KR20140097205A - Therapeutic combinations and methods of treating melanoma - Google Patents

Therapeutic combinations and methods of treating melanoma Download PDF

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KR20140097205A
KR20140097205A KR1020147013878A KR20147013878A KR20140097205A KR 20140097205 A KR20140097205 A KR 20140097205A KR 1020147013878 A KR1020147013878 A KR 1020147013878A KR 20147013878 A KR20147013878 A KR 20147013878A KR 20140097205 A KR20140097205 A KR 20140097205A
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antibody
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etbr
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폴 폴라키스
지요티 아순디
수잔나 클라크
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제넨테크, 인크.
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Priority to PCT/US2012/061533 priority patent/WO2013063001A1/en
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445Non condensed piperidines, e.g. piperocaine
    • A61K31/4523Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869Immunoglobulins [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
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [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
    • C07K16/3053Skin, nerves, brain
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
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Abstract

The present invention provides therapeutic combinations of anti-ETBR antibodies and MAP kinase inhibitors and methods of using the same to treat melanoma.
[Representative figure]
4A

Description

[0001] THERAPEUTIC COMBINATIONS AND METHODS OF TREATING MELANOMA [0002]

Related application

This application claims priority to U.S. Provisional Application No. 61 / 552,893, filed October 28, 2011, and U.S. Provisional Application No. 61 / 678,978, filed August 2, 2012 under 35 USC 119 (e) Are included by reference.

Field of invention

The present invention generally relates to the treatment of melanoma by the use of certain antibody and small molecule drug combinations.

Melanoma is an aggressive form of skin cancer that has recently increased in incidence (Thompson JF et al., Cutaneous melanoma in the era of molecular profiling. Lancet 2009; 374: 362-5). Although healing can be achieved using surgical resection of localized lesions, advanced stage melanomas respond only poorly to currently approved therapies. The 5-year survival rate for stage IV metastatic melanoma is approximately 10% (Thompson, Lancet 2009). A novel therapeutic approach, including antisense to Bcl2, antibodies to CTLA4, small molecule RAF kinase inhibitors and adoptive immunotherapy is currently in clinical trials for metastatic melanoma (Ascierto PA et al., Melanoma: a model for testing new agents in combination therapies. J Transl Med 2010; 8: 38-45). The results from some of these recent studies may seem encouraging, but a sustained impact on overall survival may require therapeutic combinations that include additional new agents.

However, it is recognized that melanoma can prove molecular changes, for example, in a specific signal transduction pathway essential for cellular reactivity to growth factors. Thus, rather than treating melanoma as a single disease, there have been attempts to stratify each subtype into molecular subtypes for treatment using the most appropriate therapy.

One subtype of melanoma possesses an abnormality in the MAP kinase pathway. The MAPK pathway is a phosphorylated-driven signaling cascade that links the intracellular response to the binding of growth factors to cell surface receptors. These pathways regulate many processes, including cell proliferation and differentiation, and are often deregulated 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 the RAF / MEK / ERK kinase complex and then drives the transcription of key modulators through protein phosphorylation. Inhibition of MAPK signaling from the pathway to the targeting agent towards the critical protein has the potential to inhibit growth in a variety of tumor types (Wong KK et al., Recent developments in anti-cancer agents targeting the Ras / Raf / MEK / ERK pathway. Recent Pat Anticancer Drug Discov. 2009; 4: 28-35).

Improper activation of the MEK / ERK pathway promotes cell growth in the absence of exogenous growth factors. GDC-0973 (aka 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. Presented at: AACR-NCI-EORTC Symposium on Molecular Targets and Cancer Therapeutics; October 22, 2007; San Francisco, CA. Abstract C209] ). As a result, tumorigenic signals from the cell surface, Ras and Raf to ERK are terminated. Continuous inhibition of ERK activation translates into reduced proliferation and induction of apoptosis. In multiple preclinical studies, GDC-0973 has been shown to inhibit cell growth and induce tumor regression.

Recently, B-Raf enzyme inhibitor Bemura penip (also known as Zelboraf®) has also been approved by the US Food and Drug Administration for the treatment of late melanoma. It has been shown that bemurafenid induces programmed cell death in melanoma cell lines (Sala E, et al., BRAF silencing by short hairpin RNA or chemical blockade by PLX4032 in different responses in melanoma and thyroid carcinoma cells Cancer Res. 6 (5): 751-9 (May 2008)]). Bemula penumb interrupts the B-Raf / MEK step on the B-Raf / MEK / ERK pathway when B-Raf has the normal V600E mutation. Bemura penip is effective in melanoma patients whose cancer has a V600E BRAF mutation (i.e., the normal valine at amino acid position 600 on the B-RAF protein is replaced by glutamic acid). Approximately 60% of melanomas have the V600E BRAF mutation. These non-mutated melanoma cells do not appear to be inhibited by bemurafenid; This can paradoxically stimulate normal BRAF and promote tumor growth (Hatzivassiliou G et al., RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464 (7287): 431-5 ; 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 23 (2): 190-200 2010)]). Clinical trials showed improved survival, improved objective response rate, and improved progression-free survival in patients treated with bemura-penip compared with DTIC, but there was a likelihood of disease recurrence (Nazarian R. et al. , Melanomas acquire resistance to B-RAF (V600E) inhibition by RTK or N-RAS upregulation. Nature Vol: 468, Pages: 973-977 (16 December 2010)).

Despite advances in melanoma therapies, there is a great need for additional healing therapies that can effectively inhibit neoplastic cell growth. It is therefore an object of the present invention to identify combinations of therapeutic agents for the production of compositions of matter useful for the curative treatment of melanoma cancers.

The invention provides a method of inhibiting tumor growth in a subject suffering from a melanoma, comprising administering to a subject suffering from a melanoma 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). ≪ / RTI >

In one aspect, the combination of an anti-ETBR antibody drug conjugate and a MAP kinase inhibitor is synergistic. In another aspect, with respect to the synergistic combination, TGI is greater than the TGI exhibited using the anti-ETBR antibody drug conjugate alone, or greater than the TGI exhibited using the MAP kinase inhibitor alone. In a further aspect, with respect to a synergistic combination, TGI is about 10% greater, or about 15% greater, or about 20% greater, or about 25% greater than the sole use of an anti-ETBR antibody drug conjugate Or greater than or about 30% greater, or about 35% greater, or about 40% greater, or about 45% greater, or about 50% greater, or about 55% Or about 60% greater, or about 65% greater, or about 70% greater, or the TGI is about 10% greater, or about 15% greater, or greater than the sole use of a MAP kinase inhibitor , 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.

In another aspect of the invention described above, the anti-ETBR antibody specifically binds to an ETBR epitope consisting of amino acids 64-101 of SEQ ID NO: 10. In another aspect of the method of the invention, the anti-ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs wherein the VH CDR1 is SEQ ID NO: 1, the VH CDR2 is SEQ ID NO: 2, the VH CDR3 is SEQ ID NO: 3 , VL CDR1 is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, and VL CDR3 is SEQ ID NO: 6. In another aspect of the method of the invention, the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or SEQ ID NO: 9. A further aspect of this method also includes an anti-ETBR antibody that also has a VL of SEQ ID NO: 8.

In one aspect of the inventive method described above, the anti-ETBR antibody is conjugated to a cytotoxin, wherein said cytotoxin is a cytotoxic agent selected from the group consisting of a toxin, an antibiotic, a radioactive isotope and a nucleic acid degrading enzyme, Toxins are toxins. In another aspect of the invention, the toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin. In another aspect of the invention, the toxin is a maytansinoid.

In one aspect of the inventive method described above, the MAP kinase inhibitor is a BRAF inhibitor. In another aspect of the inventive method described above, the BRAF inhibitor is selected from the group consisting of propane-1-sulfonic acid {3- [5- (4- chlorophenyl) -lH- pyrrolo [2,3- b] pyridine- ] -2,4-difluoro-phenyl} -amide. In another aspect, a BRAF inhibitor has the following chemical structure:

Figure pct00001

In yet another aspect of the invention described above, the MAP kinase inhibitor is a MEK inhibitor. In another aspect of the inventive method described above, the MEK inhibitor is (S) - (3,4-difluoro-2- ( Hydroxy-3- (piperidin-2yl) azetidin-1-yl) methanone. In another aspect of the inventive method described above, the MEK inhibitor has the following chemical structure.

Figure pct00002

In one aspect of the invention, it is contemplated to describe 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. In another aspect of the invention described above, the melanoma is ETBR-positive. In another aspect of the method of the invention, said melanoma is metastatic. In a further aspect of the method of the invention, the subject has not received prior therapy with MAP kinase inhibitors. In a further aspect of the method of the invention, the subject is a BRAF wild type having a V600E BRAF gene mutation or the subject having no V600E BRAF mutation. In a further aspect of the method of the invention, the subject has not received prior therapy with MAP kinase inhibitors.

In another aspect of the invention described above, the combination of an anti-ETBR antibody and a MAP kinase inhibitor is synergistic. In another aspect, with respect to the synergistic combination, TGI is greater than the TGI exhibited using the anti-ETBR antibody alone, or greater than the TGI exhibited using the MAP kinase inhibitor alone. In a further aspect, with respect to the synergistic combination, TGI is about 10% greater, or about 15% greater, or about 20% greater, or about 25% greater than the sole use of anti-ETBR antibody , 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, or TGI is about 10% greater, or about 15% greater, than the sole use of a MAP kinase inhibitor, or Greater than about 20% greater, or greater than about 25% greater, or greater than about 30% greater, or greater than about 35% greater, or greater than about 40% greater, or greater than about 45% greater, Or greater, or about 55% greater, or about 60% greater, or about 65% greater, or about 70% greater.

In another aspect of the invention described above, the anti-ETBR antibody specifically binds to an ETBR epitope consisting of amino acids 64-101 of SEQ ID NO: 10. In another aspect of the method of the invention, the anti-ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs wherein the VH CDR1 is SEQ ID NO: 1, the VH CDR2 is SEQ ID NO: 2, the VH CDR3 is SEQ ID NO: 3 , VL CDR1 is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, and VL CDR3 is SEQ ID NO: 6. In another aspect of the method of the invention, the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or SEQ ID NO: 9. A further aspect of this method also includes an anti-ETBR antibody that also has a VL of SEQ ID NO: 8.

In one aspect of the inventive method described above, the anti-ETBR antibody is conjugated to a cytotoxin, wherein said cytotoxin is a cytotoxic agent selected from the group consisting of a toxin, an antibiotic, a radioactive isotope and a nucleic acid degrading enzyme, Toxins are toxins. In another aspect of the invention, the toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin. In another aspect of the invention, the toxin is a maytansinoid.

In one aspect of the inventive method described above, the MAP kinase inhibitor is a BRAF inhibitor. In another aspect of the above described method, the BRAF inhibitor is selected from the group consisting of propane-1-sulfonic acid {3- [5- (4- chlorophenyl) -lH- pyrrolo [2,3- b] pyridine- , 4-difluoro-phenyl} -amide. In another aspect, a BRAF inhibitor has the following chemical structure:

Figure pct00003

In yet another aspect of the invention described above, the MAP kinase inhibitor is a MEK inhibitor. In another aspect of the inventive method described above, the MEK inhibitor is (S) - (3,4-difluoro-2- ( Hydroxy-3- (piperidin-2yl) azetidin-1-yl) methanone. In another aspect of the inventive method described above, the MEK inhibitor has the following chemical structure.

Figure pct00004

In one aspect of the invention, it is contemplated to describe a method of treating melanoma, comprising administering a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody drug conjugate to a subject in need of treatment of the melanoma, Wherein the MAP kinase inhibitor is first administered to the subject in need thereof. In another aspect of the invention, the anti-ETBR antibody drug conjugate is administered after administration of the MAP kinase inhibitor. Alternatively, the methods contemplated by the present invention include the simultaneous administration of an anti-ETBR antibody and a MAP kinase inhibitor.

In one aspect of the invention, it is contemplated to describe a method of treating melanoma, comprising administering a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody drug conjugate to a subject in need of treatment of the melanoma, Wherein the anti-ETBR antibody drug conjugate and the MAP kinase inhibitor are administered sequentially, wherein the anti-ETBR antibody drug conjugate is first administered to the subject, and the MAP kinase inhibitor is administered to the subject after administration of the anti-ETBR antibody drug conjugate.

In one aspect of the invention, it is contemplated to describe a method of treating melanoma, comprising administering a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody drug conjugate to a subject in need of treatment of the melanoma, Wherein the MAP kinase inhibitor is first administered to the subject and the anti-ETBR antibody drug conjugate is administered to the subject after administration of the MAP kinase inhibitor.

There is provided a method of treating melanoma comprising administering to said subject a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody drug conjugate, in a subject in need of treatment of melanoma, , Wherein said anti-ETBR antibody drug conjugate is administered intravenously. In addition, in the methods of the invention, the anti-ETBR antibody drug conjugate can be administered at a dose of 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, 15 mpk, or about 20 mpk, or about 25 mpk, or about 30 mpk.

There is provided a method of treating melanoma comprising administering to said subject a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody drug conjugate, in a subject in need of treatment of melanoma, , Wherein the MAP kinase inhibitor is administered orally. It is also contemplated that in the methods of the present invention the BRAF inhibitor may be administered 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, 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 one aspect of the invention, it is contemplated that an article of manufacture comprising a package comprising an anti-ETBR antibody drug conjugate composition and a MAP kinase inhibitor composition is used for TGI in a subject suffering from a melanoma. It is further contemplated that the anti-ETBR antibody drug conjugate specifically binds to an ETBR epitope consisting of amino acids 64-101 of SEQ ID NO: 10. Alternatively, the anti-ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs wherein the VH CDRl is SEQ ID NO: 1, the VH CDR2 is SEQ ID NO: 2, the VH CDR3 is SEQ ID NO: 3, SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, and VL CDR3 is SEQ ID NO: 6. Additional alternatives are contemplated wherein the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or 9. In another alternative, the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or 9 and VL is SEQ ID NO: 8. In another aspect of the article of manufacture, the anti-ETBR antibody is conjugated to a cytotoxin, wherein the cytotoxin is a cytotoxic agent selected from the group consisting of a toxin, an antibiotic, a radioactive isotope and a nucleic acid degrading enzyme. In a further aspect, the cytotoxin is a toxin, wherein the toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin. In one aspect, the toxin is a maytansinoid.

In one aspect of the method of treating the melanoma described above, it is also contemplated that the MAP kinase inhibitor is a BRAF inhibitor. In another aspect of the above described method, the BRAF inhibitor is selected from the group consisting of propane-1-sulfonic acid {3- [5- (4- chlorophenyl) -lH- pyrrolo [2,3- b] pyridine- , 4-difluoro-phenyl} -amide. It is also contemplated that the BRAF inhibitor has the following chemical structure.

Figure pct00005

In yet another aspect of the invention described above, the MAP kinase inhibitor is a MEK inhibitor. In another aspect of the inventive method described above, the MEK inhibitor is (S) - (3,4-difluoro-2- ( Hydroxy-3- (piperidin-2yl) azetidin-1-yl) methanone. In another aspect of the inventive method described above, the MEK inhibitor has the following chemical structure.

Figure pct00006

In one aspect of the invention, an article of manufacture for treating melanoma in a subject is contemplated, comprising a package comprising an anti-ETBR antibody drug conjugate composition and a MAP kinase inhibitor composition. It is further contemplated that the anti-ETBR antibody specifically binds to an ETBR epitope consisting of amino acids 64-101 of SEQ ID NO: 10. Alternatively, the anti-ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs wherein the VH CDRl is SEQ ID NO: 1, the VH CDR2 is SEQ ID NO: 2, the VH CDR3 is SEQ ID NO: 3, SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, and VL CDR3 is SEQ ID NO: 6. Additional alternatives are contemplated wherein the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or 9. In another alternative, the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or 9 and VL is SEQ ID NO: 8. In another aspect of the article of manufacture, the anti-ETBR antibody is conjugated to a cytotoxin, wherein the cytotoxin is a cytotoxic agent selected from the group consisting of a toxin, an antibiotic, a radioactive isotope and a nucleic acid degrading enzyme. In a further aspect, the cytotoxin is a toxin, wherein the toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin. In one aspect, the toxin is a maytansinoid.

In one aspect of the above-described article of manufacture, it is contemplated that the MAP kinase inhibitor is also a BRAF inhibitor. In another aspect of the above described article of manufacture, the BRAF inhibitor is selected from the group consisting of propane-1-sulfonic acid {3- [5- (4- chlorophenyl) -lH- pyrrolo [2,3- b] pyridine- , 4-difluoro-phenyl} -amide. It is also contemplated that the BRAF inhibitor has the following chemical structure.

Figure pct00007

In another aspect of the above-described article of manufacture, the MAP kinase inhibitor is a MEK inhibitor. In another aspect of the above described article of manufacture, the MEK inhibitor is (S) - (3,4-difluoro-2- ( 3- (piperidin-2yl) azetidin-1-yl) methanone. In another aspect of the above described article of manufacture, the MEK inhibitor has the following chemical structure:

Figure pct00008

It is contemplated that one aspect of the present invention is the use of an anti-ETBR antibody drug conjugate and a MAP kinase inhibitor in the manufacture of a medicament for TGI of melanoma. It is further contemplated that the anti-ETBR antibody specifically binds to an ETBR epitope consisting of amino acids 64-101 of SEQ ID NO: 10. Alternatively, the anti-ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs wherein the VH CDRl is SEQ ID NO: 1, the VH CDR2 is SEQ ID NO: 2, the VH CDR3 is SEQ ID NO: 3, SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, and VL CDR3 is SEQ ID NO: 6. Additional alternatives are contemplated wherein the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or 9. In another alternative, the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or 9 and VL is SEQ ID NO: 8. In another aspect of the article of manufacture, the anti-ETBR antibody is conjugated to a cytotoxin, wherein the cytotoxin is a cytotoxic agent selected from the group consisting of a toxin, an antibiotic, a radioactive isotope and a nucleic acid degrading enzyme. In a further aspect, the cytotoxin is a toxin, wherein the toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin. In one aspect, the toxin is a maytansinoid.

In one aspect of the use of the medicament described above, it is also contemplated that the MAP kinase inhibitor is a BRAF inhibitor. In another aspect of the use of the medicament described above, the BRAF inhibitor is selected from the group consisting of propane-1-sulfonic acid {3- [5- (4- chlorophenyl) -1H- pyrrolo [2,3- b] pyridine- Difluoro-phenyl} -amide. ≪ / RTI > It is also contemplated that the BRAF inhibitor has the following chemical structure.

Figure pct00009

In another aspect of the use of the medicament described above, the MAP kinase inhibitor is a MEK inhibitor. In another aspect of the use of the medicament described above, the MEK inhibitor is selected from the group consisting of (S) - (3,4-difluoro-2- ( 3- (piperidin-2yl) azetidin-1-yl) methanone. In yet another aspect of the use of the medicament described above, the MEK inhibitor has the following chemical structure.

Figure pct00010

It is contemplated that one aspect of the present invention is the use of an article of manufacture comprising an anti-ETBR antibody drug conjugate composition and a MAP kinase inhibitor composition in the manufacture of a medicament for TGI of melanoma. It is further contemplated that the anti-ETBR antibody specifically binds to an ETBR epitope consisting of amino acids 64-101 of SEQ ID NO: 10. Alternatively, the anti-ETBR antibody has three variable heavy CDRs and three variable light chain CDRs wherein the VH CDR1 is SEQ ID NO: 1, the VH CDR2 is SEQ ID NO: 2, the VH CDR3 is SEQ ID NO: 3, 4, VL CDR2 is SEQ ID NO: 5, and VL CDR3 is SEQ ID NO: 6. Additional alternatives are contemplated wherein the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or 9. In another alternative, the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is SEQ ID NO: 7 or 9 and VL is SEQ ID NO: 8. In another aspect of the use of the article of manufacture, the anti-ETBR antibody is conjugated to a cytotoxin, wherein the cytotoxin is a cytotoxic agent selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleic acid degrading enzymes. In a further aspect, the cytotoxin is a toxin, wherein the toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin. In one aspect, the toxin is a maytansinoid.

In one aspect of the use of the above-described article of manufacture, it is also contemplated that the MAP kinase inhibitor is a BRAF inhibitor. In another aspect of the use of the above-described article of manufacture, the BRAF inhibitor is selected from the group consisting of propane-1-sulfonic acid {3- [5- (4- chlorophenyl) -lH- pyrrolo [2,3- b] pyridine- Difluoro-phenyl} -amide. ≪ / RTI > It is also contemplated that the BRAF inhibitor has the following chemical structure.

Figure pct00011

In another aspect of the use of the above-described article of manufacture, the MAP kinase inhibitor is a MEK inhibitor. In another aspect of the use of the above described article of manufacture, the MEK inhibitor is selected from the group consisting of (S) - (3,4-difluoro-2- ( 3- (piperidin-2yl) azetidin-1-yl) methanone. In another aspect of the use of the above-described article of manufacture, the MEK inhibitor has the following chemical structure.

Figure pct00012

Figure 1 is a schematic diagram of a MAP kinase pathway.
Figure 2 demonstrates receptor-level relationships to ADC cell death in vitro. The indicated number of receptor copies / wells were estimated by Scatchard analysis. Panel A shows cell death by anti-ET B R ADC titration against melanoma cell line UACC-257X2.2, and Panel B shows melanoma cell line A2058. Equivalent concentrations of anti-ET B R ADC (Hu5E9v1-vc-MMAE), control IgG-vc-MMAE, or equivalent PBS vehicle controls were incubated with the cells for 5 days and relative cell viability (y- (CellTiter-Glo).
Figure 3 shows the in vivo efficacy of anti-ETBR ADC in a xenograft mouse model. Subcutaneous tumors were established in mice inoculated with UACC-257X2.2 (panel A) or A2058 (panel B) cells. A single IV injection of the indicated dose of control ADC (control-vc-MMAE) or anti-ETBR ADC (Hu5E9v1-vc-MMAE) was given to the animal when the tumor volume reached approximately 200 mm 3 (day 0) Respectively. The mean tumor volume was determined from 10 animals per group with standard deviation (shown on the graph).
Figure 4 shows ET B R expression in UACC-257X2.2 melanoma cells treated with varying concentrations of BRAFi-945 for 24 hours. Panel A shows a normalized ETBR transcript for the RPL19 transcript. Panel B shows the expression of total ETBR and GAPDH (control) protein in 50 μg whole cell lysate. Panel C shows surface ETBR protein expression in living cells as observed by flow cytometry, wherein the first peak represents the treated cells for the second detection reagent alone and the middle peak represents cells that have not been treated with the BRAF inhibitor , And the last peak represents BRAF inhibitor treated cells.
Figure 5 shows the in vivo combinatorial efficacy of anti-ET B R ADC (Hu5E9v1-vc-MMAE) and BRAFi-945 on a changing dose of the UACC-257X2.2 melanoma xenograft mouse model. Subcutaneous tumors were established in mice inoculated with the UACC-257X2.2 cell line. When the tumor volume reached approximately 200 mm 3 (day 0), the animals were orally administered BRAFi-945 or vehicle control once daily for 21 days. On day 1 (after two doses of BRAFi-945), animals were given a single dose IV vehicle or a single IV injection of anti-ET B R ADC. Mean tumor volume was determined from 10 animals per group with standard deviation. Drug and dose information is as indicated: Panel A shows a combination of 1 mpk BRAFi-945 and 1 mpk anti-ET B R-ADC (Hu5E9v1-vc-MMAE); Panel B shows the combination of 1 mpk BRAFi-945 and 3 mpk anti-ET B R-ADC (Hu5E9v1-vc-MMAE); Panel C shows the combination of 6 mpk BRAFi-945 and 1 mpk anti-ET B R-ADC (Hu5E9v1-vc-MMAE); Panel D shows the combination of 6 mpk BRAFi-945 and 3 mpk anti-ET B R-ADC (Hu5E9v1-vc-MMAE); Panel E shows a combination of 20 mpk BRAFi-945 and 3 mpk anti-ET B R-ADC (Hu5E9v1-vc-MMAE).
Figure 6 shows ETBR expression in COLO 829 melanoma cells treated with varying concentrations of the BRAF inhibitor RG7204 for 24 hours. Panel A shows a normalized ETBR transcript for the RPL19 transcript. Panel B shows the expression of total ETBR and GAPDH (control) protein in 50 μg whole cell lysate. Panel C shows surface ETBR protein expression in live cells observed by flow cytometry where the first peak represents the treated cells for the second detection reagent alone and the second peak represents the untreated BRAF inhibitor Cells, and the third peak represents BRAF inhibitor treated cells.
Figure 7 demonstrates the in vivo combinatorial efficacy of the anti-ET B R ADC and BRAF inhibitor RG7204 on the COLO 829 melanoma xenograft mouse model. Subcutaneous tumors were established in mice inoculated with COLO 829 melanoma cell line. When the tumor volume reached approximately 200 mm 3 (day 0), the animals were orally administered RG7204 twice daily for 21 days. On day 1 (after 3 doses of RG7204), the animals were given a single IV injection of vehicle or doses of anti-ET B R ADC (Hu5E9v1-vc-MMAE) indicated. Mean tumor volume was determined from 9 animals per group with standard deviation. Drug and dose information is as indicated: Panel A shows 3 mpk of anti-ET B R-ADC (Hu5E9v1-vc-MMAE) combined with 30 mpk of RG7204; Panel B shows 1 mpk of anti-ET B R-ADC (Hu5E9v1-vc-MMAE) combined with 30 mpk of RG7204; Panel C shows 1 mpk of anti-ET B R-ADC (Hu5E9v1-vc-MMAE) combined with 10 mpk of RG7204; Panel D shows a 3 mpk anti-ET B R-ADC (Hu5E9v1-vc-MMAE) in combination with RG7204 at 10 mpk.
Figure 8 shows ETBR expression in A2058 melanoma cells treated with varying concentrations of the BRAF inhibitor RG7204 for 24 hours. Panel A shows the normalized ETBR transcript for the RPL19 transcript; Panel B shows the expression of total ETBR and GAPDH (control) protein in 100 ug total cell lysate; Panel C shows surface ETBR protein expression in living cells as observed by flow cytometry. The first peak represents the cells treated for the second detection reagent alone, the cells not treated with the second peak BRAF inhibitor and the third peak represents the BRAF inhibitor treated cells.
Figure 9 demonstrates the in vivo combinatorial efficacy of the anti-ET B R ADC (Hu5E9v1-vc-MMAE) and the BRAF inhibitor RG7204 on the A2058 melanoma xenograft mouse model. Subcutaneous tumors were established in mice inoculated with A2058 melanoma cell line. When the tumor volume reached approximately 200 mm 3 (day 0), the animals were orally administered RG7204 twice daily for 21 days. On day 1 (after 3 doses of RG7204), the animals were given a single IV injection of vehicle or doses of anti-ET B R ADC (Hu5E9v1-vc-MMAE) indicated. Mean tumor volume was determined from 10 animals per group with standard deviation. Drug and dose information is shown on each graph as follows: Panel A shows 6 mpk of anti-ET B R-ADC (Hu5E9v1-vc-MMAE) combined with 10 mpk of RG7204; Panel B shows 6 mpk of anti-ET B R-ADC (Hu5E9v1-vc-MMAE) combined with 30 mpk of RG7204; Panel C shows 3 mpk of anti-ET B R-ADC (Hu5E9v1-vc-MMAE) in combination with 10 mpk of RG7204; Panel D shows a 3 mpk anti-ET B R-ADC (Hu5E9v1-vc-MMAE) combined with 30 mpk of RG7204.
Figure 10 shows Western blots performed with BRAFi RG7204 showing the expression of total ETBR, Perk and erk proteins in 25-100 microgram total cell lysates from IPC-298 melanoma cells and the control proteins GAPDH and [beta] -tubulin Show the experiment.
Figure 11 shows surface ETBR protein expression in IPC-298 live cells observed by flow cytometry after incubation with 0.1 [mu] M, 1 [mu] M and 10 [mu] M BRAFi RG7204 (Panels A, B and C, respectively) The first peak represents the cells treated for the second detection reagent alone, the second peak represents the BRAF inhibitor treated cells, and the third peak represents cells that were not treated with the BRAF inhibitor.
Figure 12 shows the expression of total ETBR, Perk and erk proteins and control proteins GAPDH and [beta] -tubulin at 50 [mu] g total cell lysates from COLO829 melanoma cells at concentrations of 0, 0.01, 0.1 and 1 [mu] M Western blot experiments were performed using MEKi-623 (Panel A) and MEKi-973 (Panel B).
FIG. 13 shows the results of the MEKi-623 (panels A, B and C, respectively) or MEKi-973 (panels D and D) of 0.01 μM (panels A and D), 0.1 μM (panels B and E) and 1 μM , ≪ / RTI > E, and F). ≪ tb >< TABLE > The first peak represents the cells treated for the second detection reagent alone, the second peak represents cells not treated with the MEK inhibitor, and the third peak represents the cells treated with the MEK inhibitor.
Figure 14 shows ETBR mRNA expression in A2058 melanoma cells treated with varying concentrations of MEKi-623 (Panel A) or MEKi-973 (Panel B) normalized to RPL19 transcripts for 24 hours.
Figure 15 shows the expression of total ETBR, Perk and erk proteins in 50-100 μg total cell lysates from A2058 melanoma cells and the expression of 0 μM, 0.01 μM, 0.1 μM and 1 μM of erk protein and control proteins GAPDH and β- Lt; RTI ID = 0.0 > MEKi-623 < / RTI > (Panel A) and MEKi-973 (Panel B).
Fig. 16 shows the results of the MEKi-623 (panels A, B and C, respectively) or MEKi-973 (panels D and B) of 0.01 uM (panels A and D), 0.1 uM (panels B and E) , ≪ / RTI > E, and F) of the surface ETBR protein in A2058 live cells observed by flow cytometry after incubation. The first peak represents the cells treated for the second detection reagent alone, the second peak represents cells not treated with the MEK inhibitor, and the third peak represents the cells treated with the MEK inhibitor.
Figure 17 demonstrates the in vivo combinatorial efficacy of anti-ET B R ADC (Hu5E9v1-vc-MMAE) and MEKi-973 on A2058 melanoma xenograft mouse models. Subcutaneous tumors were established in mice inoculated with A2058 melanoma cell line. When the tumor volume reached approximately 200 mm 3 (day 0), the animals were orally administered MEKi-973 once daily for 21 days. On day 1 (after two doses of MEKi-973), the animals were given a single IV injection of vehicle of the indicated dose or anti-ET B R ADC (Hu5E9v1-vc-MMAE). Mean tumor volume was determined from 9 animals per group with standard deviation. Drug and Dose information is shown on each graph as follows: Panel A is a 7.5 mk anti-gD ADC (control) in combination with MEKi-973 at 7.5 mpk compared to the vehicle control and anti-gD ADC alone Lt; / RTI > Panel B vehicle control and 7.5 mpk MEKi-973 alone (GDC-0973), or wherein the R 6 mpk -ET B-ADC and compare solely by the terms of the 6 mpk combination with MEKi-973 of 7.5 mpk -ET B R- ADC (Hu5E9v1-vc-MMAE).
Figure 18 shows ET B R transcript expression in SK23-MEL melanoma cells treated for 24 hours with varying concentrations of MEKi-623 (panel A) or MEKi-973 (panel B) normalized to RPL19 transcript Lt; / RTI >
Figure 19 shows the expression of total ETBR, Perk and erk proteins in 50 占 퐂 whole cell lysates from SK23-MEL melanoma cells and 0 占 0.01, 0.01 占,, 0.1 占 및 and 1 占 M Lt; RTI ID = 0.0 > MEKi-623 < / RTI > (Panel A) and MEKi-973 (Panel B).
FIG. 20 shows the results of the MEKi-623 (panels A, B and C, respectively) or MEKi-973 (panels D and D) of 0.01 μM (panels A and D), 0.1 μM (panels B and E) and 1 μM , ≪ / RTI > E, and F), following surface incubation with the SK23-MEL cells. The first peak represents the cells treated for the second detection reagent alone, the second peak represents cells not treated with the MEK inhibitor, and the third peak represents the cells treated with the MEK inhibitor.
Figure 21 demonstrates the in vivo combinatorial efficacy of anti-ET B R ADC (Hu5E9v1-vc-MMAE) and MEKi-973 on SK23-MEL melanoma xenograft mouse models. Drug and dose information is shown on each graph as follows: Panel A shows the results of a 6 mpk combined with 7.5 mpk MEKi-973 compared to vehicle control, 7.5 mpk MEKi-973 and 6 mpk anti- Of the anti-gD ADC (control group); Panel B vehicle control and 7.5 mpk MEKi-973 alone or 6 mpk wherein R B -ET-ADC and compare itself to a section of 6 mpk combination with MEKi-973 of 7.5 mpk -ET B R-ADC ( Hu5E9v1-vc of -MMAE) ("combination"). Panel C shows the results of a 3-kk anti-ET B R combined with 3 mpk MEKi-973 compared to vehicle control, 3 mpk anti-ET B R-ADC (Hu5E9v1-vc-MMAE) or 3 mpk MEKi- -ADC (Hu5E9v1-vc-MMAE) ("combination"). Panel D is the vehicle control and 7.5 mpk MEKi-973 alone or 3 mpk wherein R B -ET-ADC compared to the sole, wherein the combination of the MEKi-973 of 7.5 mpk 3 mpk -ET B R- ADC (Hu5E9v1-vc of -MMAE) ("combination"). Panel E is a vehicle control and 3 mpk MEKi-973 alone or 6 mpk wherein R B -ET-ADC and compare itself to a section of 6 mpk combination with MEKi-973 for 3 mpk -ET B R-ADC ( Hu5E9v1-vc of -MMAE) ("combination").
Figure 22 shows the expression of total ETBR, Perk and erk proteins and control proteins GAPDH and beta -tubulin in 25-100 microgram total cell lysates from IPC-298 melanoma cells at 0, 0.01, 0.1, and 0.1 (Panel A) and MEKi-973 (Panel B) at a concentration of < RTI ID = 0.0 > 2 M. < / RTI >
FIG. 23 shows the results of the MEKi-623 (panels A, B and C, respectively) or MEKi-973 (panels D and B) at 0.01 μM (panels A and D), 0.1 μM (panels B and E) and 1 μM , ≪ / RTI > E, and F). ≪ tb >< tb >< / TABLE > The first peak represents the cells treated for the second detection reagent alone, the second peak represents cells not treated with the MEK inhibitor, and the third peak represents the cells treated with the MEK inhibitor.
Figure 24 demonstrates the in vivo combinatorial efficacy of anti-ET B R ADC (Hu5E9v1-vc-MMAE) and MEKi-623 on IPC-298 melanoma xenograft mouse models. Drug and dose information is shown on each graph as follows: Panel A shows 6 mpk in combination with 1 mpk of MEKi-623 compared to vehicle control, 1 mpk MEKi-623 and 6 mpk anti-gD ADC alone Of the anti-gD ADC (control group); Panel B is the vehicle control group and 1 mpk MEKi-623 alone or 6 mpk wherein R B -ET-ADC compared to the sole, wherein the 1 mpk and in combination with 6-MEKi 623 mpk of -ET B R-ADC (Hu5E9v1- vc of -MMAE) ("combination").
Figure 25 demonstrates the in vivo combinatorial efficacy of anti-ET B R ADC (Hu5E9v1-vc-MMAE) and MEKi-973 on IPC-298 melanoma xenograft mouse models. Drug and dose information is shown on each graph as follows: Panel A shows the results of a 6 mpk combined with 7.5 mpk MEKi-973 compared to vehicle control, 7.5 mpk MEKi-973 and 6 mpk anti- Of the anti-gD ADC (control group); Panel B vehicle control and 7.5 mpk MEKi-973 alone or 6 mpk wherein R B -ET-ADC and compare itself to a section of 6 mpk combination with MEKi-973 of 7.5 mpk -ET B R-ADC ( Hu5E9v1-vc of -MMAE) ("combination").
Figure 26 shows the expression of phosphorylated erk and total erk protein in COLO 829 tumors treated with vehicle or 30 mpk BRAFi RG7204.
Figure 27 shows the expression of ETBR transcripts in COLO 829 tumors treated with BRAFi RG7204 (Panel A) and A2058 tumors treated with MEKi-973 (Panel B) for 3 days. Panel A shows normalized ETBR transcripts for control GAPDH in COLO 829 cell line in COLOR 892 tumors treated with vehicle control or 10 mpk or 30 mpk RG7204. Panel B shows the ETBR transcript normalized to control Hprt1 in A2058 tumors treated with vehicle control or 5 or 10 mpk of MEKi-973.

I. Definition

For the purposes of the present application, the term " recipient human framework "refers to a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework amino acid sequence derived from a human immunoglobulin framework or human consensus framework, ≪ / RTI > sequence. An "acceptor human framework ", derived from a human immunoglobulin framework or human consensus framework, may comprise its identical amino acid sequence or may contain amino acid sequence alterations. In some embodiments, the number of amino acid changes is 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. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

"Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an endogenous binding affinity that reflects a 1: 1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of the molecule X for its partner Y can generally be expressed as the dissociation constant (Kd). The affinity can be measured by conventional methods known in the art including the methods described herein. Specific illustrative and representative embodiments for binding affinity measurement are described below.

An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) as compared to a parental antibody that does not alter the affinity of the antibody to the antigen.

The terms "anti-ETBR antibody" and "antibody binding to ETBR" refer to an antibody capable of binding to ETBR with sufficient affinity for the antibody to be useful as a diagnostic agent and / or therapeutic agent in targeting the endothelin B receptor do. In one embodiment, the degree of binding of the anti-ETBR antibody to a non-related, non-ETBR protein is less than about 10% of the binding of the antibody to the ETBR, e.g., as measured by a radioimmunoassay (RIA) . In certain embodiments, antibodies that bind to ETBR is ≤ 1μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM , or ≤ 0.001 nM (e.g., 10 -8 M or less, e. (10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M) dissociation constants (Kd). In certain embodiments, the anti-ETBR antibody binds to an epitope of an ETBR conserved between ETBRs from different species.

The term "antibody" is used herein in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e. G., Bispecific antibodies), and antibody fragments But are not limited to, various antibody structures.

"Antibody fragment" refers to a molecule other than an intact antibody, including a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab ', Fab'-SH, F (ab') 2 ; Diabody; Linear antibodies; Single-chain antibody molecules (e. G., ScFv); And multispecific antibodies formed with antibody fragments.

"Antibody that binds to the same epitope" as the reference antibody blocks more than 50% of the binding of the reference antibody to its antigen in the competition assay and, conversely, in the competition assay, the reference antibody blocks the binding of the antibody to its antigen by more than 50% Lt; / RTI > An exemplary competitive assay is provided herein.

As used herein, the term "BRAF" is a protein encoded by the BRAF gene in humans and also includes a serine / threonine-protein kinase B-Raf known as the germline B-Raf or v-Raf murine sarcoma virus tumor gene homolog B1. Raf. B-Raf proteins are involved in signal transduction and cell growth in cells.

As used herein, the term "BRAF inhibitor" or "BRAFi" refers to any number of known small molecule drug compounds capable of inhibiting or stopping the B-Raf / MEK step on the B-Raf / MEK / ERK pathway. Examples of suitable BRAFi are International Patent Application PCT / US2010 / 047007 filed on August 27, 2010, International Patent Application PCT / US2010 / 046975 filed on August 27, 2010; International patent application PCT / US2010 / 046952 filed on August 27, 2010; International patent application PCT / US2010 / 046955 filed on August 27, 2010; And International Patent Application No. PCT / US2006 / 024361, filed on June 21, 2006, all of which are incorporated herein by reference. Another example is CAS Registry number 405554-55-4, and also has the name of 5- [2- [4- [2- (dimethylamino) ethoxy] phenyl] -5- (4-pyridinyl) -4-yl] -2,3-dihydro-1H-inden-1-one oxime.

The term "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 chain and / or light chain is derived from another source or species.

A "class" of an antibody refers to a type of constant domain or constant region retained by its heavy chain. Of the main class of the five kinds of antibodies: IgA, IgD, IgE, IgG and IgM are present, some of which are a subclass (isotype), such as IgG 1, IgG 2, IgG 3 , IgG 4, IgA 1 , and IgA < / RTI > 2 . The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively.

The term "cytotoxic agent" as used herein refers to a substance that causes inhibition or inhibition of cellular function and / or cell death or destruction. The cytotoxic agent may be a radioactive isotope (eg At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , Zr 89 , element); A chemotherapeutic agent or a drug (e.g., methotrexate, adriamycin, vinca alkaloid (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, ); Growth inhibitors; Enzymes and fragments thereof, such as nucleic acid degrading enzymes; Antibiotic; Toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin (including fragments and / or variants thereof); And various antineoplastic or anti-cancer agents disclosed below.

"Effector function" refers to the biological activity attributable to the Fc region of an antibody, which depends on the antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; Antibody-dependent cell-mediated cytotoxicity (ADCC); Phagocytic action; Down regulation of cell surface receptors (e. G., B cell receptors); And B cell activation.

An "effective amount" of an agonist, e. G., A pharmaceutical formulation, refers to an amount effective to achieve the desired therapeutic or prophylactic result for the required period of time at the requisite dosage.

As used herein, the term "ETBR" refers to any natural (non-human) animal from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) Endothelial < / RTI > B receptor (ETBR). The term "full-length" encompasses non-processed ETBR as well as any form of ETBR produced from processing in cells. The term also encompasses naturally occurring variants of ETBR, such as splice variants or allelic variants. The amino acid sequence of an exemplary human ETBR is shown in SEQ ID NO: 10 (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 1): 34-9).

The term " anti-ETBR antibody-ADC "as used herein refers to any anti-ETBR antibody described herein conjugated to a toxin. Such toxins include, but are not limited to, maytansinoids or, in particular, monomethylauristatin (MMAE). Anti-ETBR antibody-ADC is considered a species of "anti-ETBR antibody of the invention ".

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region is extended from Cys226, or Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. According to the EU numbering system, also referred to as the EU index, as described in the Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. FRs in a variable domain generally consist of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.

The terms "full-length antibody "," intact antibody "and" whole antibody "are used interchangeably herein and refer to a heavy chain that has a structure substantially similar to that of a native antibody construct or that contains an Fc region as defined herein ≪ / RTI >

As used herein, the term "945" or BRAFi-945 "refers to 4-amino-N- (6-chloro-2-fluoro-3- (3- fluoropropylsulfonamido) phenyl) thieno [ -d] pyrimidine-7-carboxamide, as disclosed in Example 15 of International Patent Application No. PCT / US2010 / 046955, filed August 27, 2010, the disclosure of which is incorporated herein by reference, ≪ / RTI > refers to a B-Raf enzyme inhibitor having the structure of < RTI ID = 0.0 >

Figure pct00013

The terms "host cell," " host cell strain "and" host cell culture "are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" which include progeny derived therefrom irrespective of the number of primary transformed cells and subculture. The offspring may not have exactly the same nucleic acid content as the parent cell, but may contain mutations. Mutant descendants having the same function or biological activity screened or selected for the originally transformed cells are included herein.

A "human antibody" is an antibody that retains an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or that is derived from a non-human source using a human antibody repertoire or other human antibody-coding sequence. Humanized antibodies comprising non-human antigen-binding moieties in this definition of human antibodies are expressly excluded.

The "human consensus framework" is a framework for the most common amino acid residues occurring in the selection of human immunoglobulin VL or VH framework sequences. Generally, selection of a human immunoglobulin VL or VH sequence is from a subgroup of variable domain sequences. Generally, subgroups of sequences are described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vol. 1-3]. In one embodiment, in the case of VL, the subgroup is subgroup kappa I as in Kabat et al., Supra. In one embodiment, subgroups in the case of VH are subgroup III as in Kabat et al., Supra.

"Humanized" antibody refers to a chimeric antibody comprising an amino acid residue from a non-human HVR and an amino acid residue from a human FR. In certain embodiments, the humanized antibody will comprise substantially more than one, typically two, variable domains, wherein all or substantially all of the HVRs (e. G., CDRs) correspond to those of non- All or substantially all FRs correspond to those of a human antibody. Humanized antibodies may optionally comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e. G., A "humanized form," of a non-human antibody refers to an antibody that has undergone humanization.

The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain in which the sequence forms a hypervariable and / or structurally defined loop ("hypervariable loop"). In general, the native four-chain antibody has six HVRs; (Hl, H2, H3) in the VH and three (L1, L2, L3) in the VL. HVRs generally include amino acid residues from hypervariable loops and / or "complementarity determining regions" (CDRs), with the latter being the most highly sequence variant and / or associated with antigen recognition. Exemplary hypervariable loops are found at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 Occurs. L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-L3 of the exemplary CDRs (Chothia and Lesk, J. Mol. Biol. 196: 901-917 H3) occurs at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2 and 95-102 of H3. Except for CDR1 in VH, the CDRs are generally hypervariable, with the exception of CDR1 in VH (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., National Institutes of Health, Bethesda, Lt; / RTI > amino acid residues that form a loop. CDRs also include "specificity crystal moieties" or "SDRs" which are residues that contact the antigen. SDRs are contained within the region of the CDRs referred to as short-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR- , 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) Kabat et al., Supra).

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule (s), including but not limited to cytotoxic agents.

An "individual" or "subject" is a mammal. Mammals include rabbits and rodents (e.g., mice and rats), rabbits, rabbits, rabbits, and rabbits, including, but not limited to, livestock (e.g., cows, sheep, cats, dogs and horses), primates (e. G., Human and non-human primates such as monkeys) But is not limited thereto. In certain embodiments, the subject or subject is a human.

An "isolated" antibody is isolated from its natural environment components. In some embodiments, the antibody may be conjugated to an antibody, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) Gt; 95% < / RTI > or greater than 99%. For a review of methods of assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule isolated from a component of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain nucleic acid molecules, but nucleic acid molecules are present at chromosomal locations other than or at a different chromosomal location than their natural chromosomal location.

An "isolated nucleic acid encoding an anti-ETBR antibody" refers to a nucleic acid molecule comprising one or more nucleic acid molecules that encode an antibody heavy chain and a light chain (or fragment thereof) (present in one or more positions in a single vector or in a separate vector, (S) of such nucleic acid molecule (s).

As used herein, the term " mitogen-activated protein kinase "(MAP kinase) refers to a serine / threonine-specific protein kinase belonging to the CMGC (CDK / MAPK / GSK3 / CLK) kinase family. The ERK1 / 2 pathway in mammals is probably the best characterized MAPK system. The most important upstream activator of this pathway is the Raf protein (A-Raf, B-Raf or c-Raf), the key mediator of the response to growth factors (EGF, FGF, PDGF, etc.).

As used herein, the term "MAPK / ERK kinase" (MEK) refers to a tyrosine kinase that plays a pivotal role in the MAPK pathway. Expression of constitutively active forms of MEK leads to transformation of the cell line.

The term "MEK inhibitor" (MEKi) as used herein refers to any number of known small molecule drug compounds capable of inhibiting or stopping the MEK stage of the MAP kinase pathway. Examples of suitable MEKi may include, but are not limited to, those described as MEKi-623, MEKi-973 or GSK1120212.

As used herein, the term "MEKi-973" refers to a MEK inhibitor (S) - (3,4-difluoro-2- 3-hydroxy-3- (piperidin-2yl) azetidin-1-yl) methanone.

Figure pct00014

As used herein, the term "monoclonal antibody" is used to refer to an antibody obtained from a population of substantially homogeneous antibodies, that is, individual antibodies that make up such a population are, for example, Or bind to the same and / or the same epitope, except for possible mutant antibodies that contain mutations or are produced during the production of monoclonal antibody formulations. In contrast to polyclonal antibody preparations which typically contain different antibodies directed against different crystallizers (epitopes), each monoclonal antibody of the monoclonal antibody preparation is directed against a single crystallizer on the antigen. Thus, the modifier "monoclonal" refers to the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be regarded as requiring antibody production by any particular method. For example, a monoclonal antibody used in accordance with the present invention includes a hybridoma method, a recombinant DNA method, a phage-display method, and a method using transgenic animals containing all or part of a human immunoglobulin locus But not limited to, and other exemplary methods for producing such methods and monoclonal antibodies are described herein.

"Naked antibody" refers to a heterologous moiety (e. G., A cytotoxic moiety) or an antibody that is not conjugated to a radioactive label. Naked antibodies can be present in pharmaceutical preparations.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a varying structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons consisting of two identical light chains and two identical heavy chains that are disulfide-linked. From the N-terminus to the C-terminus, each heavy chain has three constant domains (CH1, CH2 and CH3) following the variable region (VH) (also referred to as the variable heavy chain or heavy chain variable domain). Similarly, from the N-terminus to the C-terminus, each light chain has a constant light chain (CL) domain following the variable region (VL) (also referred to as the variable light chain or light chain variable domain). The light chain of an antibody can be assigned to one of two types, referred to as kappa (?) And lambda (?), Based on the amino acid sequence of its constant domain.

The term "package insert" is intended to encompass a range of instructions that are typically included in commercial packages of therapeutic products, including information about indications, usage, dosage, administration, combination therapy, contraindications, and / . ≪ / RTI >

"Amino acid sequence identity percent (%)" for a reference polypeptide sequence refers to the number of amino acid residues in the reference polypeptide (s), without aligning the sequence and, if necessary, introducing a gap to achieve maximum sequence identity percent, Is defined as the percentage of amino acid residues in the same candidate sequence as the amino acid residue in the sequence. Alignment to determine percent amino acid sequence identity can be accomplished using a variety of methods within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software can do. Those skilled in the art will be able to determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment for the full-length sequence to be compared. However, for purposes of this disclosure,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is owned by Genentech, Inc. and the source code is submitted as a user's document to the US Copyright Office (Washington, DC 20559), with US copyright registration number TXU510087 It is registered. The ALIGN-2 program was developed by Genentech, Inc. (South San Francisco, Calif.), Or compiled from source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

In a given amino acid sequence B, in a situation where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B, or to a given amino acid sequence B (alternatively, B may be described with the given amino acid sequence B, or with a given amino acid sequence A that has or has a specified percentage amino acid sequence identity to a given amino acid sequence B) is calculated as follows.

Fraction of X / Y x 100

Where X is the number of amino acid residues scored in the same match by the program at the time of program alignment of A and B by the sequence alignment program ALIGN-2 and Y is the total number of amino acid residues of B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of A to B will not be equal to the% amino acid sequence identity of B to A. Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained as described in the above paragraph using the ALIGN-2 computer program.

The term "pharmaceutical formulation" refers to a formulation that exists in a form such that the biological activity of the active ingredients contained therein is effective, and that the formulation does not contain additional ingredients that are unacceptable toxicity to the subject to which it is to be administered.

"Pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The term "RG7204" refers to a B-Raf inhibitor that has the molecular formula and structure: C 23 H 18 CIF 2 N 3 O 3 S.

Figure pct00015

As used herein, "treatment" (and its grammatical variation, such as "treating" or "treating ") refers to the clinical intervention to alter the natural course of the subject being treated, Can be performed during the process. A preferred therapeutic effect is to prevent the occurrence or recurrence of the disease, alleviate the symptoms, reduce any direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, improve or improve the disease state, Including but not limited to prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of the disease or to slow the progression of the disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in binding of an antibody to an antigen. The variable domains of the heavy and light chains of the native antibody (VH and VL, respectively) generally have a similar structure, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6 th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen- have. In addition, antibodies that bind to a particular antigen can be isolated using a VH or VL domain from an antibody that binds to the antigen to screen for a library of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which the vector is linked. The term encompasses not only the vector as a self-replicating nucleic acid construct but also a vector into which the vector is integrated into the genome of the host cell into which it is introduced. A particular vector may direct expression of the nucleic acid to which the vector is operatively linked. Such vectors are referred to herein as "expression vectors ".

II. Composition and method

In one aspect, the invention is based in part on an antibody that binds to an ETBR. The antibodies of the invention are useful, for example, in the treatment of melanoma.

Exemplary anti-ETBR antibodies

In one aspect, the invention provides an isolated antibody that binds to an ETBR. (B) CDR-L2 (LVSKLDS, SEQ ID NO: 8), (c) CDR-L3 (WQGTHFPYT; SEQ ID NO: 9), at least 1, 2, 3, 4, or 5 selected from CDR-H1 (GYTFTSYWMQ; SEQ ID NO: 1), (e) CDR-H2 (TIYPGDGDTSYAQKFKG; SEQ ID NO: 2), and (f) CDR- Or six CDRs.

In any of the above embodiments, the anti-ETBR antibody is humanized. In one embodiment, the anti-ETBR antibody comprises a CDR as in any of the above embodiments and additionally comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another aspect, the invention provides an isolated anti-ETBR antibody having a VL amino acid sequence of SEQ ID NO: 8 and a VH amino acid sequence of SEQ ID NO: 7. In yet another aspect, 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.

In yet another aspect, the anti-ETBR antibody comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% And a heavy chain variable domain (VH) sequence having 100% sequence identity. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, For example, conservative substitutions), insertions or deletions, but the anti-ETBR antibody comprising this sequence retains the ability to bind to the ETBR. In certain embodiments, a total of from 1 to 10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 7 or SEQ ID NO: 9. In certain embodiments, substitutions, insertions, or deletions occur in the region outside the CDRs (i.e., in FRs).

In yet another aspect, a polypeptide 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: An anti-ETBR antibody comprising a light chain variable domain (VL) is provided. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is replaced For example, conservative substitutions), insertions or deletions, but the anti-ETBR antibody comprising this sequence retains the ability to bind to the ETBR. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 8. In certain embodiments, substitution, insertion, or deletion occurs in the region outside the HVR (i. E., At the FR).

In another aspect, there is provided an anti-ETBR antibody comprising a VH as in any of the provided embodiments described above and a VL as in any of the provided embodiments. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 7 or 9 and SEQ ID NO: 8, respectively, including post-translational modifications of these sequences.

In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-ETBR antibody provided herein. For example, in certain embodiments, an antibody that binds to the same epitope as an anti-ETBR antibody comprising the VH sequence of SEQ ID NO: 7 or 9 and the VL sequence of SEQ ID NO: 8 is provided. In certain embodiments, an anti-ETBR antibody is provided that binds to an epitope within the N-terminal extracellular domain # 1 fragment of an ETBR consisting of amino acid Nos. 64-101 of SEQ ID NO: 10.

In a further aspect of the invention, the anti-ETBR antibody according to any of the above embodiments is a monoclonal antibody comprising a chimeric, humanized or human antibody. In one embodiment, the anti-ETBR antibody is an antibody fragment, such as an Fv, Fab, Fab ', scFv, diabody or F (ab') 2 fragment. In another embodiment, the antibody is a full-length antibody, e. G. An intact IgGl antibody, or another antibody class or isotype as defined herein.

In a further aspect, the anti-ETBR antibodies according to any of the above embodiments may comprise any feature as described in sections 1-7 below alone or in combination.

Antibody affinity

In certain embodiments, the antibody provided herein is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM , or ≤ 0.001 nM (e.g., 10 -8 M or less, e. (10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M) dissociation constants (Kd).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed using the Fab version of the antibody of interest and its antigen as described in the following assays. The solution binding affinity of the Fab for the antigen was determined by equilibrating the Fab with a minimal concentration of ( 125 I) -labeled antigen in the presence of a titration series of unlabeled antigens and then conjugating the bound antigen to the anti-Fab antibody-coated plate (See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish assay conditions, a MICROTITER 占 multi-well plate (Thermo Scientific) was incubated with 5 占 퐂 / ml of capture anti-Fab antibody (Capellaps (pH7.6) in 50 mM sodium carbonate Cappel Labs) overnight and then blocked with 2% (w / v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (approximately 23 ° C). In a non-adsorptive plate (Nunc # 269620), 100 pM or 26 pM [ 125 I] -antigen is mixed with serial dilutions of the Fab of interest (see, for example, Presta et al., Cancer Res. 57 : 4593-4599 (1997)], an estimate of the anti-VEGF antibody, Fab-12). Then, the Fab of interest is incubated overnight; Can be incubated for a longer period of time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (for example, for 1 hour). The solution was then removed and the plate was washed 8 times with 0.1% polysorbate 20 in PBS (TWEEN-20). 150 μl / well scintillant (MICROSCINT-20 ™; Packard) was added to the plate and the plate was incubated with a TOPCOUNT ™ gamma counter (Packard) for 10 minutes Lt; / RTI > The concentration of each Fab providing less than 20% of the maximal binding is selected and used for competitive binding assays.

According to another embodiment, Kd can be measured at 25 ° C using BIACORE®-2000 or ViaCore®-3000 (Biacore®-3000, manufactured by Biacore, Inc.) using an antigen CM5 chip immobilized, for example, (BIAcore, Inc., Piscataway, NJ) using a surface plasmon resonance assay. Briefly, a carboxymethylated dextran biosensor chip (CM5, Biacore, Inc.) Is mixed with N-ethyl-N '- (3- dimethylaminopropyl) -carbodiimide hydrochloride (EDC) Is activated with N-hydroxysuccinimide (NHS). The antigen is diluted to 5 μg / ml (~0.2 μM) using 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to achieve approximately 10 reaction units (RU) of the coupled protein. After injection of the antigen, 1 M ethanolamine is injected to block the unreacted group. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were run in PBS (PBS) with 0.05% polysorbate 20 (Tween-20 ™) surfactant at 25 ° C. at a flow rate of approximately 25 μl / PBST). The association rate (k on ) and dissociation rate (k off ) are calculated by simultaneously fitting association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (Biacore® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the ratio of k off / k on . For example, Chen et al., J. Mol. Biol. 293: 865-881 (1999). If the association rate due to the surface-plasmon resonance assay is greater than 10 6 M -1 s -1 , the association rate may be measured using a spectrophotometer, such as a stationary-flow setup spectrophotometer (Aviv Instruments) or a stirred cuvette Antigens antibodies in PBS (pH 7.2) in the presence of increasing concentrations of antigen when measured on an 8000-series SLM-AMINCO ™ spectrophotometer (ThermoSpectronic) Can be determined using a fluorescence quenching technique to measure the increase or decrease of the fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band-pass) at 25 캜.

Antibody fragment

In certain embodiments, the 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 review of specific antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For review of scFv fragments, see, for example, Pluckthuen, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); WO 93/16185; And U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F (ab ') 2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that can 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).

A single-domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain of an antibody, or all or part of a light chain variable domain. In certain embodiments, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, Mass., E.g., U.S. Patent No. 6,248,516 B1).

Antibody fragments may be produced by various techniques including, but not limited to, production by recombinant host cells (e. G. E. coli or phage) as well as proteolytic digestion of intact antibodies as described herein ≪ / RTI >

Chimeric and humanized antibodies

In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent Nos. 4,816,567; And Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, 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. In a further example, a chimeric antibody is a "class switching" antibody in which the class or subclass is changed from that of the parent antibody. A chimeric antibody comprises its antigen-binding fragment.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains from which an HVR, e.g., a CDR (or portion thereof), is derived from a non-human antibody and FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody may also optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody may be replaced with corresponding residues from a non-human antibody (e. G., An antibody from which the HVR residue is derived), for example to restore or enhance antibody specificity or affinity .

Humanized antibodies and methods for their preparation are described, for example, in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)), for example in Riechmann et al., Nature 332: 323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86: 10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; [Kashmiri et al., Methods 36: 25-34 (2005)] (SDR (a-CDR) grafting substrate); [Padlan, Mol. Immunol. 28: 489-498 (1991) (described "rescaling"); [Dall'Acqua et al., Methods 36: 43-60 (2005)] (referred to as "FR shuffling"); And Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br. J. Cancer, 83: 252-260 (2000) (describing a "guide selection" approach to FR shuffling).

The human framework region that can be used for humanization can be selected from framework regions selected using the " best-fit "method (see, for example, Sims et al. J. Immunol. 151: 2296 (1993)); A framework region (e. G., Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992)) derived from the consensus sequence of human subclasses of a particular subgroup of light or heavy chain variable regions Presta et al. J. Immunol., 151: 2623 (1993)); Human maturation (somatic cell maturation) framework regions or human wiring framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); (Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

Human antibody

In certain embodiments, the antibody provided herein is a human antibody. Human antibodies can be generated using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

Human antibodies can be produced by administering an immunogen to a transgenic animal that has been modified to produce an intact human antibody or an intact antibody with a human variable region in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or that is extrachromosomally or randomly integrated into the chromosome of the animal. In these transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (described in XENOMOUSE) technology; U.S. Patent No. 5,770,429 (described in HuMab® technology); See U.S. Patent No. 7,041,870 (K-M MOUSE) technology description, and U.S. Patent Application Publication No. US 2007/0061900 (VelociMouse® technology description). The human variable region from an intact antibody produced by such an animal may be further modified, for example, in combination with a different human constant region.

Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human xenogeneic myeloma cell lines for the production of human monoclonal antibodies are described. (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 produced through human B-cell hybridoma techniques have also been described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods are described, for example, in U.S. Patent No. 7,189,826 (monoclonal human IgM antibody production from a hybridoma cell line) and in Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006) - human hybridoma substrate). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27 (3): 185-91 (2005).

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the preferred human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

Library-derived antibodies

The antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies having the desired activity. Various methods are known in the art for generating phage display libraries, for example, and screening such libraries for antibodies with the desired binding properties. Such a method is described, for example, in Hoogenboom et al. for example, in McCafferty et al., Nature 348: 552-7 (1986)), which has been reviewed in < RTI ID = 0.0 &554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338 (2): 299-310 (2004); Lee et al., J. Mol. Biol. 340 (5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); And Lee et al., J. Immunol. Methods 284 (1-2): 119-132 (2004).

In a particular phage display method, the repertoires of VH and VL genes are individually cloned by polymerase chain reaction (PCR) and randomly recombined into a phage library, which is described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically displays antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need to build hybridomas. Alternatively, a naïve repertoire may be cloned (e.g., from a human) to produce a wide variety of non-magnetic and / or non-magnetic immunoglobulins, such as those described in Griffiths et al., EMBO J, 12: 725-734 It can also provide a single source of antibodies to the self antigens. Finally, as described in Hoogenboom and Winter, J. Mol. (SEQ ID NO: 2), cloning V rearranged V-gene fragments from stem cells, coding for highly variable CDR3 regions and in vitro sequencing to achieve in vitro rearrangement, as described in Biol., 227: 381-388 Lt; RTI ID = 0.0 > a < / RTI > PCR primer. Patent publications describing human antibody phage libraries are described, for example, in U.S. Patent Nos. 5,750,373 and U.S. 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 herein considered human antibodies or human antibody fragments.

Multispecific antibody

In certain embodiments, the antibodies provided herein are multispecific antibodies, e. G. Bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificity for two or more different sites. In certain embodiments, one of the binding specificities is for an ETBR and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of the ETBR. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing ETBR. Bispecific antibodies can be produced as whole antibody or antibody fragments.

Techniques for producing multispecific antibodies include recombinant co-expression of two immunoglobulin heavy-chain pairs with different specificity (Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, And "knob-in-hole" manipulations (see, for example, U.S. Patent No. 5,731,168), and [Traunecker et al., EMBO J. 10: 3655 But is not limited thereto. Multispecific antibodies also include manipulating electrostatic steering effects to produce antibody Fc-heterodimeric molecules (WO 2009 / 089004A1); Cross-linking of two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); The use of leucine zipper to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992)); (See, for example, Hollinger et al., Proc. Nat'l Acad. Sci. USA, 90: 6444-6448 (1993)) for the preparation of bispecific antibody fragments ; And the use of single-chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol., 152: 5368 (1994)); And for example, Tutt et al. J. Immunol. 147: 60 (1991)). ≪ RTI ID = 0.0 >

Engineered antibodies having three or more functional antigen binding sites, including "octopus antibody ", 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 an ETBR as well as another, different antigen (see, e.g., US 2008/0069820).

Antibody variant

In certain embodiments, 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 the antibody may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct so that the final construct retains the desired properties, e. G., Antigen-binding.

Substitution, insertion and deletion mutants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Interest regions for inducing replacement mutations include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Conservative substitutions ". More substantial changes are provided as further described below with respect to the amino acid side chain classes in Table 1 under the heading "Exemplary Substitutions ". Amino acid substitutions are introduced into the antibody of interest and the product can be screened for the desired activity, e. G., Maintenance / improved antigen binding, reduced immunogenicity or improved ADCC or CDC.

Figure pct00016

Amino acids can be classified according to their common side chain properties.

Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

Acid: Asp, Glu;

Basic: His, Lys, Arg;

Residues affecting chain orientation: Gly, Pro;

Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will involve exchanging members of one of these classes for another.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e. G., A humanized or human antibody). Generally, the generated variant (s) selected for further study will have a modification (e. G., Improvement) of specific biological properties (e. G., Increased affinity, reduced immunogenicity) Or substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies that can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated, variant antibodies are displayed on a phage and screened for a particular biological activity (e. G., Binding affinity).

Alterations (e. G., Substitutions) can be made in the HVR, for example, to improve antibody affinity. These changes may result in an HVR "hotspot, " a binding affinity during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008) In a residue encoded by a codon that performs mutation at a high frequency with the resulting variant VH or VL tested against B. Affinity maturation by construction and reselection from a secondary library is described, (O'Brien et al., Ed., Human Press, Totowa, NJ, (2001)]. In some embodiments of affinity maturation, , Diversity is introduced into a variable gene selected for maturation by any of a variety of methods (e. G., Error-triggered PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Secondary libraries are then generated . Then, any term with the desired affinity Screening the library for screening for variants. Another method of introducing diversity is associated with the HVR-induced approach where multiple HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues associated with antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitution, insertion or deletion can occur within one or more HVRs, so long as such alteration does not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e. G., Conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the HVR. These changes may be outside the HVR "hotspot" or the SDR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered, or contains no more than 1, 2, or 3 amino acid substitutions.

A useful method for identifying a residue or region of an antibody that can be targeted for mutagenesis, as described in Cunningham and Wells (1989) Science, 244: 1081-1085, is referred to as "alanine scanning mutagenesis. &Quot; In this method, a group of residues or target residues is identified (e.g., charged residues such as arg, asp, his, lys and glu), neutral or negatively charged amino acids (e.g., alanine or polyalanine ) To determine whether it affects the interaction of the antibody with the antigen. Additional substitutions may be introduced at amino acid positions that demonstrate functional susceptibility to initial substitution. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to determine the contact point between the antibody and the antigen. Such contact residues and neighboring residues can be targeted or removed as candidates for substitution. Variants can be screened to determine whether they contain the desired characteristics.

Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length ranging from one residue to more than 100 residues in the polypeptide, as well as sequential insertion of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertional variants of the antibody molecule include those in which an enzyme (e.g., in the case of ADEPT) or a polypeptide that increases the serum half-life of the antibody is fused to the N- or C-terminus of the antibody.

Glycosylation variant

In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of the glycosylation site to the antibody can conveniently be accomplished by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically include a branched double-antenna oligosaccharide that is typically attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15: 26-32 (1997). Oligosaccharides may include fucose attached to GlcNAc in the "stem" of a dual-antenna oligosaccharide structure as well as various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid. In some embodiments, modification of the oligosaccharides in the antibodies of the invention can be made to produce antibody variants with certain improved properties.

In one embodiment, antibody variants having a fucose-deficient carbohydrate structure attached (directly or indirectly) to the Fc region are provided. For example, the amount of fucose in such antibodies may be between 1% and 80%, between 1% and 65%, between 5% and 65%, or between 20% and 40%. The amount of fucose is compared to the sum of all sugar structures (e.g., complexes, hybrids and gonorrhea structures) attached to Asn 297 as measured by MALDI-TOF mass spectroscopy as described, for example, in WO 2008/077546 Is determined by calculating the average amount of fucose in the sugar chains in Asn297. Asn297 represents an asparagine residue located at the weak position 297 of the Fc region (Eu numbering of Fc region residues); Asn297 may also be located upstream or downstream of about +/- 3 amino acids of position 297, i.e., between positions 294 and 300, by additional sequence variations of the antibody. Such fucosylation variants may have improved ADCC function. For example, U.S. Patent Publication No. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of references to "tamofucosyl" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005 / 053742; 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 the dedufucosylated antibody include Lecl3 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986)); U.S. Patent Application No. US 2003 Such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e. G. (For example, Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94 (4): 680-688 (2006); And WO 2003/085107).

An antibody variant with bisecting oligosaccharides is additionally provided, for example a dual-antenna oligosaccharide attached to the Fc region of an antibody is bisected by GlcNAc. Such antibody variants can reduce fucosylation and / or improve ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); And US 2005/0123546 (Umana et al.). Antibody variants having 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, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); And WO 1999/22764 (Raju, S.).

Fc region variants

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein to generate Fc region variants. Fc region variants may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the present invention is not all effector functions, but it has several effector functions, so that although the half-life of the antibody in vivo is important, certain effector functions (such as complement and ADCC) ≪ / RTI > antibody variants that are preferred candidates for the antibody. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor binding (FcR) binding assay can be performed to confirm that the antibody lacks Fc [gamma] R binding (thus, perhaps lacking ADCC activity) and retains FcRn binding capacity. NK cells that are primary cells mediating ADCC express only Fc [gamma] RIIII whereas monocytes express Fc [gamma] RI, Fc [gamma] RII and Fc [gamma] RIII. FcR expression on hematopoietic cells is described in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991), page 464, Table 3. A non-limiting example of an in vitro assay for assessing ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83: 7059 -7063 (1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985)); 5,821,337 (Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, for example, ACTI) non-radioactive cytotoxicity assays for flow cytometry (CellTechnology, Inc., Mountain, CA And CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, Wis.)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be determined in vivo, for example, in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). In addition, a C1q binding assay can be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs of WO 2006/029879 and WO 2005/100402. CDC assays can be performed to assess complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045 -1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo removal rate / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18 (12): 1759 -1769 (2006)).

Antibodies with reduced effector function include those having one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (US Patent No. 6,737,056). Such an Fc mutant has an Fc mutant having a substitution at two or more of the amino acid positions 265, 269, 270, 297 and 327 (including the so-called "DANA" Fc mutant with substitution with alanine at residues 265 and 297) (U.S. Patent No. 7,332,581).

Certain antibody variants having improved or decreased binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004/056312; and Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001)).

In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333 and / or 334 (EU numbering of residues) of the Fc region.

In some embodiments, for example, U.S. Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164, 4178-4184 (2000)), changes are made in the Fc region that represent altered (i.e., improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC).

Newborn Fc receptors (FcRn) (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24 : 249 (1994)) are described in US 2005/0014934 A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such an Fc variant may be selected from the group consisting of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 434, e.g. having a substitution at the Fc region residue 434 (U. S. Patent No. 7,371, 826).

Other examples of Fc region variants are also described in Duncan & Winter, Nature 322: 738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; And WO 94/29351.

Cysteine engineered antibody variants

In certain embodiments, it may be desirable to prepare a cysteine engineered antibody, e.g., a "thio MAb," in which one or more residues of the antibody have been replaced with a cysteine residue. In certain embodiments, such substituted moieties occur at accessible sites of the antibody. Substituting these residues for cysteine places the reactive thiol group at the accessible site of the antibody, which can be used to conjugate the antibody to another moiety, such as a drug moiety or linker-drug moiety, as further described herein A conjugate can be generated. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 of the heavy chain (EU numbering); And S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies can be generated, for example, as described in U.S. Patent No. 7,521,541.

Antibody derivative

In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties that are well known in the art and readily available. Suitable moieties for the derivatization of antibodies include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly- Ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycols, propylene glycol homopolymers, But are not limited to, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, the polymers can be the same or different molecules. In general, the number and / or type of polymer used in the derivatization will depend on a number of factors including, but not limited to, the particular characteristics or function of the antibody to be ameliorated, whether the antibody derivative will be used in therapy under defined conditions, . ≪ / RTI >

In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety that can be selectively heated by radiation exposure is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, including, but not limited to, wavelengths that heat non-proteinaceous moieties to a temperature that does not harm normal cells but which is close to the antibody-non-proteinaceous moiety.

Recombinant methods and compositions

Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acids encoding the anti-ETBR antibodies described herein are provided. Such a nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH (e.g., the light and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e. G., Expression vectors) comprising such nucleic acids are provided. In a further embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the amino acid sequence comprising the VL of the antibody and the VH of the antibody, or (2) a vector comprising the amino acid sequence comprising the VL of the antibody (E. G., Transfected with) a first vector comprising a nucleic acid encoding a VH of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, for example Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a host cell comprising a nucleic acid encoding an anti-ETBR antibody as provided above is cultivated under conditions suitable for expression of the antibody, and the antibody is isolated from the host cell (or host cell culture medium) Lt; RTI ID = 0.0 > anti-ETBR < / RTI > antibody.

For recombinant production of an anti-ETBR antibody, for example, a nucleic acid encoding an antibody as described above is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e. G., By using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of the antibody-coding vector include the prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, especially when glycosylation and Fc effector function is not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli ). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains, which cause the glycosylation pathway to "humanize" to produce antibodies in partial or total human glycosylation patterns, Suitable for host expression. Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used to transfect insect cells, particularly Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (describing PLANTIBODIES ™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growing in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 cell line (COS-7) transformed by SV40; Human embryonic kidney cell lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); Fetal hamster kidney cells (BHK); Mouse Sertoli cells (for example, TM4 cells as described in Mather, Biol. Reprod. 23: 243-251 (1980)); 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 breast tumor (MMT 060562); See, e.g., Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982); MRC 5 cells; And FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, such as DHFR - CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); And myeloma cell lines such as Y0, NS0 and Sp2 / 0. For review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

black

The anti-ETBR antibodies provided herein can be identified by various assays known in the art, screened, or characterized for their physical / chemical properties and / or biological activity.

Combination Tests and Other Tests

In one aspect, the antibodies of the invention are tested for their antigen binding activity by known methods such as, for example, ELISA, Western blot, and the like.

In another aspect, competition assays can be used to identify antibodies that compete with, for example, Hu5E9v.1 or Hu5E9v.2 for binding to an ETBR. In certain embodiments, such competing antibodies bind to the same epitope (e. G., A linear or conformational epitope) bound by Hu5E9v.1 or Hu5E9v.2. Details of exemplary methods for mapping epitopes to which antibodies bind are described in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ). In one aspect of the invention, the anti-ETBR antibody described herein specifically binds to an ETBR epitope consisting of amino acid numbers 64 to 101 of SEQ ID NO: 10.

In an exemplary competition assay, an immobilized ETBR is tested for its ability to compete with a first antibody for binding to a first labeled antibody (e.g., Hu5E9v.1 or Hu5E9v.2) and ETBR binding to an ETBR Lt; RTI ID = 0.0 > unlabeled < / RTI > antibody. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized ETBR is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permitting binding of the first antibody to the ETBR, an excess amount of unbound antibody is removed and the amount of label associated with the immobilized ETBR is determined. If the amount of label associated with the immobilized ETBR is substantially reduced in the test sample relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to the ETBR. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Active black

In one aspect, assays are provided to ascertain whether anti-ETBR antibodies and / or BRAFi compounds have biological activity. Biological activities may include those described in the Examples, e. G. In vitro melanoma cell viability assays or in vivo xenograft models in which melanoma cell lines are implanted in nude mice and tumor growth inhibition (TGI) is assessed.

Immunoconjugate

The present invention also relates to a method of treating or preventing a disease or disorder associated with one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., an enzyme active toxin of bacterial, fungal, plant or animal origin or a fragment thereof) Lt; RTI ID = 0.0 > anti-ETBR < / RTI > antibody.

The present invention also relates to a pharmaceutical composition comprising at least one cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzyme active toxin of bacterial, fungal, plant or animal origin, ("Antibody-drug conjugate" or "ADC") comprising an antibody conjugated to an element (i.e., a radioactive conjugate).

Immunoconjugates have been used in the treatment of cancer for cytotoxic agents, local delivery of drugs that kill or inhibit growth or proliferation of cells (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; (2005) Curr Opinion in Pharmacology 5: 543-549; Wu et al. (2005) Nature Biotechnology 23 (9): 1137-1146; Payne, G. (2003) i 3: 207-212; Syrigos and Epenetos 1999) Anticancer Research 19: 605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Deliv. Rev. 26: 151-172; U.S. Patent No. 4,975,278). Immunoconjugates enable the targeted delivery of a drug moiety to the tumor and the intracellular accumulation therein, wherein systemic administration of the unconjugated drug results in toxicity that is not acceptable to normal cells as well as tumor cells to be removed Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological < RTI ID = 0.0 & And Clinical Applications (A. Pinchera et al., Eds) pp. 475-506). Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother. 21: 183-87). Drugs used in the above methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986), supra). The toxins used in the antibody-toxin conjugate include bacterial toxins such as diphtheria toxin, plant toxins such as lysine, small molecule toxins such as geladenamycin (Mandler et al. (2000) J. Nat. Cancer Inst. ; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate Chem. 13: 786-791), maytansinoids (EP 1391213 (1996) Proc Natl Acad Sci USA 93: 8618-8623) and calicheamicin (Lode et al. (1998) Cancer Res. 58: 2928; Hinman et al. et al. (1993) Cancer Res. 53: 3336-3342). Efforts to design and refine ADCs have focused on the selectivity of monoclonal antibodies (mAbs) and also on the mechanism of action, drug-binding and drug / antibody ratio (loading) and drug-releasing properties of drugs [ Erickson et al. (2006) Cancer Res. 66 (8): 1-8; Sanderson et < RTI ID = 0.0 > Cancer Res. 11: 843-852; Jeffrey et al. (2005) J. Med. Chem. 48: 1344-1358; Hamblett et al. -7070]). The toxin can exert cytotoxic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or reduced in activity when conjugated to large antibody or protein receptor ligands.

ZEVALIN® (ibritumomabi cocetane, Biogen / Idec) is bound to the CD20 antigen found on the surface of normal and malignant B lymphocytes bound by a thiourea linker-chelating agent Is an antibody-radioactive isotope conjugate composed of a murine IgG1 kappa monoclonal antibody designated for the human IgG1 and a 111In or 90Y radioisotope directed against the antibody (Wiseman et al. (2000) Eur. Jour. Nucl. (2002) Blood 99 (12): 4336-42; Witzig et al. (2002) J. Clin. Oncol. 20 (10): 2453-63; Witzig et al. Clin. Oncol. 20 (15): 3262-69). Zevalin is active against B-cell non-Hodgkin's lymphoma (NHL), but administration causes severe and prolonged cytopenias in most patients. MYLOTARG ™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody-drug conjugate composed of huCD33 antibodies linked to calicheamicin, has been used for treatment of acute myelogenous leukemia (Drugs of the Future (2000) 25 (7): 686; U.S. Pat. Nos. 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). Antibody-drug conjugates (ADCs) composed of maytansuid DM1 linked to trastuzumab have potent antitumor activity in HER2-overexpressing Trastuzumab-sensitive and Trastuzumab-resistant tumor cell lines, and xenograft human cancer models . Trastuzumab-MCC-DM1 (T-DM1) is currently being evaluated in Phase II clinical trials for patients refractory to HER2-induced therapy (Beeram et al. (2007) A phase I study of trastuzumab-MCC-DM1 (T-DM1), a first-in-class HER2 antibody-drug conjugate (ADC), in patients (pts) with HER2 + metastatic breast cancer Oncology 43rd: June 02 (Abs 1042; Krop et al., European Cancer Conference ECCO, Poster 2118, September 23-27, 2007, Barcelona); US 7097840; US 2005/0276812; US 2005/0166993) The peptides auristatin E (AE) and monomethylauristatin (MMAE) (synthetic analogs of dolastatin) were found to bind chimeric monoclonal antibody cBR96 (specific for Lewis Y on carcinoma) and cAC10 (Doronina et al. (2003) Nature Biotechnol. 21 (7): 778-784)) and are being developed for therapeutic use.

In certain embodiments, the immunoconjugate comprises an antibody and a chemotherapeutic agent or other toxin. Chemotherapeutic agents useful for the generation of immunoconjugates are described herein (e. G., Above). Enzyme active toxins and fragments thereof can also be used and are described herein.

In certain embodiments, the immunoconjugate comprises an antibody and one or more small molecule drug moieties (calicheamicin, maytansinoid, dolastatin, auristatin, anthracycline, taxane, tricothecene and CC1065, But are not limited to, small molecule drugs such as derivatives of these drugs with activity. Examples of such immunoconjugates are discussed in more detail below.

Exemplary immunoconjugates

(Or "antibody-drug conjugate" ("ADC")) of the present invention is characterized in that the antibody is conjugated (i.e., covalently attached) to one or more drug moieties (D) via any linker I can be of.

(I)

Figure pct00017

Thus, the antibody can be conjugated to the drug either directly or through a linker. In Formula I, p is the average number of drug moieties per antibody, and can be, for example, from about 1 to about 20 drug moieties per antibody, and in certain embodiments from 1 to about 8 drug moieties per antibody.

Exemplary linker

The linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine- (2-pyridylthio) pentanoate ("SPP"), N-succinimidyl 4- (N- (&Quot; SMAB ") and N-succinimidyl (4-iodo-acetyl) aminobenzoate (" SIAB "). A variety of linker components are known in the art, some of which are described below.

The linker may be a "cleavable linker" that facilitates release of the drug in the cell. For example, linkers containing acid-labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photoreactive linkers, dimethyl linkers or disulfide linkers , Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).

In certain embodiments, the linker is as shown in Formula II below.

≪

Figure pct00018

Wherein A is a stretcher unit and a is an integer from 0 to 1; W is an amino acid unit; w is an integer from 0 to 12; Y is a spacer unit, y is 0, 1, or 2; Ab, D and p are as defined above for formula (I). Exemplary embodiments of such linkers are described in US 2005-0238649 Al, which is expressly incorporated herein by reference.

In some embodiments, the linker component may comprise a "stretcher unit" that links the antibody to another linker moiety or moiety. Exemplary stretcher units are shown below (where the wave line represents the covalent attachment site for the antibody).

Figure pct00019

Figure pct00020

In some embodiments, the linker component may comprise amino acid units. In one such embodiment, the amino acid unit allows cleavage of the linker by the protease, thereby facilitating the release of the drug from the immunoconjugate upon exposure to an intracellular protease, e.g., a rysosomal enzyme. See, for example, Doronina et al. (2003) Nat. Biotechnol. 21: 778-784. Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. 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-glycine. Amino acid units may include naturally occurring amino acid residues 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 terms of their selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C and D, or plasmin proteases.

In some embodiments, the linker component may comprise a "spacer" unit linking the antibody directly to the drug moiety or by a stretcher unit and / or an amino acid unit. The spacer unit may be "self-sacrificing" or "non-self-sacrificing". A "non-self sacrificial" spacer unit is one in which some or all of the spacer units are in association with the drug moiety upon enzymatic (eg, proteolytic) cleavage of the ADC. Examples of non-self sacrificial spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. Other combinations of peptide spacers that are susceptible to sequence-specific enzymatic cleavage are also contemplated. For example, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by a tumor cell-associated protease will release the glycine-glycine-drug moiety from the remainder of the ADC. In one such embodiment, the glycine-glycine-drug moiety is applied to the individual hydrolysis step in the tumor cells to cleave the glycine-glycine spacer unit from the drug moiety.

The "self-sacrificial" spacer unit allows release of the drug moiety without a separate hydrolysis step. In certain embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In one such embodiment, the p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond, and a carbamate, methylcarbamate or carbonate is formed between the benzyl alcohol and the cytotoxic agent. See, for example, Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15: 1087-1103. In one embodiment, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In certain embodiments, p- amino benzyl unit in the phenylene portion is substituted with Qm, wherein, Q is -C 1 -C 8 alkyl, -O- (C 1 -C 8 alkyl), -halogen, - nitro or - cyano; m is an integer of 0-4. Examples of self-sacrificial spacer units include aromatic compounds which are electrically similar to p-aminobenzyl alcohol (see, for example, US 2005/0256030 A1), such as 2-aminoimidazole-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett., 9: 2237) and ortho- or para-aminobenzyl acetals. Spacers that undergo cyclization during amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223); Suitable substituted bicyclo [2.2.1] and bicyclo [2.2.2] ring systems (Storm, et al., J. Amer. Chem. Soc., 1972, 94, 5815); Aminophenylpropionic acid amide (Amsberry, et al., J. Org. Chem., 1990, 55, 5867) can be used. Removal of amine-containing drugs substituted at the a-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) is also an example of a self-sacrificial spacer useful in ADC.

In one embodiment, the spacer unit is a branched bis (hydroxymethyl) styrene (BHMS) unit as shown below, which can be used to incorporate and release multiple drugs.

Figure pct00021

Wherein, Q is -C 1 -C 8 alkyl, -O- (C 1 -C 8 alkyl), -halogen, - nitro or - cyano; m is an integer of 0-4; n is 0 or 1; and p ranges from 1 to about 20.

In another embodiment, linker L can be a dendritic type linker for covalently attaching more than one drug moiety to an antibody via a branched multifunctional linker moiety (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12: 2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11: 1761-1768). The dendritic linker can increase the molar ratio, or loading, of drug-antibody associated with the effect of the ADC. Thus, when the cysteine engineered antibody has only one reactive cysteine thiol group, a number of drug moieties can be attached through the dendritic linker.

Exemplary linker components and combinations thereof are presented below in connection with ADCs of formula II.

Figure pct00022

Figure pct00023

Linker components including stretchers, spacers and amino acid units can be synthesized by methods known in the art, such as those described in US 2005-0238649 A1.

Exemplary drug moiety

Maytansine and maytansinoid

In some embodiments, the immunoconjugate comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansin was first isolated from East Africa shrub Maytenus serrata (US Patent No. 3896111). Subsequently, certain microorganisms have also been found to produce maytansinoids such as maytansinol and C-3 maytansinol ester (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; And 4,371,533.

The maytansinoid drug moiety may be selected from the group consisting of (i) relatively available for preparation by fermentation or chemical modification, or derivatization of the fermentation product, (ii) suitable for conjugation to the antibody via a non-disulfide linker (Iii) is stable in plasma, and (iv) is effective against a variety of tumor cell lines, which is an attractive drug moiety in antibody-drug conjugates.

Maytansine compounds suitable for use as maytansinoid drug moieties are well known in the art and can be isolated from natural sources or produced using genetic engineering techniques according to known methods (Yu et al. 2002) PNAS 99: 7968-7973). In addition, maytansinol and maytansinol analogs can be prepared in a synthetic manner by known methods.

Exemplary maytansinoid drug moieties have the following modified aromatic rings: C-19-dechloro (U.S. Patent No. 4256746) (prepared by lithium aluminum hydride reduction of ansamitocin P2); C-20- hydroxy (or C-to 20- methyl) +/- C-to 19- chloro (U.S. Pat. No. 4.36165 million and 4,307,016) (MRS Streptomyces (Streptomyces) or bad Martino demethylation or using MRS (Actinomyces) Prepared by dechlorination with LAH); And C-20-demethoxy, C-20-acyloxy (-OCOR), +/- dechloro (U.S. Patent No. 4,294,757) (prepared by acylation using acyl chloride) .

Exemplary maytansinoid drug moieties may also be C-9-SH (US Patent 4424219) (prepared by 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) (U.S. Patent No. 4450254) (manufactured by Nocardia); C-15-hydroxy / acyloxy (US 4364866) (prepared by conversion of maytansinol by streptomyces); C-15-methoxy (U.S. Patent Nos. 4313946 and 4315929) (isolated from Trewia nudlflora); C-18-N-demethyl (US Patent Nos. 4362663 and 4322348) (prepared by demethylation of maytansinol by streptomycetes); And 4,5-deoxy (US 4371533) (prepared by titanium trichloride / LAH reduction of maytansinol).

Many sites on the maytansin compound are known to be useful as linking sites, depending on the type of linkage. For example, to form an ester linkage, a C-20 position with a hydroxyl group, a C-14 position with a hydroxymethyl, a C-15 position with a hydroxyl group and a C-20 position with a hydroxyl group Are all suitable.

The maytansinoid drug moiety includes those having the following structure.

Figure pct00024

Wherein the wave line represents the covalent attachment of the sulfur atom of the maytansinoid drug moiety to the linker of the ADC. R can be independently H or C 1 -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).

For any of the compounds of the present invention, any stereoisomer of the maytansinoid drug moiety, any combination of the R and S configurations in the chiral carbon of D is contemplated (US 7276497; US 6913748; US 6441163; US 633410 (RE 39151) ; US 5208020; Widdison et al. (2006) J. Med. Chem. 49: 4392-4408, the disclosures of which are incorporated herein by reference). In one embodiment, the maytansinoid drug moiety will have the following stereochemical structure.

Figure pct00025

Exemplary embodiments of the maytansinoid drug moiety include DM1 having the structure: DM3; And DM4.

Figure pct00026

Figure pct00027

In the above formula, the wave line indicates the covalent attachment of the sulfur atom of the drug to the linker (L) of the antibody-drug conjugate. HERCEPTIN® (trastuzumab) linked to DM1 by SMCC has been reported (WO 2005/037992; US 2005/0276812; US 2005/016993).

Other exemplary maytansinoid antibody-drug conjugates have the following structures and abbreviations (where Ab is an antibody and p is 1 to about 8).

Figure pct00028

Exemplary antibody-drug conjugates in which DMl is linked to the thiol group of the antibody via a BMPEO linker have the following structure and abbreviations.

Figure pct00029

Ab is an antibody; n is 0, 1 or 2; p is 1, 2, 3 or 4;

Immunoconjugates containing maytansinoids, methods for their production, and therapeutic uses thereof are described, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, US 2005/0276812 A1, and European Patent EP 0 425 235 B1. Liu et al. Proc. Natl. Acad. Sci. USA 93: 8618-8623 (1996) describes an immunoconjugate comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic to cultured colon cancer cells and showed antitumor activity in in vivo tumor growth assays. See Chari et al. Cancer Research 52: 127-131 (1992)) discloses that murine monoclonal antibody TA7, which binds to murine antibody A7, which binds an antigen on human colon cancer cell line, or which binds to HER-2 / neu oncogene, An immunoconjugate conjugated through a disulfide linker is described. The cytotoxicity of the TA.1-maytansinoid conjugate was tested in vitro for the human breast cancer cell line SK-BR-3 expressing 3 x 10 5 HER-2 surface antigens per cell. The drug conjugate achieved a similar level of cytotoxicity as the free maytansinoid drug, which can be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

The antibody-maytansinoid conjugate is prepared by chemically linking the antibody to the maytansinoid molecule without significantly decreasing the biological activity of the antibody or maytansinoid molecule. See, for example, U.S. Patent No. 5,208,020, the disclosure of which is expressly incorporated herein by reference. Maytansinoid molecules conjugated to an average of 3-4 per antibody molecule showed the ability to enhance cytotoxicity of target cells without adversely affecting the function or solubility of the antibody, but the use of naked antibodies To increase cytotoxicity. Maytansinoids are well known in the art and may be synthesized by known techniques and isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Pat. No. 5,208,020 and other patents and non-patent publications referred to hereinabove. The preferred maytansinoids are maytansinol, and maytansinol analogs modified at the aromatic ring or other position of the maytansinol molecule, such as various maytansinol esters.

U.S. Patent No. 5208020 or EP Patent 0 425 235 B1, the disclosure of which is hereby expressly incorporated by reference; See Chari et al. Cancer Research 52: 127-131 (1992); And US 2005/016993 Al, a number of linking groups for the production of antibody-maytansinoid conjugates are known in the art. Antibody-maytansinoid conjugates containing the linker component SMCC can be prepared as disclosed in US 2005/0276812 Al ("Antibody-drug conjugates and Methods"). Linkers include disulfide groups, thioether groups, acid labile groups, photo labile groups, peptidase labile groups or esterase labile groups as disclosed in the above identified patents. Additional linkers are described and illustrated herein.

Conjugates of antibodies and maytansinoids may be conjugated to various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl- (DMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidylsuberate), cyclohexane-1-carboxylate (P-diazonium benzoyl) -ethylenediamine (e.g., bis- (p-diazonium benzoyl) hexanediamine), aldehydes (e.g., glutaraldehyde), bis-azido compounds ), Diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-activated fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). In certain embodiments, the coupling agent to provide a disulfide linkage 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).

The linker may be attached to the maytansinoid molecule at various positions depending on the type of linkage. For example, ester linkages can be formed by reaction with hydroxyl groups using conventional coupling techniques. The reaction may take place at the C-3 position with a hydroxyl group, the C-14 position with a hydroxymethyl, the C-15 position with a hydroxyl group, and the C-20 position with a hydroxyl group. In one embodiment, the linking group is formed at the C-3 position of the maytansinol or maytansinol analog.

Auristatin and dolastatin

In some embodiments, the immunoconjugate comprises an antibody conjugated to dolastatin or a dolastatin peptide analog or derivative, such as auristatin (US Patent No. 5635483; 5780588). Dolastatin and auristatin inhibit microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45 (12): 3580-3584) (US Patent No. 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42: 2961-2965). The dolastatin or auristatin drug moiety can be attached to the antibody via the N (amino) terminal or the C (carboxyl) terminal of the peptide drug moiety (WO 02/088172).

Exemplary auristatin embodiments include N-terminal linked monomethyl auristatin drug moieties DE and DF (US 7498298).

The peptide drug moiety may be selected from the following formulas: D E and D F.

Figure pct00030

Wherein the wave lines of D E and D F represent the covalent attachment sites for the antibody or antibody-linker moiety, and independently at each position:

R 2 is selected from H and C 1 -C 8 alkyl;

R 3 is selected from the group consisting of H, C 1 -C 8 alkyl, C 3 -C 8 carbocycle, aryl, C 1 -C 8 alkyl-aryl, C 1 -C 8 alkyl- (C 3 -C 8 carbocycle) C 3 -C 8 heterocycle and C 1 -C 8 alkyl- (C 3 -C 8 heterocycle);

R 4 is selected from the group consisting of H, C 1 -C 8 alkyl, C 3 -C 8 carbocycle, aryl, C 1 -C 8 alkyl-aryl, C 1 -C 8 alkyl- (C 3 -C 8 carbocycle) C 3 -C 8 heterocycle and C 1 -C 8 alkyl- (C 3 -C 8 heterocycle);

R < 5 > is selected from H and methyl; Or R 4 and R 5 are connected to form a carbocyclic ring and have the formula - (CR a R b ) n -, wherein R a and R b are independently H, C 1 -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 1 -C 8 alkyl;

R 7 is selected from the group consisting of H, C 1 -C 8 alkyl, C 3 -C 8 carbocycle, aryl, C 1 -C 8 alkyl-aryl, C 1 -C 8 alkyl- (C 3 -C 8 carbocycle) C 3 -C 8 heterocycle and C 1 -C 8 alkyl- (C 3 -C 8 heterocycle);

Each R 8 is independently selected from H, OH, C 1 -C 8 alkyl, C 3 -C 8 carbocycle and O- (C 1 -C 8 alkyl);

R 9 is selected from H and C 1 -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 1 -C 8 alkyl;

R 11 is H, C 1 -C 20 alkyl, aryl, C 3 -C 8 heterocycle, - (R 13 O) m -R 14 or - (R 13 O) m -CH (R 15) is selected from 2 ;

m is an integer ranging from 1 to 1000;

R 13 is C 2 -C 8 alkyl;

R 14 is H or C 1 -C 8 alkyl;

With R 15 for each occurrence are independently H, COOH, - (CH 2 ) n -N (R 16) 2, - (CH 2) n -SO 3 H or - (CH 2) n -SO 3 -C 1 - C 8 alkyl;

R 16 in each occurrence is independently H, C 1 -C 8 alkyl or - (CH 2 ) n -COOH;

R 18 is -C (R 8) 2 -C ( R 8) 2 - aryl, -C (R 8) 2 -C (R 8) 2 - (C 3 -C 8 heterocycle), and -C (R 8 ) 2 -C (R 8 ) 2 - (C 3 -C 8 carbocycle); n is an integer ranging from 0 to 6;

In one embodiment, R 3 , R 4 and R 7 are independently isopropyl or sec-butyl and R 5 is -H or methyl. In an exemplary embodiment, R 3 and R 4 are each isopropyl, R 5 is -H, and R 7 is sec-butyl. In another embodiment, R 2 and R 6 are each methyl and R 9 is -H. In another embodiment, R 8 in each case 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, and R 8 in each case is -OCH 3 and R < 9 > is -H. In one embodiment, Z is -O- or -NH-. In one embodiment, R < 10 > is aryl. In an exemplary embodiment, R < 10 > is -phenyl. In an exemplary embodiment, when Z is -O-, R < 11 > is -H, methyl or t-butyl. In one embodiment, when Z is -NH, R 11 is -CH (R 15) 2, wherein R 15 is - (CH 2) n -N ( R 16) 2 , and R 16 is -C 1 -C 8 alkyl or - (CH 2) n is -COOH. In another embodiment, when Z is -NH, R 11 is -CH (R 15 ) 2 , wherein R 15 is - (CH 2 ) n -SO 3 H.

An exemplary auristatin embodiment of the formula D E is MMAE and the wave line indicates the covalent attachment of the antibody-drug conjugate to the linker (L).

Figure pct00031

Exemplary auristatin embodiments of the formula D F are MMAF, wherein the pharmeric line indicates the covalent attachment of the antibody-drug conjugate to the linker (L) (US 7498298 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124).

Figure pct00032

Another exemplary embodiment is a monomethyl valine compound having a phenylalanine carboxy modification at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008848), and a phenylalanine at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008603). ≪ / RTI >

Other drug moieties include the following MMAF derivatives, and the wave line represents the covalent attachment of the antibody-drug conjugate to the linker (L).

Figure pct00033

Figure pct00034

Figure pct00035

In one aspect, hydrophilic groups, including but not limited to triethylene glycol esters (TEG) as indicated above, may be attached to the drug moiety at R < 11 >. Without being bound to any particular theory, a hydrophilic group aids in the internalization and non-aggregation of drug moieties.

Exemplary embodiments of ADCs of formula I comprising auristatin / dolastatin or derivatives thereof are described in US 7498298, which is expressly incorporated herein by reference, and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. Exemplary embodiments of ADCs of formula I comprising MMAE or MMAF and various linker components have the following structure and abbreviations wherein "Ab" is an antibody, p is from 1 to about 8, and & Valine-citrulline dipeptide; "S" is a sulfur atom.

Figure pct00036

Figure pct00037

Exemplary embodiments of ADCs of formula I, including MMAF and various linker components, further comprise Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Interestingly, an immunoconjugate containing MMAF attached to an antibody by a non-proteolytically cleavable linker has an activity similar to that of an immunoconjugate containing MMAF attached to the antibody by a proteolytically cleavable linker appear. Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. In this case, drug release is believed to occur by antibody degradation in the cell. The same document is referred to.

Typically, the peptide-based drug moiety may be prepared by peptide bond formation between two or more amino acids and / or peptide fragments. Such peptide bonds can be obtained, for example, by liquid phase synthesis methods (E. Schroeder and K. Luebke, "The Peptides ", volume 1, pp. 76-136, 1965, Academic Press) well known in the field of peptide chemistry, . ≪ / RTI > Auristatin / dolastatin drug moiety is described in US 2005-0238649 A1; U.S. Patent No. 5635483; U.S. Patent 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.

In particular, the auristatin / dolastatin drug moiety of formula D F , such as MMAF and its derivatives, is described in US 7498298 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. The auristatin / dolastatin drug moiety of formula D E , such as MMAE and its derivatives, is described in Doronina et al. (2003) Nat. Biotech. 21: 778-784. ≪ / RTI > The drug-linker moieties MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF and MC-vc-PAB-MMAE are described, for example, by Doronina et al. (2003) Nat. Biotech. 21: 778-784, and US 7498298, and can then be conjugated to the antibody of interest.

Calicheamicin

In another embodiment, the immunoconjugate comprises an antibody conjugated to one or more calicheamicin molecules. Antibiotics of the calicheamicin family can produce double-stranded DNA cuts at concentrations below picomolar. See US 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 American Cyanamid Company) for the preparation of conjugates of the calicheamicin family. Structural analogs of calicheamicin that can be used include, but are not limited to, gamma 1 I , alpha 2 I , alpha 3 I , N-acetyl- gamma 1 I , PSAG and θ I 1 (Hinman et al. , Cancer Research 53: 3336-3342 (1993), Lode et al., Cancer Research 58: 2925-2928 (1998)], and the above-mentioned americanian cyanamide. Another anti-tumor drug to which antibodies can be conjugated is QFA, an anti-factor. Both calicheamicin and QFA have intracellular action sites and do not readily pass through the plasma membrane. Thus, the intracellular uptake of these materials through antibody-mediated internalization greatly enhances their cytotoxic effects.

Other cytotoxic agents

Other anti-tumor agents that can be conjugated to antibodies include antracycline (Kratz et al. (2006) Current Med. Chem. 13: 477-523; Jeffrey et al. (2006) Bioorganic & 16: 717-721; Nagy et al. (2000) Proc Natl Acad Sci. 97: 829-834; Dubowchik et al. (2002) (2002) J. Med. Chem. 45: 4336-4343; US 6630579), BCNU, streptozocin, vincristine, and 5-fluoro (US Pat. No. 5,877,296), as well as lowacyl (family of agonists known collectively as LL-E33288 complex, described in U.S. Patent No. 5,053,394, 5,770,710).

The enzyme active toxins and fragments thereof that can be used are selected from the group consisting of diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, Aleurites fordii protein, Dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), Momordica charantia Inhibitors, curcine, crotin, sapaonaria officinalis inhibitors, gelonin, mitogelin, resorcinosine, penomycin, enomycin and tricothecene. See, for example, WO 93/21232, published October 28, 1993.

The present invention further contemplates an immunoconjugate formed between an antibody and a compound having a nucleic acid degrading activity (e. G., Ribonuclease or DNA endonuclease such as deoxyribonuclease, DNase).

In certain embodiments, the immunoconjugate can comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radiolabeled antibodies. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 Zr 89 , and radioactive isotopes of Lu. The immunoconjugate can be used for detection if it contains a radioactive atom for a cytotoxic study, for example tc 99m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) For example, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radioactive- or other label may be incorporated into the immunoconjugate in a known manner. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using a suitable amino acid precursor including fluorine-19 instead of, for example, hydrogen. tc 99m or I 123 , Re 186 , Re 188 , Zr 89 and In 111 may be attached via cysteine residues in the peptide. Yttrium-90 can be attached through the lysine residue. Iodine-123 can be incorporated using the IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57). Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

In certain embodiments, the immunoconjugate can 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, have. Such immunoconjugates are useful in antibody-dependent enzyme-mediated prodrug therapy ("ADEPT"). Enzymes that can be conjugated to antibodies include alkaline phosphatase useful for converting a phosphate-containing prodrug into a free drug; Aryl sulfatase useful for converting a sulfate-containing prodrug into a free drug; Cytosine deaminase useful for converting non-toxic 5-fluorocytosine to the anticancer drug 5-fluorouracil; Proteases useful for converting a peptide-containing prodrug into a free drug, such as serratia protease, thermolysin, subtilisin, carboxypeptidase and cathepsin (e.g., cathepsins B and L); Alanylcarboxypeptidase useful for converting a prodrug containing a D-amino acid substituent; Carbohydrate-cleaving enzymes such as beta -galactosidase and neuraminidase useful for converting glycosylation prodrugs into free drugs; beta -lactamase useful for converting a drug derivatized with? -lactam into a free drug; And penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting a drug derivatized with a phenoxyacetyl or phenylacetyl group at its amine nitrogen, respectively, into a free drug. Enzymes can be covalently linked to antibodies by recombinant DNA techniques well known in the art. See, for example, Neuberger et al., Nature 312: 604-608 (1984).

Drug loading

Drug loading is expressed as p, the average number of drug moieties per antibody in the molecule of formula (I). The drug loading may be from 1 to 20 drug moieties (D) per antibody. The ADC of formula I comprises a collection of antibodies conjugated to from 1 to 20 drug moieties. The average number of drug moieties per antibody in the preparation of ADC by conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA analysis, and HPLC. In addition, the chronological distribution of the ADC according to p may be determined. In some cases, the isolation, purification and characterization of a homogeneous ADC, where p is a specific value from an ADC with a different drug loading value, can be achieved by means such as reverse phase HPLC or electrophoresis.

In some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, if the attachment is cysteinyl thiol as in the exemplary embodiment, the antibody may have only one or several cysteine thiol groups, or only one fully reactive thiol group to which the linker may be attached Or several. In certain embodiments, greater drug loading, such as p > 5, can cause aggregation, insolubility, toxicity, or loss of cell permeability of a particular antibody-drug conjugate. In certain embodiments, the drug loading for the ADC of the invention is from 1 to about 8; About 2 to about 6; Or from about 3 to about 5. Indeed, in certain ADCs, the optimal ratio of drug moiety per antibody may be less than 8, and has been found to be from about 2 to about 5. See US 2005-0238649 A1

In certain embodiments, less than the theoretical maximum drug moiety is conjugated to the antibody during the conjugation reaction. The antibody may comprise, for example, a lysine residue that does not react with the drug-linker intermediate or linker reagent, as discussed below. In general, antibodies do not contain many free and reactive cysteine thiol groups that can be linked to drug moieties, and in fact most cysteine thiol moieties in the antibody are present as disulfide bridges. In certain embodiments, the antibody may be reduced to a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or complete reduction conditions to produce a reactive cysteine thiol group. In certain embodiments, the antibody is subjected to denaturing conditions to reveal a reactive nucleophilic group, e. G. Lysine or cysteine.

The loading of the ADC (drug / antibody ratio) can be measured in different ways, for example, (i) restriction of the molar excess of the drug-linker intermediate or linker reagent relative to the antibody, (ii) restriction of the conjugation reaction time or temperature, and (iii) ) Cysteine thiol modification by partial or limited reduction conditions.

When more than one nucleophilic group is reacted with a drug-linker intermediate or linker reagent followed by a drug moiety reagent, the product obtained is understood to be a mixture of ADC compounds having a distribution with one or more drug moieties attached to the antibody shall. The average number of drugs per antibody can be calculated from the mixture by antibody-specific and drug-specific double ELISA antibody analysis. The individual ADC molecules can be identified in the mixture by mass spectrometry and separated by HPLC, for example by hydrophobic interaction chromatography (see, for example, McDonagh et al. (2006) Prot. Engr. Design ≪ / RTI > < RTI ID = 0.0 > Hamblett, < / RTI > KJ, et al. 2004, Proceedings of the AACR, Volume 45, March 2004, Alley, SC, et al., "An anti-CD30 antibody-drug conjugate," Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004), which is incorporated herein by reference in its entirety. In certain embodiments, a homogeneous ADC with a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.

Specific methods of producing immunoconjugates

The ADC of formula (I) can be prepared by several routes using organic chemistry reactions, conditions and reagents known to those skilled in the art, including: (1) reacting the nucleophilic group of the antibody with a divalent linker reagent to form a covalent bond To form Ab-L and then reacting it with the drug moiety D; And (2) reacting the nucleophilic group of the drug moiety with a divalent linker reagent to form a D-L via a covalent bond and then reacting it with the nucleophilic group of the antibody. Exemplary methods for preparing the ADC of Formula I through the latter route are described in US 2005-0238649 Al, which is expressly incorporated herein by reference.

The nucleophilic group on the antibody can be a nucleophilic group on the antibody, such as (i) an N-terminal amine group, (ii) a side chain amine group such as lysine, (iii) a side chain thiol group such as cysteine, and (iv) Groups, or amino groups. The amine, thiol, and hydroxyl groups are nucleophilic and include electrophilic groups on linker moieties and linker reagents such as (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as halo acetamide; (iii) react with aldehydes, ketones, carboxyls and maleimide groups to form covalent bonds. Certain antibodies have reducible interstrand disulfides, i.e., cysteine bridges. The antibody may be rendered reactive for conjugation with the linker reagent by treating the antibody with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) so that the antibody is completely or partially reduced. Thus, each cysteine bridge would theoretically form two reactive thiol nucleophiles. Through the modification of the lysine residues, additional nucleophilic groups can be introduced into the antibody by reaction of a lysine residue and a 2-iminothiolane (Trout reagent), for example, converting an amine to a thiol. The reactive thiol group may be introduced into the antibody by introducing one, two, three, four, or more cysteine residues (e. G., By producing a variant antibody comprising one or more non-natural cysteine amino acid residues) .

The antibody-drug conjugates of the invention may also be produced by reaction between a linker reagent or a nucleophilic group on the drug and an electrophilic group on the antibody, such as an aldehyde or ketone carbonyl group. Useful nucleophilic groups on linker reagents include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazinecarboxylate, and arylhydrazide. In one embodiment, the antibody is modified to introduce an electrophilic moiety capable of reacting with a linker reagent or a nucleophilic substituent on the drug. In another embodiment, the sugar of the glycosylated antibody can be oxidized, for example, with a periodate oxidation reagent to form an aldehyde or ketone group capable of reacting with the linker reagent or the amine group of the drug moiety. The resulting imine Schiff base may form a stable linkage, or may be reduced, for example, by a borohydride reagent to form a stable amine linkage. In one embodiment, by reacting the carbohydrate portion of the glycosylated antibody with galactose oxidase or sodium meta-periodate, carbonyl (aldehyde and ketone) groups capable of reacting with the appropriate group on the drug can be produced in the antibody Hermanson, Bioconjugate Techniques). In another embodiment, an antibody containing an N-terminal serine or threonine residue is reacted with sodium meta-periodate to generate an aldehyde instead of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3: 138-146; US 5362852). These aldehydes can react with drug moieties or linker nucleophiles.

The nucleophilic group on the drug moiety may be an electrophilic group in the linker moiety and the linker reagent such as (i) an active ester such as an NHS ester, an HOBt ester, a haloformate and an acid halide, (ii) (Iii) amines capable of forming a covalent bond with aldehydes, ketones, carboxyls and maleimide groups, thiols, hydroxyls, hydrazides, oximes, hydrazines, thiosemicarbazones, hydrazines But are not limited to, carboxylate, and arylhydrazide groups.

The compounds of the present invention are commercially available cross-linker reagents such as: BMPS, EMCS, GMBS, HBVS, LC-MS (from Pierce Biotechnology, Inc., Rockford, Ill. SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMSC, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIB, sulfo-SMB, and SVSB Succinimidyl- (4-vinylsulfone) benzoate), but the present invention is not limited thereto.

Various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane- (E.g. dimethyidipimidate HCl), active esters (e. G., Disuccinimidyl beverages), aldehydes (e. G., Glutaraldehyde) , Bis-azido compounds (e.g., bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p- diazoniumbenzoyl) -ethylenediamine), diisocyanates (E.g., toluene 2,6-diisocyanate), and a bis-active fluorine compound (such as 1,5-difluoro-2,4-dinitrobenzene) can be used to create an immunoconjugate of an antibody and a cytotoxic agent have. For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies. See WO94 / 11026.

Alternatively, fusion proteins comprising antibodies and cytotoxic agents can be prepared, for example, by recombinant techniques or peptide synthesis. The recombinant DNA molecule may comprise a region that is separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate, or that encodes an antibody and a cytotoxic portion of the conjugate that are adjacent to each other.

In another embodiment, the antibody can be conjugated to a "receptor" (e.g., streptavidin) for utilization in tumor preliminary targeting, and the antibody-receptor conjugate is administered to a patient, The conjugate is removed from the circulatory system and then a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e. G., A radionucleotide) is administered.

Pharmaceutical preparation

In one aspect, the invention further provides pharmaceutical agents comprising one or more antibodies of the invention and / or one or more immunoconjugates thereof. In some embodiments, the pharmaceutical agent comprises 1) an antibody of the invention and / or an immunoconjugate thereof, and 2) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical agent comprises 1) an antibody of the invention and / or an immunoconjugate thereof, and optionally 2) one or more additional therapeutic agents. Additional therapeutic agents include, but are not limited to, those described below.

An antibody or immunoconjugate having a desired degree of purity of a pharmaceutical agent comprising an antibody or immunoconjugate of the invention may be combined with any physiologically acceptable carrier, excipient or stabilizer (Remington ' s Pharmaceutical Sciences 16th edition, Osol, A. Ed (1980)]) for storage in the form of aqueous solutions or lyophilized or other dry formulations. Acceptable carriers, excipients or stabilizers are nontoxic to the recipient 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 (e.g., octadecyldimethylbenzylammonium 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 immunoglobulin; 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 TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG). Pharmaceutical preparations used for in vivo administration are generally sterilized. This is easily accomplished by filtration through a sterile filtration membrane.

The active ingredient may also be incorporated into microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively, May be encapsulated in a delivery system (e. G., Liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington ' s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of hydrophobic solid polymers containing the antibodies or immunoconjugates of the invention, which are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (US Patent No. 3,773,919), L- And copolymers of γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ™ (lactic acid-glycolic acid copolymer and leuprolide acetate Injectable microspheres), and poly-D - (-) - 3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, while certain hydrogels release proteins for shorter periods of time. If the encapsulated antibody or immunoconjugate remains in the body for an extended period of time, they may denature or aggregate as a result of exposure to water below 37 ° C, resulting in loss of biological activity and changes in immunogenicity. A reasonable strategy for stabilization can be devised depending on the relevant mechanism. For example, if the aggregation mechanism is found to be an intermolecular SS bond formation via thio-disulfide interchange, stabilization can be achieved by modifying the sulfhydryl moiety, lyophilizing from an acidic solution, controlling moisture content, Can be achieved by the development of certain polymer matrix compositions.

Antibodies can be formulated in any suitable form for delivery to the target cell / tissue. For example, the antibody can be formulated as an immunoliposome. "Liposome" is a small vesicle composed of various types of lipids, phospholipids and / or surfactants useful for delivering a drug to a mammal. Typically, the components of the liposome are arranged in a bilayer form similar to the lipid array of the biological membrane. Liposomes containing antibodies have been described by Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77: 4030 (1980); U.S. Patent Nos. 4,485,045 and 4,544,545; And WO 97/38731 (published October 23, 1997). Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

Particularly useful liposomes can be produced by reverse-phase evaporation methods using lipid compositions comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extracted through a filter of defined pore size to produce liposomes with the desired diameter. Fab 'fragments of the antibodies of the present invention can be made by disulfide interconversion using Martin et al., J. Biol. Chem. 257: 286-288 (1982)). Optionally, a chemotherapeutic agent is included in the liposome. Gabizon et al., J. National Cancer Inst. 81 (19): 1484 (1989).

In another embodiment, the immunoconjugate comprises an enzyme active toxin or a fragment thereof (diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, (PAPI, PAPII and PAP-S), Momordica carantia Inhibitors, Curcumin, Crocine, Sapa, < RTI ID = 0.0 > Including, but not limited to, an immunosuppressive agent, an immunosuppressive agent, an angiopathicin inhibitor, gelonin, mitogelin, resorcinosine, penomycin, enomycin and tricothecene.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioactive isotopes are available for the production of radioactive conjugates. Examples include Zr 89 , At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. The radioactive conjugate may comprise a radioactive atom, for example tc99m or I123, for use in detection when used for detection, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) Iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Antibodies and cytotoxic agents may be conjugated to various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl- Methyl dicyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (P-diazonium benzoyl) -ethylenediamine (e.g., bis- (p-diazonium benzoyl) hexanediamine), aldehydes (e.g., glutaraldehyde), bis-azido compounds ), Diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-activated fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies. See WO94 / 11026. The linker may be a " cleavable linker "that facilitates release of the cytotoxic drug from the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photodegradable linker, a dimethyl linker or a disulfide-containing linker (Chari et al., Cancer Res. 52: 127-131 Patent No. 5,208,020) can be used.

The immunoconjugates or ADCs of the present disclosure can be used in combination with commercially available (e.g. Pierce Biotechnology, Inc.) (from BMX, EMCS, GMBS, HBVS, LC-SMCC, MBS , MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMSC, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIB, sulfo-SMCC and sulfo-SMPB, (4-vinylsulfone) benzoate)), but are not limited to these conjugates.

Pharmaceutical preparation

A pharmaceutical formulation of an anti-ETBR antibody as described herein may be prepared by mixing the antibody having the desired degree of purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ) In the form of a lyophilized preparation or an aqueous solution. Pharmaceutically acceptable carriers are nontoxic to the recipient at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; Antioxidants such as ascorbic acid and methionine; A preservative such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexane 3-pentanol; and m-cresol); Low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; Monosaccharides, disaccharides and other carbohydrates such as glucose, mannose or dextrin; 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 polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 HYLENEX (R), Baxter International, Inc.). Specific illustrative sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase.

Exemplary lyophilized antibody preparations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent Nos. 6,171,586 and WO2006 / 044908, and the latter formulations include histidine-acetate buffer.

In addition, the formulations herein may contain more than one active ingredient required for the particular indication being treated, preferably those that have a complementary activity that does not deleteriously affect each other. For example, it may be desirable to additionally provide BRAF inhibitors, MEK inhibitors or anti-CTLA-4 antibodies, eicilimumab. These active ingredients are suitably present in combination in amounts effective for their intended purpose.

The active ingredient may be in the form of, for example, microcapsules prepared by coacervation techniques or interfacial polymerization in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) For example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively. Such techniques are described in Remington ' s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of shaped articles, such as films or microcapsules.

Preparations used for in vivo administration are generally sterilized. Sterilization can be easily accomplished, for example, by filtration through a sterile filtration membrane.

Therapeutic methods and compositions

Any of the anti-ETBR antibodies provided herein can be used in therapeutic methods.

In one aspect, an anti-ETBR antibody is provided for use as a medicament. In yet another aspect, the method provides an anti-ETBR antibody in combination with a pharmaceutically useful BRAF inhibitor. In a further aspect, this combination is useful for treating melanoma and / or metastatic melanoma. In certain embodiments, an anti-ETBR antibody in combination with a BRAF inhibitor for use in a therapeutic method is provided. In certain embodiments, the invention provides a method of treating an individual having melanoma and / or metastatic melanoma with an effective amount of an anti-ETBR antibody and an effective amount of a BRAF inhibitor, Lt; / RTI > antibody for use in a method for the treatment of cancer. In one such embodiment, the method further comprises administering to the subject in combination, for example, an effective amount of one or more additional therapeutic agents as described below, as described. In a further embodiment, the invention provides an anti-ETBR antibody in combination with a BRAF inhibitor for use in tumor growth inhibition (TGI). In certain embodiments, the invention provides a method of inhibiting tumor growth in a subject, comprising administering to the subject an anti-ETBR antibody in combination with a BRAF inhibitor effective to inhibit tumor growth. -ETBR < / RTI > The "subject" according to any of the above embodiments is preferably a human.

In a further aspect, the invention provides the use of an anti-ETBR antibody in combination with a BRAF inhibitor in the manufacture or manufacture of a medicament. In one embodiment, the medicament is for the treatment of melanoma and / or metastatic melanoma. In a further embodiment, the medicament is for use in a method of treating melanoma and / or metastatic melanoma, comprising administering an effective amount of a medicament to a subject having melanoma and / or metastatic melanoma. In one such embodiment, the method further comprises administering to the subject an effective amount of, for example, one or more additional therapeutic agents as described below. In a further embodiment, the medicament is for inhibiting tumor growth. In a further embodiment, the medicament is for use in a method of inhibiting tumor growth in a subject, comprising administering to the subject an amount of a medicament effective to inhibit tumor growth. An "entity" according to any of the above embodiments may be a human.

In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-ETBR antibodies provided herein, for example, in combination with a BRAF inhibitor for use in any of the above therapeutic methods. In one embodiment, the pharmaceutical agent comprises any anti-ETBR antibody provided herein in combination with a BRAF inhibitor and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical agent comprises any anti-ETBR antibody provided herein combined with a BRAF inhibitor and one or more additional therapeutic agents such as, for example, those described below.

The antibodies of the present invention may be used in therapy alone or in combination with other agents. For example, an antibody of the invention can be co-administered with one or more additional therapeutic agents. In certain non-limiting embodiments, the additional therapeutic agent is a BRAF inhibitor, a MEK inhibitor or an anti-CTLA-4 antibody, such as eicilimumab.

Such combination therapy as mentioned above includes combination administration wherein the two or more therapeutic agents are included in the same or separate agent, and individual administration, wherein administration of the antibody of the present invention comprises administration of an additional therapeutic agent and / May be performed before, concurrently with, and / or after administration. The antibodies of the present invention may also be used in combination with radiation therapy.

The antibodies (and any additional therapeutic agents such as BRAF inhibitors) of the invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and may be administered by any suitable means, ≪ / RTI > Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by injection, such as intravenous or subcutaneous injection, depending on any suitable route, for example partly whether the administration is short-term or long-term. A variety of dosing schedules are contemplated herein, including, but not limited to, single doses or multiple doses over time, bolus doses, and pulse injections. Alternatively, the BRAF inhibitor may be administered orally in tablet or capsule or liquid form.

The antibodies of the invention will be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered 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 delivery site of the agent, the method of administration, the scheduling of the administration, and other factors known to the clinician. Antibodies need not be, but are optionally formulated with one or more agents currently used to prevent or treat the disorder. 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. It is generally used by the same dosage and route of administration as described above, or by about 1 to 99% of the dosages described herein, or by any dose and route determined to be experimentally / clinically appropriate.

(For use alone or in combination with one or more other therapeutic agents) for the prevention or treatment of disease 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 prophylactic or therapeutic purposes, the prognosis, the patient's clinical history and response to the antibody, and the judgment of the clinician. 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, for example, from about 1 [mu] g / kg to 15 mg / kg (e.g., from 0.1 mg / kg to 10 mg / kg) of the antibody may be an initial candidate dose for administration to a patient. One typical daily dosage may range from about 1 [mu] g / kg to 100 mg / kg or more, depending on the factors mentioned above. In the case of repeated administrations over several days, the treatment will generally continue until the desired inhibition of the disease symptoms occurs, depending on the condition. An exemplary antibody dose will range from about 0.05 mg / kg to about 10 mg / kg. Thus, a dose of at least 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 a patient. Such a dose may be administered intermittently, e. G. Every week or every three weeks (e. G., From about 2 to about 20, or for example about 6 doses of the antibody administered to the patient) . After administration of a higher initial loading dose, one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of such therapy is readily monitored by conventional techniques and assays.

Manufactured goods

In another aspect of the invention there is provided an article of manufacture containing a substance useful for the treatment, prevention and / or diagnosis of the disorders described above. The article of manufacture comprises a container, and a label or package insert on or in combination with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags and the like. The container may be formed from a variety of materials, such as glass or plastic. The container accepts the composition itself, or accepts the composition in combination with another composition effective for the treatment, prevention and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be an intravenous infusion bag It may be a vial having a stopper that can be pierced with an injection needle). One or more active agents in the composition are the antibodies of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. Also, the article of manufacture comprises: (a) a first container containing therein a composition comprising an antibody of the invention; And (b) a second container containing therein a composition comprising an additional cytotoxic agent or other therapeutic agent. An article of manufacture in this embodiment of the invention may further comprise a package insert that indicates that the composition may be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, for example, a bacterial infusion water (BWFI), a phosphate-buffered saline solution, a Ringer's solution and a dextrose solution . This may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Figure pct00038

Figure pct00039

Example

The following are examples of the methods and compositions of the present invention. It is understood that various other embodiments may be practiced in accordance with the general description provided above.

Example 1: In vitro evaluation of specific cell death by anti-ETBR ADC

The anti-ETBR antibody-ADC candidate Hu5E9v1-ADC was found to have a relatively low ETBR copy number in the case of cell line A2058 (obtained from the American Type Culture Collection) or a high ETBR copy number in the case of the cell line UACC-257X2.2 Lt; RTI ID = 0.0 > cell line expressing < / RTI > The UACC-257X2.2 cell line is a derivative of the parental UACC-257 cell line (NCI-Frederick's cancer DCT tumor reservoir) optimized for in vivo growth. The parent UACC-257 cells were subcutaneously injected into the right flank of female NCr nude mice, one tumor was harvested, dissociated and grown in vitro to produce the UACC-257X1.2 cell line. The UACC-257X1.2 cell line was subcutaneously injected into the right flank of female NCr nude mice in an effort to improve the growth of the cell line again. Tumors from these studies were collected and again adapted to in vitro growth to generate the UACC-257X2.2 cell line. This cell line expresses high levels of ET B R when determined by flow cytometry. The relationship of receptor levels to Hu5E9v1-vc-MMAE cell death in these cell lines was evaluated as follows.

Melanoma cell lines A2058 and UACC-257X2.2 were grown in appropriate media at 37 ° C and 5% CO 2 . To evaluate the effect of Hu5E9v1-ADC on cell viability, cells were plated at 1,500 cells / well in 50 [mu] L of normal growth medium in 96-well clear-bottomed black plates. After 24 hours, an additional 50 μL of culture medium with a series of dilutions of the Hu5E9v1-ADC concentrate was added to the triplet well. After 5 days, cell viability was determined with an EnVision 2101 multi-label reader (Perkin-Elmer) using a celite-glow-emitting cell viability reagent (G7572; Promega Corporation) Respectively.

(Scatchard analysis): The affinity constant for each antibody and the number of cell surface binding sites were determined by incubating melanoma cells with a fixed concentration of 125 I-labeled < RTI ID = 0.0 > Hu5E9v1-ADC and an increasing concentration of unlabeled Hu5E9v1-ADC. Data were analyzed by nonlinear curve fit using analytical programming method.

As shown in Figures 2A and 2B, by Scatchard analysis, the number of Hu5E9v1-ADC binding sites on A2058 and UACC-257X2.2 were estimated to be 1,582 sites and 33,939 sites, respectively, per cell. Titration of these cell lines to the anti-ETBR ADC candidates showed specific cell death in association with a control ADC, which is generally proportional to the level of ETBR expression.

Example 2: In vivo evaluation of specific tumor killing by anti-ETBR ADC

Based on the studies described in Example 1 above, melanoma cell lines A2058 and UACC-257X2.2 were selected as models suitable for in vivo anti-tumor activity studies showing extensive 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 in vivo growth. Specifically, parent UACC-257 cells were subcutaneously injected into the right flank of female NCr nude mice, one tumor was harvested and grown in vitro to produce a UACC-257X1.2 cell line. The UACC-257X1.2 cell line was subcutaneously injected into the right flank of female NCr nude mice in an effort to improve the growth of the cell line again. From these studies, tumors were harvested and adapted for in vitro growth again to generate the UACC-257X2.2 cell line. These cell lines and tumors derived from these cell lines express ETBR in parallel with the parental cell line UACC-257 (data not shown).

Next, efficacy studies were performed using melanoma cell lines in the xenograft mouse model described above. All studies were performed according to guidelines for laboratory animal care and use (see: Institute of Laboratory Animal Resources (NIH publication no. 85-23), Washington, DC: National Academies Press; 1996). 10- to 14-week old female CRL Nu / Nu or NCr nude mice from Charles River Laboratories were injected with 5 X 10 6 UACC-257X2.2 cells or Matrigel in HBSS containing Matrigel Were injected subcutaneously into the right flank of the dorsal 5 x 10 6 A2058 cells. When the tumor volume reached approximately 200 mm 3 (day 0), the animals were randomly grouped into 10 groups each.

In the case of a single agent efficacy study, the anti-ETBR ADC candidate Hu5E9v1-ADC was administered as a single intravenous (IV) injection at day 0 at 1 mpk, 3 mpk or 6 mpk (mg / kg). Control ADC antibodies and vehicle controls were also administered. Mean tumor volume was determined from 10 animals per group with standard deviation. Tumor volume was measured twice a week to the study endpoint.

The results are shown in Figure 3A for high ETBR copy number UACC-257X2.2 cell line and in 3B for low ETBR copy number cell line A2058. Consistent with the in vitro cell death experiments described in Example 1, UACC-257X2.2 xenograft tumors were more reactive to the Hu5E9v1-ADC. Although the efficacy was not apparent in the 1 mg / kg dose group, sustained tumor regression was observed in response to a single dose of 3 and 6 mg / kg Hu5E9v1-ADC (Fig. 3A).

3 and 6 mg / kg Hu5E9v1-ADC, 6 mg / kg control ADC or vehicle control were administered to animals bearing low ETBR copy number A2058 tumors. A partial reduction in tumor burden was observed in a high dose Hu5E9v1-ADC of 6 mpk compared to a corresponding dose of control ADC or vehicle. The efficacy was not evident in the group receiving the Hu5E9v1-ADC at 3 mg / kg. Thus, a reduction in tumor burden (Asundi et al., 2011) in the A2058 xenograft model showing the lower end of the spectrum of ETBR expression in human melanoma (Asundi et al., 2011) Suggesting that efficacy can be achieved with the candidate Hu5E9v1-ADC as a single agonist.

Example 3: Effect of BRAF inhibitor drugs on the expression level of ETBR

The effect of BRAF inhibitor drugs on the expression levels of ETBR transcripts and proteins (total protein and cell surface protein) was tested against a variety of gene backgrounds of melanomas, such as mutants for BRAF (V600E), wild-type and RAS (Q61L) for BRAF, Lt; RTI ID = 0.0 > melanoma cells. ≪ / RTI >

("BRAFi"), especially RG7204, at various concentrations for 24 hours on a 4-well plate were cultured for 24 hours in a culture medium containing the appropriate concentrations of the melanoma cell lines UACC-257X2.2, A2058, COLO 829, IPC- Lt; RTI ID = 0.0 > vol. ≪ / RTI >

To determine the effect of BRAFi on ETBR and control ribosomal protein L19 (RPL19) transcript levels, the following experiments were performed. Cells treated with RG7204 for 24 hours were harvested from the plates by scraping and processed for total RNA using Qiashredder and RNeasy mini kits (79654, 74104, Qiagen, Valencia, Calif.) Respectively. Taqman assays were established using reagents from Applied Biosystems (ABI, Foster City, CA) and assayed using 7500 real-time PCR instrument and software from ABI. The primer-probe set was designed as a primer flanking a double-labeled fluorescent probe with a reporter dye FAM and a quencher dye TAMRA.

The primer-probe set for RPL19 is as follows:

Forward primer-5 'AGC GGA TTC TCA TGG AAC A (SEQ ID NO: 11); 5 'CTG GTC AGC CAG GAG CTT (SEQ ID NO: 12) and probe 5' TCC ACA AGC TGA AGG CAG ACA AGG (SEQ ID NO: 13).

The primer-probe set for ETBR is as follows:

5'TCA CTG AAT TCC TGC ATT AAC C (SEQ ID NO: 14), reverse primer-5'GCA TAA GCA TGA CTT AAA GCA GTT (SEQ ID NO: 15) and probe-5 'AAT TGC TCT GTA TTT GGT GAG CAA AAG ATT CAA (SEQ ID NO: 16).

Results for UACC-257X2.2 are shown in Figure 4A, results for A2058 are shown in Figure 8A, and results for COLO 829 are shown in Figure 6A. These results demonstrate that treatment with BRAFi RG7204 for 24 hours was shown to increase ETBR transcripts in all cell lines tested compared to control cells without BRAFi RG7204.

Western blot experiments were performed on the same cell line treated with BRAFi RG7204 as described above to test whether the increase in ETBR transcripts due to BRAFi treatment also caused any changes in ETBR total protein levels. In the Western blotting, the following reagents were used: in the case of the detection of the protein: anti-EBR in-house generated monoclonal antibody 1H1.8.5, anti-phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 (Cell signaling technology), anti-p44 / 42 MAPK (Erk1 / 2) antibody (9102, cell signaling technology), and as a control, rabbit polyclonal anti-GAPDH (PA1-987; Affinity Bioreagents) and mouse monoclonal anti-β-tubulin antibody (556321, BD Farmingen). The results for UACC-257X2.2 are shown in FIG. 4B, the results for A2058 are shown in FIG. 8B, the results for COLO 829 are shown in FIG. 6B, and the results for IPC-298 are shown in FIG. These results demonstrate that treatment with BRAFi RG7204 for 24 hours resulted in increased total protein levels of ETBR in the UACC-257X2.2, A2058 and COLO 829 cell lines compared to control cells without BRAFi. However, with respect to the wild-type and BRAF mutant cell line IPC-298 for RAS (Q61L), BRAFi did not appear to increase ETBR levels over a range of BRAFi dose levels, Activated the level of POR-ERK.

Fluorescence-activated cell sorting (FACS) analysis was performed to determine whether the observed increase in total ET B R protein levels due to BRAFi treatment also resulted in an increase in ET B R surface protein levels. Cells were harvested in PBS containing 2.5 mmol / L EDTA and washed in PBS buffer containing 1% FBS. All subsequent steps were carried out at 4 ° C. Cells were incubated with anti-human IgG fluorescence detection reagent (A11013; Invitrogen) following 3 μg / mL anti-ET B B antibody Hu5E9v1 for 1 hour. Cells were then analyzed with a FACS Calibur flow cytometer (BD Biosciences). The results for UACC-257X2.2 are shown in Figure 4C, the results for A2058 are shown in Figure 8C, and the results for COLO 829 are shown in Figure 6C. These results demonstrate that treatment with BRAFi RG7204 for 24 hours has increased surface levels of ET B R protein expressed in all cell lines tested compared to control cells without BRAFi. However, with respect to cell line IPC-298, BRAFi RG7204 was shown to reduce ETBR levels at all dose levels tested, as shown in FIG. 11A-C.

Example 4: Effect of BRAF inhibitor drugs on the in vivo efficacy of anti-ETBR ADC

In view of the results demonstrated in Example 3 above, the effect of the BRAF inhibitor drug on the in vivo efficacy of anti-ET B R ADC was tested. To do this, the in vivo potency against various combinations of Hu5E9v1-ADC and BRAFi-945 was evaluated for the UACC-257X2.2 melanoma model described above. Tumors were grown to an average size of approximately 200 mm 3 , at which time the animals were randomly grouped into 10 groups. A suitable vehicle control (Klucel LF) or BRAFi-945 at doses of 1, 6, or 20 mpk was administered orally once daily x 21 days starting on study day 0. A single 1 mpk or 3 mpk dose of the Hu5E9v1-ADC or the control, histidine Buffer # 8 was administered intravenously (after 2 doses of 945) via the tail vein on day 1 of the study.

The average tumor volume was determined from 10 animals per group. Tumor volume was measured twice a week to the study endpoint. Tumor volumes were measured in two dimensions (length and width) using an UltraCal IV caliper (Model 54 10 111, Fred V. Fowler Company, Newton, Mass.). Tumor volume was calculated using the following equation: Excel (version 12.2.8, Microsoft, Redmond, Wash.): Tumor volume (mm 3) = (length x width 2 ) x 0.5

A mixed-modeling linear blending effect (LME) approach was used to analyze repeated measures of tumor volume from the same animal over time (Pinheiro et al. 2009). This approach can handle both repeated measures and intermediate dropout rates due to non-treatment-related closure of animals throughout the study endpoint. Linear regression spline was used to fit the non-linear profile to the log 2 tumor volumes over time at each dose level. This non-linear profile was then associated with capacity in the mixing model. The following equation can be used to calculate tumor growth inhibition (TGI) as a percentage of vehicle as percent area under curve (AUC) fitted to the vehicle on day 1.

Figure pct00040

Using this formula, a TGI value of 100% indicates tumor stasis,> 1% but <100% indicates tumor growth delay and> 100% indicates tumor degeneration. To determine the Uncertain Interval (UI) for% TGI, a fitted sample was generated as an approximation to the distribution of% TGI using fitted curves and a fitted covariance matrix. A random sample consists of 1000 simulated implementations of a fitted-mixed model, where% TGI is recalculated for each implementation. Here, the recorded UI is a value for 95% of the time, and the recomputed value of% TGI will be included in this area given to the fitted model. The 2.5 and 97.5 percentiles of the mock distribution were used as upper and lower UIs.

The results are shown in Figures 5A, 5B, 5C, 5D and 5E. All combinations of Hu5E9v1-ADC and BRAFi-945 demonstrated superior efficacy over drugs as single agent alone. The two drugs were combined at the lowest tested level to provide combined potency almost indistinguishable from that achieved at the highest dose tested.

Example 5: Capacity testing of in vivo anti-ETBR ADC and BRAFi combinations in COLO 829 xenografts

The study described in this example allowed the refinement of the assessment of the in vivo combinatorial efficacy of the Hu5E9v1-ADC with the BRAF inhibitor drug RG7204. The antagonism between the drugs was preceded by the loss of the antagonism, and hence the combination efficacy of the drug was tested at a lower dose. The COLO 829 xenograft model was selected as representative of the media level of ET B R expression and further increased the rigor of the combination studies. Tumors were grown to an average size of approximately 200 mm 3 , at which time the animals were randomly grouped into nine groups. A suitable vehicle control (Clucel LF) or G00044364.1-12 (RG7204) was orally administered at a dose of 10 mpk or 30 mpk starting on day 0 and twice daily for 21 days. A single dose of Hu5E9v1-ADC of 1 mpk or 3 mpk or control, histidine Buffer # 8 was administered intravenously (after 3 doses of RG7204) on day 1. The results are shown in Figs. 7A, 7B, 7C and 7D.

Both mid-dose dosing drugs (30 mg / kg RG7204 and Hu5E9v1-ADC at 3 mg / kg) were combined together to provide greater combined efficacy than drug alone. Other combinations of the smaller doses of the two drugs showed similar tendencies except for the lowest dose tested in the combination.

Example 6: Capacity testing of in vivo anti-ETBR ADC and BRAFi combinations in A2058 xenografts

The efficacy of the Hu5E9v1-ADC and RG7204 in the A2058 xenograft model was tested. This model was of particular interest because of the high level of rigidity it represents. The A2058 xenograft model represents the lower end of the ETBR expression spectrum found in melanoma patients, thereby becoming a challenging model to achieve anti-ETBR ADC efficacy. In addition, despite his BRAF V600E mutation status, this model was demonstrated to be non-reactive to RG7204 with an in vitro kill efficacy of > 20 μM (data not shown).

Tumors were grown to an average size of approximately 200 mm 3 , at which time the animals were randomly grouped into 10 groups. A suitable vehicle control (Clucel LF) or RG7204 was orally administered at doses of 10 mpk or 30 mpk starting on day 0 and twice daily for 21 days. A single dose of Hu5E9v1-ADC of 3 mpk or 6 mpk or control, histidine Buffer # 8 was administered intravenously (after 3 doses of RG7204) via tail vein on day 1. The results are shown in Figs. 9A, 9B, 9C and 9D.

The results show that, in all cases tested, the combination of Hu5E9v1-ADC and RG7204 demonstrated greater efficacy than any single agent alone. RG7204 alone at a dose of 10 mg / kg did not show a single agonist efficacy against the A2058 model (see Figures 9A and 9C). However, when combined with a Hu5E9v1-ADC at a dose of 6 mg / kg, superior efficacy was achieved over agonist alone (see Figures 9A and 9C). The combination of the 10 mg / kg RG7204 with the Hu5E9v1-ADC at 6 mg / kg (Fig. 9A) is almost indistinguishable from the highest dose level tested, i.e. the combination efficacy achieved with the 30 mpk RG7204 and 6 mpk Hu5E9v1-ADC shown in Fig. 9B Lt; / RTI &gt; combination efficacy.

Table 3 summarizes the three melanoma xenograft models tested at varying doses as described above to compare the percent TGI of an anti-ETBR ADC as a single agonist or the percent TGI of a BRAF inhibitor as a single agonist, As the percent delta (last column) in combination with the BRAF inhibitor. Percent TGI was calculated using a linear mixing effect (LME) modeling approach as described above.

Example 7 Effect of MEK Inhibitor Drugs on the Level of Expression of ETBR

(BRAF V600E ), A2058 (BRAF V600E ), SK23-B, and BRAF V600E , as well as the effect of MEK inhibitor drugs on the expression levels of ETBR transcripts and proteins (total protein and cell surface protein) MEL (BRAF WT / RAS WT ) or IPC-298 (BRAF WT / RAS C61L ).

MEK inhibitor drugs ("MEKi-973" or "MEKi-623") at varying concentrations (0 μM, 0.01 μM, 0.1 μM or 1 μM) were administered to melanoma cell lines A2058, COLO 829, SK23-MEL and IPC- ") By adding the appropriate drug volume to the cells in the culture for 24 hours on a 4-well plate.

To determine the effect of MEKi on the ETBR transcript level, the following experiment was performed as described above in Example 3. Cells treated with MEKi-973 for 24 hours were harvested from the plates by scraping and treated with quiesced leather and RNeasy mini kits (79654, 74104, Qiagen, Valencia, Calif.) For total RNA. The taxane assay was established using reagents from Applied Biosystems (ABI, Foster City, Calif.) And assayed using 7500 real-time PCR instrument and software from ABI. The primer-probe set was designed as a primer flanking a double-labeled fluorescent probe with a reporter dye FAM and a quencher dye TAMRA.

The primer-probe set for RPL19 is as follows:

Forward primer-5 'AGC GGA TTC TCA TGG AAC A (SEQ ID NO: 11); 5 'CTG GTC AGC CAG GAG CTT (SEQ ID NO: 12) and probe 5' TCC ACA AGC TGA AGG CAG ACA AGG (SEQ ID NO: 13).

The primer-probe set for ETBR is as follows:

5'TCA CTG AAT TCC TGC ATT AAC C (SEQ ID NO: 14), reverse primer-5'GCA TAA GCA TGA CTT AAA GCA GTT (SEQ ID NO: 15) and probe-5 'AAT TGC TCT GTA TTT GGT GAG CAA AAG ATT CAA (SEQ ID NO: 16).

The results for A2058 are shown in Figure 14A (treated with indicated dose of MEKi-623) and 14B (treated with indicated dose of MEKi-973). These results demonstrate that treatment with MEK inhibitor for 24 hours has increased ETBR transcripts compared to control cells without MEK inhibitor.

To test whether the increase in ETBR transcript due to MEKi treatment also caused any change in the total protein level of the ETBR, Western blot experiments were performed with the cell line COLO829 (BRAF V600E ) treated with MEKi-973 as described above, A2058 (BRAF V600E ), SK23-MEL (BRAF WT / RAS WT ) or IPC-298 (BRAF WT / RAS C61L ). In Western blotting, the following reagents were used: in the case of protein detection: anti-EBR-in-house generated monoclonal antibody 1H1.8.5, anti-phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / GAPDH (glyceraldehyde-3-phosphate) as a control and anti-p44 / 42 MAPK (Erk1 / 2) antibody (9102, cell signaling technology) (PA1-987; Affinity Bioreergens) and mouse monoclonal anti-beta-tubulin antibody (556321, BD Farming). The results for A2058 are shown in FIG. 15A (treated with MEKi-623) and 15B (treated with MEKi-973) and the results for COLO 829 are shown in FIG. 12A (treated with MEKi-623) (Treated with MEKi-623) and 19B (treated with MEKi-973), and results for IPC-298 are shown in Fig. 22A (treated with MEKi-623) Treated with MEKi-973). These results demonstrate that treatment with MEKi-623 or MEKi-973 for 24 h has increased total protein levels of ETBR in all cell lines tested compared to control cells without MEKi.

Fluorescence-activated cell sorting (FACS) assays were performed as described above to determine whether the observed increase in total ET B R protein levels due to MEKi treatment also resulted in an increase in ET B R surface protein levels . Cells were harvested in PBS supplemented with 2.5 mmol / L EDTA and washed in PBS containing 1% FBS. All subsequent steps were carried out at 4 ° C. Cells were incubated with anti-human IgG fluorescence detection reagent (A11013; Invitrogen) following 3 μg / mL anti-ET B B R antibody Hu5E9v1 for 1 hour. The cells were then analyzed with a FACS caliber flow cytometer (BD Biosciences). The results for A2058 are shown in Figs. 16A-F, the results for COLO829 are shown in Figs. 13A-F, the results for SK23-MEL are shown in Figs. 20A-F and the results for IPC-298 are shown in Figs. 23A-F . These results demonstrate that treatment with MEKi-623 or MEKi-973 for 24 h has increased surface levels of ET B R protein expressed in all cell lines tested compared to control cells without MEK inhibitor do.

Example 8: Effect of MEK inhibitor drugs on the in vivo efficacy of anti-ETBR ADC

Taking into account the results demonstrated in Example 7 above, the effect of the MEK inhibitors described herein on the in vivo efficacy of anti-ET B R ADC was tested. In order to accomplish this, the in vivo efficacy against various combinations of Hu5E9v1-ADC and MEKi-623 and / or MEKi-973 was compared with that of A2058 and SK-MEL-23 and IPC-298 melanoma The in vivo model was evaluated. The appropriate methylcellulose twin-vehicle control (0.5% methylcellulose, 0.2% tween-80 (MCT) or MEK inhibitor at 1 mpk, 3 mpk or 7.5 mpk doses, starting on study day 0, A single 3 mpk or 6 mpk dose of Hu5E9v1-ADC or control, histidine buffer # 8 was administered intravenously (after two doses of MEK inhibitor) via tail vein on day 1 of the study.

The results are shown in Fig. 17A-B, Fig. 21, Fig. 24 and Fig. Surprisingly, all combinations of tested Hu5E9v1-ADC and MEK inhibitors demonstrated greater efficacy than the additional potency of the drug as a single agent alone.

Example 9: PD studies of A2058 and COLO 829 melanoma xenografts

The tumors collected at the end of the study shown in Figure 7 (COLO 829 versus the combination anti-ETBR-ADC and BRAFi RG7204) did not show an increase in ETBR. This may be due to the fact that the time point of tumor collection (day 34) has passed well through the wash period of the BRAFi drug administered. Whether the in vitro effects of BRAFi / MEKi on the cell line (i. E., Increase in ETBR and decrease in Perk) also occur in vivo, thus allowing a much greater efficacy of the anti-ETBR ADC and BRAFi / MEKi in the combination To evaluate, the following experiment was performed.

A2058 or COLO 829 tumors were grown to an average size of approximately 200 mm 3 , in which animals were randomly grouped into 5-6 groups. In the case of the BRAFi PD study, the appropriate vehicle control (Clucel) LF) or RG7204 was dosed twice daily for three days at a dose of 10 mpk or 30 mpk (Fig. 27A). For the MEKi PD study, the appropriate vehicle control or MEKi-973 was orally administered once daily for three days at a dose of 5 mpk and 10 mpk (FIG. 27B). Rapidly frozen tumors harvested at the end of the study were homogenized and processed for RNA and / or protein. The taxane assay was established using reagents from Applied Biosystems (ABI, Foster City, Calif.) And assayed using 7500 real-time PCR instrument and software from ABI. The primer-probe set was designed as a primer flanking a double-labeled fluorescent probe with a reporter dye FAM and a quencher dye TAMRA. The ETBR transcript levels in tumors are specific for the human homologues of these genes for transcript levels of the reference genes, such as Hprt1 (hypoxanthine phospholiposyltransferase 1) or GAPDH (glyceraldehyde 3 phosphate dehydrogenase) Lt; / RTI &gt; primer and probe set.

The primer-probe set for the reference gene Hprt1 (hypoxanthine phosphoribosyl transferase 1) is as follows:

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:

5'TCA CTG AAT TCC TGC ATT AAC C (SEQ ID NO: 14), reverse primer-5'GCA TAA GCA TGA CTT AAA GCA GTT (SEQ ID NO: 15) and probe-5 'AAT TGC TCT GTA TTT GGT GAG CAA AAG ATT CAA (SEQ ID NO: 16).

The primer-probe set for the reference gene GAPDH (glyceraldehyde 3 phosphate dehydrogenase) is as follows:

5 'GAA GAT GGG GAT GGG ATT TC (SEQ ID NO: 20), reverse primer -5' GAA GGT GAA GGT CGG AGT C (SEQ ID NO: 21), and probe 5 'CAA GCT TCC CGT TCT CAG CC .

Figure 27A shows that BRAFi induces ETBR mRNA in vivo compared to a control vehicle. Figure 27B shows that MEKi-973 also induces ETBR mRNA in vivo compared to a control vehicle.

Phosphorylated erk and total erk protein levels were assessed by Western blot in tumors using the following reagents: For detection of protein: anti-phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (Fig. 26) as anti-p44 / 42 MAPK (Erk1 / 2) antibody (9102, cell signaling technology) and as a control mouse monoclonal anti-beta-tubulin antibody (556321, BD Pharmingen) . Here, BRAFi appears to inhibit Perk in vivo compared to the control.

Figure pct00041

While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it should be understood that the description and examples are not intended to limit the scope of the invention. The disclosures of all patents and scientific publications cited herein are expressly incorporated by reference in their entirety.

                              SEQUENCE LISTING <110> GENENTECH, INC. ET AL. <120> THERAPEUTIC COMBINATIONS AND METHODS OF TREATING MELANOMA <130> P4777R1-WO <141> &Lt; 150 > US 61 / 552,893 <151> 2011-10-28 &Lt; 150 > US 61 / 678,978 <151> 2012-08-02 <160> 22 <210> 1 <211> 10 <212> PRT <213> Mus musculus <400> 1  Gly Tyr Thr Phe Thr Ser Tyr Trp Met Gln                    5 10 <210> 2 <211> 17 <212> PRT <213> Mus musculus <400> 2  Thr Ile Tyr Pro Gly Asp Gly Asp Thr Ser Tyr Ala Gln Lys Phe    1 5 10 15  Lys Gly          <210> 3 <211> 9 <212> PRT <213> Mus musculus <400> 3  Trp Gly Tyr Ala Tyr Asp Ile Asp Asn                    5 <210> 4 <211> 16 <212> PRT <213> Mus musculus <400> 4  Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu    1 5 10 15  Asn      <210> 5 <211> 7 <212> PRT <213> Mus musculus <400> 5  Leu Val Ser Lys Leu Asp Ser                    5 <210> 6 <211> 9 <212> PRT <213> Mus musculus <400> 6  Trp Gln Gly Thr His Phe Pro Tyr Thr                    5 <210> 7 <211> 109 <212> PRT <213> Artificial sequence <220> <223> humanized variable heavy chain <400> 7  Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly    1 5 10 15  Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr                   20 25 30  Ser Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu                   35 40 45  Glu Trp Ile Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Ser Tyr                   50 55 60  Ala Gln Lys Phe Lys Gly Arg Ala Thr Leu Ser Thr Asp Lys Ser                   65 70 75  Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                   80 85 90  Thr Ala Val Tyr Tyr Cys Ala Arg Trp Gly Tyr Ala Tyr Asp Ile                   95 100 105  Asp Asn Trp Gly                  <210> 8 <211> 112 <212> PRT <213> Artificial sequence <220> <223> humanized variable light chain <400> 8  Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Val    1 5 10 15  Gly Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu                   20 25 30  Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Lys Pro                   35 40 45  Gly Lys Ala Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp                   50 55 60  Ser Gly Val Ser Ser Arg Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser                   65 70 75  Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr                   80 85 90  Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Tyr Thr Phe Gly Gln                   95 100 105  Gly Thr Lys Val Glu Ile Lys                  110 <210> 9 <211> 109 <212> PRT <213> Artificial sequence <220> <223> humanized variable heavy chain. <400> 9  Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly    1 5 10 15  Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr                   20 25 30  Ser Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu                   35 40 45  Glu Trp Ile Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Ser Tyr                   50 55 60  Ala Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser                   65 70 75  Thr Ser Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp                   80 85 90  Thr Ala Val Tyr Tyr Cys Ala Arg Trp Gly Tyr Ala Tyr Asp Ile                   95 100 105  Asp Asn Trp Gly                  <210> 10 <211> 442 <212> PRT <213> Homo sapiens <400> 10  Met Gln Pro Pro Pro Ser Leu Cys Gly Arg Ala Leu Val Ala Leu    1 5 10 15  Val Leu Ala Cys Gly Leu Ser Arg Ile Trp Gly Glu Glu Arg Gly                   20 25 30  Phe Pro Pro Asp Arg Ala Thr Pro Leu Leu Gln Thr Ala Glu Ile                   35 40 45  Met Thr Pro Pro Thr Lys Thr Leu Trp Pro Lys Gly Ser Asn Ala                   50 55 60  Ser Leu Ala Arg Ser Leu Ala Pro Ala Glu Val Pro Lys Gly Asp                   65 70 75  Arg Thr Ala Gly Ser Pro Pro Arg Thr Ile Ser Pro Pro Pro Cys                   80 85 90  Gln Gly Pro Ile Glu Ile Lys Glu Thr Phe Lys Tyr Ile Asn Thr                   95 100 105  Val Val Ser Cys Leu Val Phe Val Leu Gly Ile Ile Gly Asn Ser                  110 115 120  Thr Leu Leu Arg Ile Ile Tyr Lys Asn Lys Cys Met Arg Asn Gly                  125 130 135  Pro Asn Ile Leu Ile Ala Leu Ala Leu Gly Asp Leu Leu His                  140 145 150  Ile Val Ile Asp Ile Pro Ile Asn Val Tyr Lys Leu                  155 160 165  Asp Trp Pro Phe Gly Ala Glu Met Cys Lys Leu Val Pro Phe Ile                  170 175 180  Gln Lys Ala Ser Val Gly Ile Thr Val Leu Ser Leu Cys Ala Leu                  185 190 195  Ser Ile Asp Arg Tyr Arg Ala Val Ala Ser Trp Ser Arg Ile Lys                  200 205 210  Gly Ile Gly Val Lys Trp Thr Ala Val Glu Ile Val Leu Ile                  215 220 225  Trp Val Val Ser Val Val Leu Ala Val Pro Glu Ala Ile Gly Phe                  230 235 240  Asp Ile Ile Thr Met Asp Tyr Lys Gly Ser Tyr Leu Arg Ile Cys                  245 250 255  Leu Leu His Pro Val Gln Lys Thr Ala Phe Met Gln Phe Tyr Lys                  260 265 270  Thr Ala Lys Asp Trp Trp Leu Phe Ser Phe Tyr Phe Cys Leu Pro                  275 280 285  Leu Ala Ile Thr Ala Phe Phe Tyr Thr Leu Met Thr Cys Glu Met                  290 295 300  Leu Arg Lys Lys Ser Gly Met Gln Ile Ala Leu Asn Asp His Leu                  305 310 315  Lys Gln Arg Arg Glu Val Ala Lys Thr Val Phe Cys Leu Val Leu                  320 325 330  Val Phe Ala Leu Cys Trp Leu Pro Leu His Leu Ser Arg Ile Leu                  335 340 345  Lys Leu Thr Leu Tyr Asn Gln Asn Asp Pro Asn Arg Cys Glu Leu                  350 355 360  Leu Ser Phe Leu Leu Val Leu Asp Tyr Ile Gly Ile Asn Met Ala                  365 370 375  Ser Leu Asn Ser Cys Ile Asn Pro Ile Ala Leu Tyr Leu Val Ser                  380 385 390  Lys Arg Phe Lys Asn Cys Phe Lys Ser Cys Leu Cys Cys Trp Cys                  395 400 405  Gln Ser Phe Glu Glu Lys Gln Ser Leu Glu Glu Lys Gln Ser Cys                  410 415 420  Leu Lys Phe Lys Ala Asn Asp His Gly Tyr Asp Asn Phe Arg Ser                  425 430 435  Ser Asn Lys Tyr Ser Ser Ser                  440 <210> 11 <211> 19 <212> DNA <213> Homo sapiens <400> 11  agcggattct catggaaca 19 <210> 12 <211> 18 <212> DNA <213> Homo sapiens <400> 12  ctggtcagcc aggagctt 18 <210> 13 <211> 24 <212> DNA <213> Homo sapiens <400> 13  tccacaagct gaaggcagac aagg 24 <210> 14 <211> 22 <212> DNA <213> Homo sapiens <400> 14  tcactgaatt cctgcattaa cc 22 <210> 15 <211> 24 <212> DNA <213> Homo sapiens <400> 15  gcataagcat gacttaaagc agtt 24 <210> 16 <211> 33 <212> DNA <213> Homo sapiens <400> 16  aattgctctg tatttggtga gcaaaagatt caa 33 <210> 17 <211> 24 <212> DNA <213> Homo sapiens <400> 17  cacatcaaag acagcatcta agaa 24 <210> 18 <211> 25 <212> DNA <213> Homo sapiens <400> 18  caagttggaa aatacagtca acatt 25 <210> 19 <211> 37 <212> DNA <213> Homo sapiens <400> 19  ttttgttctg tcctggaatt attttagtag tgtttca 37 <210> 20 <211> 20 <212> DNA <213> Homo sapiens <400> 20  gaagatggtg atgggatttc 20 <210> 21 <211> 19 <212> DNA <213> Homo sapiens <400> 21  gaaggtgaag gtcggagtc 19 <210> 22 <211> 20 <212> DNA <213> Homo sapiens <400> 22  caagcttccc gttctcagcc 20

Claims (77)

  1. A method of inhibiting tumor growth (TGI) in a subject suffering from a melanoma, comprising administering to a subject suffering from a melanoma an effective amount of an anti-endothelin B receptor (ETBR) antibody in combination with an effective amount of a MAP kinase inhibitor .
  2. 2. The method of claim 1, wherein the combination is synergistic.
  3. 2. The method of claim 1, wherein the TGI is greater than the TGI exhibited using the anti-ETBR antibody alone.
  4. 2. The method of claim 1, wherein the TGI is greater than the TGI exhibited using the MAP kinase inhibitor alone.
  5. The method of claim 3, wherein the TGI is greater than about 10% greater, or greater than about 15% greater, or greater than about 20% greater, or greater than about 25% greater, or greater than about 30% greater than the sole use of anti- , Greater than 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.
  6. The method of claim 4, wherein the TGI is greater than about 10% greater, or greater than about 15% greater, or greater than about 20% greater, or greater than about 25% greater, or greater than about 30% greater than the sole use of a MAP kinase inhibitor. 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.
  7. 2. The method of claim 1, wherein said anti-ETBR antibody specifically binds to an ETBR epitope consisting of amino acids 64-101 of SEQ ID NO: 10.
  8. The method of claim 1, wherein the anti-ETBR antibody has three variable heavy CDRs and three variable light CDRs, wherein the VH CDR1 is SEQ ID NO: 1, the VH CDR2 is SEQ ID NO: 2, the VH CDR3 is SEQ ID NO: 3, Wherein the VL CDR2 is SEQ ID NO: 5 and the VL CDR3 is SEQ ID NO: 6.
  9. The method of claim 1, wherein the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein the VH is SEQ ID NO: 7 or SEQ ID NO: 9.
  10. 10. The method of claim 9, wherein the VL is SEQ ID NO: 8.
  11. 2. The method of claim 1, wherein the anti-ETBR antibody is conjugated to a cytotoxin.
  12. 12. The method of claim 11, wherein the cytotoxin is a cytotoxic agent selected from the group consisting of a toxin, an antibiotic, a radioactive isotope, and a nucleic acid degrading enzyme.
  13. 13. The method of claim 12, wherein the cytotoxin is a toxin.
  14. 14. The method of claim 13, wherein said toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin.
  15. 15. The method of claim 14, wherein the toxin is a maytansinoid.
  16. 2. The method of claim 1 wherein the MAP kinase inhibitor is a BRAF inhibitor.
  17. The method of claim 16, wherein the BRAF inhibitor is propane-1-sulfonic acid {3- [5- (4-chlorophenyl) -1H- pyrrolo [2,3- b] pyridine- -Difluoro-phenyl} -amide. &Lt; / RTI &gt;
  18. 17. The method of claim 16, wherein the BRAF inhibitor has the following chemical structure.
    Figure pct00042
  19. 2. The method of claim 1 wherein the MAP kinase inhibitor is a MEK inhibitor.
  20. 20. The method of claim 19, wherein the MEK inhibitor is selected from the group consisting of (S) - (3,4-difluoro-2 - ((2-fluoro- (Piperidin-2yl) azetidin-1-yl) methanone.
  21. 20. The method of claim 19, wherein the MEK inhibitor has the following chemical structure.
    Figure pct00043
  22. A method of treating a melanoma, comprising administering to a subject in need thereof a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody.
  23. 23. The method of claim 22, wherein the combination is synergistic.
  24. 23. The method of claim 22, wherein the TGI is greater than the TGI exhibited using the anti-ETBR antibody alone.
  25. 23. The method of claim 22, wherein the TGI is greater than the TGI exhibited using the MAP kinase inhibitor alone.
  26. 26. The method of claim 24, wherein the TGI is greater than about 10% greater, or greater than about 15% greater, or greater than about 20% greater, or greater than about 25% greater, or greater than about 30% greater than the sole use of anti- , Greater than 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.
  27. 26. The method of claim 25, wherein the TGI is greater than about 10% greater, or greater than about 15% greater, or greater than about 20% greater, or greater than about 25% greater, or greater than about 30% greater than the sole use of a MAP kinase inhibitor. 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.
  28. 23. The method of claim 22, wherein said anti-ETBR antibody specifically binds to an ETBR epitope consisting of amino acid numbers 64-101 of SEQ ID NO: 10.
  29. 23. The method of claim 22, wherein the anti-ETBR antibody has three variable heavy CDRs and three variable light CDRs, wherein the VH CDR1 is SEQ ID NO: 1, the VH CDR2 is SEQ ID NO: 2, the VH CDR3 is SEQ ID NO: 3, Wherein the VL CDR2 is SEQ ID NO: 5 and the VL CDR3 is SEQ ID NO: 6.
  30. 23. The method of claim 22, wherein the anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein the VH is sequence 7 or 9.
  31. 31. The method of claim 30, wherein said VL is sequence 8.
  32. 23. The method of claim 22, wherein the anti-ETBR antibody is conjugated to a cytotoxin.
  33. 33. The method of claim 32, wherein the cytotoxin is a cytotoxic agent selected from the group consisting of a toxin, an antibiotic, a radioactive isotope, and a nucleic acid degrading enzyme.
  34. 34. The method of claim 33, wherein said cytotoxin is a toxin.
  35. 35. The method of claim 34, wherein said toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin.
  36. 36. The method of claim 35, wherein said toxin is a maytansinoid.
  37. 23. The method of claim 22, wherein said MAP kinase inhibitor is a BRAF inhibitor.
  38. 37. The method of claim 37, wherein the BRAF inhibitor is propane-1-sulfonic acid {3- [5- (4- chlorophenyl) -lH- pyrrolo [2,3- b] pyridine- 3- carbonyl] -Difluoro-phenyl} -amide. &Lt; / RTI &gt;
  39. 38. The method of claim 37, wherein the BRAF inhibitor has the following chemical structure.
    Figure pct00044
  40. 23. The method of claim 22, wherein the MAP kinase inhibitor is a MEK inhibitor.
  41. 41. The method of claim 40, wherein the MEK inhibitor is selected from the group consisting of (S) - (3,4-difluoro-2 - ((2-fluoro- (Piperidin-2yl) azetidin-1-yl) methanone.
  42. 41. The method of claim 40, wherein the MEK inhibitor has the following chemical structure.
    Figure pct00045
  43. 23. The method of claim 22, wherein the black paper is ETBR positive.
  44. 23. The method of claim 22, wherein the black paper is metastable.
  45. 23. The method of claim 22, wherein said subject has not received prior therapy with a MAP kinase inhibitor.
  46. 23. The method of claim 22, wherein the subject has a V600E BRAF gene mutation.
  47. 23. The method of claim 22, wherein said subject is V600E wild type.
  48. 23. The method of claim 22, wherein said MAP kinase inhibitor is first administered to said subject in need thereof.
  49. 23. The method of claim 22, wherein the anti-ETBR antibody is administered after administration of the MAP kinase inhibitor.
  50. 23. The method of claim 22, wherein the anti-ETBR antibody and the MAP kinase inhibitor are administered simultaneously.
  51. 23. The method of claim 22, wherein the anti-ETBR antibody and the MAP kinase inhibitor are administered sequentially.
  52. 52. The method of claim 51, wherein the anti-ETBR antibody is first administered to the subject.
  53. 53. The method of claim 52, wherein the MAP kinase inhibitor is administered to the subject following administration of the anti-ETBR antibody.
  54. 52. The method of claim 51, wherein the MAP kinase inhibitor is first administered to the subject.
  55. 55. The method of claim 54, wherein the anti-ETBR antibody is administered to a subject after administration of a MAP kinase inhibitor.
  56. 23. The method of claim 22, wherein the anti-ETBR antibody is administered intravenously.
  57. 23. The method of claim 22, wherein said anti-ETBR antibody is administered 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 20 mpk, or about 25 mpk, or about 30 mpk.
  58. 23. The method of claim 22, wherein the MAP kinase inhibitor is administered orally.
  59. 23. The method of claim 22, wherein the MAP kinase inhibitor is administered 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, 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.
  60. An article of manufacture for TGI in a subject suffering from melanoma, comprising a package comprising an anti-ETBR antibody composition and a MAP kinase inhibitor composition.
  61. An article of manufacture for treating melanoma in a subject, comprising a package comprising an anti-ETBR antibody composition and a MAP kinase inhibitor composition.
  62. 63. The article of manufacture of claim 60 or 61, wherein said anti-ETBR antibody specifically binds to an ETBR epitope consisting of amino acid numbers 64-101 of SEQ ID NO: 10.
  63. 61. The method of claim 60 or 61 wherein said anti-ETBR antibody has three variable heavy CDRs and three variable light CDRs wherein VH CDR1 is SEQ ID NO: 1, 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, and VL CDR3 is SEQ ID NO: 6.
  64. 62. The article of manufacture of claim 60 or 61 wherein said anti-ETBR antibody has a variable heavy chain and a variable light chain, wherein said VH is sequence 7 or 9.
  65. 65. The article of manufacture of claim 64, wherein the VL is sequence 8.
  66. 62. The article of manufacture of claim 60 or 61 wherein said anti-ETBR antibody is conjugated to a cytotoxin.
  67. 66. The article of manufacture of claim 66, wherein the cytotoxin is a cytotoxic agent selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleic acid degrading enzymes.
  68. 69. The article of manufacture of claim 67, wherein the cytotoxin is a toxin.
  69. 69. The article of manufacture of claim 68, wherein said toxin is selected from the group consisting of maytansinoid, calicheamicin and auristatin.
  70. 70. The article of manufacture of claim 69, wherein said toxin is a maytansinoid.
  71. 62. The article of manufacture of claim 60 or 61 wherein said MAP kinase inhibitor is a BRAF inhibitor.
  72. 74. The method of claim 71, wherein said BRAF inhibitor is propane-1-sulfonic acid {3- [5- (4- chlorophenyl) -lH- pyrrolo [2,3- b] pyridine- -Difluoro-phenyl} -amide. &Lt; / RTI &gt;
  73. 74. The article of manufacture of claim 71, wherein the BRAF inhibitor has the following chemical structure:
    Figure pct00046
  74. 63. The article of manufacture of claim 60 or 61 wherein the MAP kinase inhibitor is a MEK inhibitor.
  75. 74. The method of claim 74, wherein said MEK inhibitor is selected from the group consisting of (S) - (3,4-difluoro-2- (Piperidin-2-yl) azetidin-1-yl) methanone.
  76. 74. The article of manufacture of claim 74, wherein said MEK inhibitor has the following chemical structure:
    Figure pct00047
  77. Use of the article of manufacture of claim 60 or 61 in the manufacture of a medicament for TGI of melanoma.
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