 255 - 268 |
YU ET AL., PNAS, vol. 99, 2002, pages 7968 - 7973 |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9724413B2 (en) | 2011-08-01 | 2017-08-08 | Genentech, Inc. | Methods of treating cancer using PD-1 axis binding antagonists and MEK inhibitors |
US10646567B2 (en) | 2011-08-01 | 2020-05-12 | Genentech, Inc. | Methods of treating cancer using PD-1 axis binding antagonists and MEK inhibitors |
US9216170B2 (en) | 2012-03-19 | 2015-12-22 | Hoffmann-La Roche Inc. | Combination therapy for proliferative disorders |
US9486445B2 (en) | 2012-03-19 | 2016-11-08 | Hoffmann-La Roche Inc. | Combination therapy for proliferative disorders |
US10946093B2 (en) | 2014-07-15 | 2021-03-16 | Genentech, Inc. | Methods of treating cancer using PD-1 axis binding antagonists and MEK inhibitors |
WO2016055907A1 (fr) * | 2014-10-10 | 2016-04-14 | Pfizer Inc. | Associations synergiques d'auristatine |
EP3549583A1 (fr) * | 2014-10-10 | 2019-10-09 | Pfizer Inc | Associations synergiques d'auristatine |
US10617670B2 (en) | 2014-10-10 | 2020-04-14 | Pfizer Inc. | Synergistic auristatin combinations |
WO2017220739A1 (fr) | 2016-06-24 | 2017-12-28 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | Anticorps dirigé contre le sous-type bêta du récepteur de l'endothéline |
FR3053042A1 (fr) * | 2016-06-24 | 2017-12-29 | Commissariat Energie Atomique | Anticorps dirige contre le sous-type b des recepteurs aux endothelines et ses utilisations |
US11312780B2 (en) | 2016-06-24 | 2022-04-26 | Commissariat à l'énergie atomique et aux énergies alternatives | Antibody directed against the endothelin receptor beta sub-type |
US11040027B2 (en) | 2017-01-17 | 2021-06-22 | Heparegenix Gmbh | Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death |
Also Published As
Publication number | Publication date |
---|---|
KR20140097205A (ko) | 2014-08-06 |
CL2014001092A1 (es) | 2014-07-25 |
JP2014532648A (ja) | 2014-12-08 |
EP2776051A4 (fr) | 2015-06-17 |
TW201325613A (zh) | 2013-07-01 |
JP6251682B2 (ja) | 2017-12-20 |
CO7151543A2 (es) | 2014-12-29 |
EA201490879A1 (ru) | 2014-08-29 |
IN2014CN03062A (fr) | 2015-07-31 |
PE20142312A1 (es) | 2015-01-25 |
CA2850034A1 (fr) | 2013-05-02 |
CN104039340A (zh) | 2014-09-10 |
AU2012328980A1 (en) | 2014-04-24 |
AR088509A1 (es) | 2014-06-11 |
CN104039340B (zh) | 2017-04-05 |
IL232135A0 (en) | 2014-05-28 |
BR112014009953A2 (pt) | 2017-12-05 |
MX2014004991A (es) | 2014-05-22 |
SG11201401815XA (en) | 2014-05-29 |
JP2018027948A (ja) | 2018-02-22 |
EP2776051A1 (fr) | 2014-09-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11352431B2 (en) | Anti-FCRH5 antibodies | |
US10653792B2 (en) | Anti-Ly6E antibodies and immunoconjugates and methods of use | |
US9175089B2 (en) | Anti-LGR5 antibodies and immunoconjugates | |
US20180312602A1 (en) | Anti-gpc3 antibodies and immunoconjugates | |
JP6251682B2 (ja) | メラノーマ治療の治療の組み合わせ及び方法 | |
US9926377B2 (en) | Anti-GPC3 antibodies and immunoconjugates | |
US20170043034A1 (en) | Methods of using anti-steap1 antibodies and immunoconjugates | |
EP3046940B1 (fr) | Procédés d'utilisation d'anticorps anti-lgr5 | |
US20140341916A1 (en) | Therapeutic combinations and methods of treating melanoma | |
NZ715125B2 (en) | Anti-fcrh5 antibodies |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12842985 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2850034 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012842985 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14083118 Country of ref document: CO |
|
WWE | Wipo information: entry into national phase |
Ref document number: 232135 Country of ref document: IL |
|
ENP | Entry into the national phase |
Ref document number: 2012328980 Country of ref document: AU Date of ref document: 20121024 Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: MX/A/2014/004991 Country of ref document: MX |
|
WWE | Wipo information: entry into national phase |
Ref document number: 000603-2014 Country of ref document: PE Ref document number: 14354362 Country of ref document: US |
|
ENP | Entry into the national phase |
Ref document number: 2014538892 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 20147013878 Country of ref document: KR Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 201490879 Country of ref document: EA |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112014009953 Country of ref document: BR |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01E Ref document number: 112014009953 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 112014009953 Country of ref document: BR Kind code of ref document: A2 Effective date: 20140425 |