JP6251682B2 - Combinations and methods of treatment for melanoma treatment - Google Patents

Description

RELATED APPLICATIONS This application was filed on August 28, 2011, and US Provisional Application No. 61 / 552,893, filed August 28, 2012, as provided for in US Patent Act 119 (e) Claims the benefit of US Provisional Application No. 61 / 678,978, the contents of which are incorporated herein by reference.

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

Background Melanoma is a high-grade form of skin cancer with a surprising increase in incidence (Thompson JF et al., Cutaneous melanoma in the era of molecular profiling. Lancet 2009; 374: 362-5). Healing can be achieved by surgical excision of localized lesions, but is currently approved in the advanced stages of melanoma and only responds slightly to treatment. Stage IV metastatic melanoma has a 5-year survival rate of approximately 10% (Thompson, Lancet 2009). New therapeutic approaches including antisense to Bcl2, antibodies to CTLA4, small molecule RAF kinase inhibitors, and adoptive immunotherapy have entered 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). Although the results from some of these recent studies seem promising, for a lasting effect on overall survival, a combination of treatments including additional new drugs may be required.

  However, it is recognized that melanoma can exhibit molecular mutations in specific signal transduction pathways necessary for, for example, cellular responses to growth factors. Thus, in order to treat each subtype with the most appropriate therapy rather than treating melanoma as a single disease, attempts have been made to stratify it into molecular subtypes.

  One subtype of melanoma has an abnormality in the MAP kinase pathway. The MAPK pathway is a phosphorylation-driven signaling cascade that links the intracellular response to growth factor binding to cell surface receptors. This pathway regulates several processes, including cell proliferation and differentiation, and is often dysregulated in various cancers (Sebolt-Leopold JS, Herrera R. Mitogen activity to treat cancer 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 induces the formation of the RAF / MEK / ERK kinase complex and then transcription of key regulators via protein phosphorylation. To drive. Inhibition of MAPK by agents targeting pathway critical proteins has the potential to inhibit the growth of various tumor types (in Wong KK et al., In anticancer agents targeting Ras / Raf / MEK / ERK pathway) Recent developments (Recent developments in anti-cancer agents targeting the Ras / Raf / MEK / ERK pathway.) Recent Pat Anticancer Drug Discov. 2009; 4: 28-35.

  Inappropriate activation of the MEK / ERK pathway promotes cell proliferation in the absence of exogenous growth factors. GDC-0973 (also known as XL518) is a potent and highly selective small molecule inhibitor of MEK1 / 2, a MAPK kinase that activates ERK1 / 2 (Johnston S. XL518 is a potent and selective , An orally bioavailable MEK1 inhibitor that down-regulates the Ras / Raf / MEK / ERK pathway in vivo leading to tumor growth inhibition and regression in preclinical models (XL518, a potent, selective, orally bioavailable MEK1 inhibitor, downregulates the Ras / Raf / MEK / ERK pathway in vivo, resulting in tumor growth inhibition and regression in preclinical models.)): AACR-NCI-EORTC Symposium on Molecular Targeting and Cancer Treatment Targets and Cancer Therapeutics); October 22, 2007; San Francisco, California. (Presentation at abstract C209). As a result, the carcinogenic signals from the cell surface, Ras and Raf to ERK are interrupted. Sustained inhibition of ERK activation leads to decreased proliferation and induction of apoptosis. In numerous preclinical studies, GDC-0973 has been shown to inhibit cell proliferation and induce tumor regression.

  Recently, Vemurafenib, an enzyme inhibitor of B-Raf, known as Zelboraf®, has been approved by the US Food and Drug Administration for the treatment of late melanoma. Vemurafenib has been shown to cause programmed cell death in melanoma cell lines (BRA silencing by chemical blockade with Sala E, et al., Short hairpin RNA or PLX4032 results in different responses in melanoma and thyroid cancer cells. (BRAF silencing by short hairpin RNA or chemical blockade by PLX4032 leads to different responses in melanoma and thyroid carcinoma cells.) Mol. Cancer Res. 6 (5): 751-9 (May 2008). -If Raf has a common V600E mutation, interrupt the B-Raf / MEK step on the B-Raf / MEK / ERK pathway Vemurafenib is a cancer that has a V600E BRAF mutation (ie, an amino acid on the B-RAF protein) At position 600, normal valine is replaced by glutamic acid. About 60% of melanomas have the V600E BRAF mutation, melanoma cells without this mutation do not appear to be inhibited by vemurafenib; Can stimulate and promote tumor growth (Hatzivassiliou G et al., RAF inhibitors stimulate wild-type RAF, activate MAPK pathway and enhance proliferation.) Nature 464 (7287): 431-5; Halaban R et al., PLX4032, a selective BRAF (V600E) kinase inhibitor activates the ERK pathway, cell migration and BRAF (WT) melanoma Increase cell proliferation (PLX4032, a Selective BRAF (V600E) Kinase Inhibitor, Activates the ERK Pathway and Enhances Cell Migration and Proliferation of BRAF (WT) Melanoma Ce lls.) Pigment Cell Melanoma Res 23 (2): 190-200 (February 2010). Clinical trials revealed improved survival, objective response, and progression-free survival in patients treated with vemurafenib compared to DTIC, but increased likelihood of disease recurrence . (Nazarian R. et al., Melanoma acquires resistance to B-RAF (V600E) inhibition by upregulation of RTK or N-RAS. (Melanomas acquire resistance to B-RAF (V600E) inhibition by RTK or N -RAS upregulation.) Nature 468, pp. 973-977 (December 16, 2010)).

  Despite advances in melanoma cancer therapy, there is a great need for additional therapeutic treatments that can effectively inhibit neoplastic cell proliferation. Accordingly, it is an object of the present invention to identify combinations of therapeutic agents to produce compositions of matter useful for therapeutic treatment of melanoma cancer.

SUMMARY The present invention relates to a tumor in a subject suffering from melanoma comprising administering to the subject an effective amount of an anti-endothelin B receptor (ETBR) antibody drug conjugate in combination with an effective amount of a MAP kinase inhibitor. Intended for growth inhibition (TGI).

  In one aspect, the combination of the anti-ETBR antibody drug conjugate and the MAP kinase inhibitor is synergistic. In another aspect, for a synergistic combination, the TGI is greater than the TGI observed using the anti-ETBR antibody drug conjugate alone, or greater than the TGI observed using the MAP kinase inhibitor alone. In yet another aspect, for a synergistic combination, the TGI is greater than about 10%, or greater than about 15%, or greater than about 20%, or greater than the use of the anti-ETBR antibody drug conjugate alone. More than 25%, or more than about 30%, or more than about 35%, or more than about 40%, or more than about 45%, or more than about 50%, or about 55% Or greater than about 60%, greater than about 65%, or greater than about 70%, or greater than about 10%, or about 15% than the use of a MAP kinase inhibitor alone Or more than about 20% or more than about 25% or more than about 30% or more than about 35% or more than about 40% or more than about 45% Or more than about 50% or more than about 55% or more than about 60% Te, or greater than about 65%, or greater than about 70% greater.

  In another aspect of the invention described above, the anti-ETBR antibody binds to an ETBR epitope consisting of amino acid numbers 64 to 101 of SEQ ID NO: 10. In yet another aspect of the claimed method, the anti-ETBR antibody has three variable heavy chain CDRs and three variable transchain CDRs, wherein VH CDR1 is SEQ ID NO: 1 and VH CDR2 is SEQ ID NO: 2. VH CDR3 is SEQ ID NO: 3, and VL CDR1 is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, and VL CDR3 is SEQ ID NO: 6. In yet another embodiment of the claimed method, the anti-ETBR antibody has a variable heavy chain and a variable transchain, wherein the VH is SEQ ID NO: 7 or 9. Further embodiments of the method also include an anti-ETBR antibody having a VL that is SEQ ID NO: 8.

  In one aspect of the claimed method described above, the anti-ETBR antibody is conjugated to a cytotoxin, wherein the cytotoxin is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes. A cytotoxic drug, and the cytotoxin is a toxin. In another aspect of the claimed invention, the toxin is selected from the group consisting of maytansinoids, calicheamicins and auristatins. In yet another aspect of the invention, the toxin is a maytansinoid.

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

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

  In one aspect of the invention, a method for treating melanoma is described that comprises 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 claimed method described above, the melanoma is ETBR positive. In another embodiment of the claimed method, the melanoma is metastatic. In yet another aspect of the claimed method, the subject has not received prior treatment with a MAP kinase inhibitor. In yet another aspect of the claimed method, the subject has a V600E BRAF genetic mutation, or the subject is a BRAF wild type that does not have a V600E BRAF mutation. In yet another aspect of the claimed method, the subject has not received prior treatment with a MAP kinase inhibitor.

  In another aspect of the claimed method described above, the combination of anti-ETBR antibody and MAP kinase inhibitor is synergistic. In another aspect, for a synergistic combination, the TGI is greater than the TGI observed using the anti-ETBR antibody alone, or greater than the TGI observed using the MAP kinase inhibitor alone. In yet another aspect, for a synergistic combination, the TGI is greater than about 10%, or greater than about 15%, or greater than about 20%, or about 25% than the use of anti-ETBR antibody alone. Greater than, or greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, or greater than about 55% Or greater than about 60%, or greater than about 65%, or greater than about 70%, or greater than about 10% or greater than about 15% than the use of a MAP kinase inhibitor alone. Or more than about 20%, or more than about 25%, or more than about 30%, or more than about 35%, or more than about 40%, or more than about 45%, or Greater than about 50%, or greater than about 55%, or greater than about 60%, or about 65% Beyond, or greater than about 70%.

  In another aspect of the invention described above, the anti-ETBR antibody binds to an ETBR epitope consisting of amino acid numbers 64 to 101 of SEQ ID NO: 10. In yet another aspect of the claimed method, the anti-ETBR antibody has three variable heavy chain CDRs and three variable transchain CDRs, wherein VH CDR1 is SEQ ID NO: 1 and VH CDR2 is SEQ ID NO: 2. VH CDR3 is SEQ ID NO: 3, and VL CDR1 is SEQ ID NO: 4, VL CDR3 is SEQ ID NO: 5, and VL CDR2 is SEQ ID NO: 6. In yet another embodiment of the claimed method, the anti-ETBR antibody has a variable heavy chain and a variable transchain, wherein the VH is SEQ ID NO: 7 or 9. Further embodiments of the method also include an anti-ETBR antibody having a VL that is SEQ ID NO: 8.

  In one aspect of the claimed method described above, the anti-ETBR antibody is conjugated to a cytotoxin, wherein the cytotoxin is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes. A cytotoxic drug, and the cytotoxin is a toxin. In another aspect of the claimed invention, the toxin is selected from the group consisting of maytansinoids, calicheamicins and auristatins. In yet another aspect of the invention, the toxin is a maytansinoid.

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

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

  In one aspect of the invention, a method for treating melanoma is described comprising administering a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody to a subject in need thereof, wherein the MAP kinase inhibitor comprises , First administered to said 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, contemplated methods of the invention include when the anti-ETBR antibody and the MAP kinase inhibitor are administered simultaneously.

  In one aspect of the invention, it is intended 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. The anti-ETBR antibody drug conjugate and the MAP kinase inhibitor are administered sequentially, 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. To be administered.

  In one aspect of the invention, it is intended 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. 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.

  To facilitate the above contemplated aspects of the invention, a method of treating melanoma comprising administering to a subject in need thereof a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody drug conjugate. Wherein the anti-ETBR antibody drug conjugate is administered intravenously. Also, in the claimed method of the invention, the anti-ETBR antibody drug conjugate is about 0.1 mpk, or about 0.2 mpk, or about 0.3 mpk, or about 0.5 mpk, or about 1 mpk, Or about 5 mpk, or about 10 mpk, or about 15 mpk, or about 20 mpk, or about 25 mpk, or about 30 mpk.

  To facilitate the above contemplated aspects of the invention, a method of treating melanoma comprising administering to a subject in need thereof a therapeutically effective amount of a MAP kinase inhibitor and an anti-ETBR antibody drug conjugate. Described, wherein the MAP kinase inhibitor is administered orally. Also, in the claimed method of the invention, the BRAF inhibitor is about 1 mpk, or about 2 mpk, or about 3 mpk, or about 4 mpk, or about 5 mpk, or about 6 mpk, or about 7 mpk, or about 8 mpk. Or about 9 mpk, or about 10 mpk, or mpk about 11, or about 12 mpk, or about 15 mpk, or about 20 mpk or about 30 mpk.

  In one aspect of the invention, the article of manufacture is intended to be used for TGI in patients with melanoma comprising a package comprising an anti-ETBR antibody drug conjugate composition and a MAP kinase inhibitor composition. . The anti-ETBR antibody drug conjugate is further intended to specifically bind to an ETBR epitope consisting of amino acid numbers 64 to 101 of SEQ ID NO: 10. Alternatively, the anti-ETBR antibody has three variable heavy chain CDRs and three variable transchain CDRs, where VH CDR1 is SEQ ID NO: 1, VH CDR2 is SEQ ID NO: 2, and VH CDR3 is SEQ ID NO: 3, and VL CDR1 is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, and VL CDR3 is intended to be SEQ ID NO: 6. A further option contemplated is that the anti-ETBR antibody has a variable heavy chain and a variable transchain, where the VH is SEQ ID NO: 7 or 9. In yet another option, the anti-ETBR antibody has a variable heavy chain and a variable transchain, wherein the 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 drug selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes. And the cytotoxin is a toxin. In another embodiment, the cytotoxin is a toxin, wherein the toxin is selected from the group consisting of maytansinoids, calicheamicins and auristatins. In one embodiment, the toxin is a maytansinoid.

In one embodiment of the method of treating melanoma described above, it is also contemplated that the MAP kinase inhibitor is a BRAF inhibitor. In one embodiment of the above-described method, the BRAF inhibitor is propane-1-sulfonic acid {3- [5- (4-chlorophenyl) -1H-pyrrolo [2,3-b] pyridine-3-carbonyl] -2. , 4-difluoro-phenyl} -amide. In addition, BRAF inhibitors are intended to have the following chemical structure:

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

  In one aspect of the invention, an article of manufacture for treating melanoma in a subject is intended to include a package comprising an anti-ETBR antibody drug conjugate composition and a MAP kinase inhibitor composition. The anti-ETBR antibody is further intended to specifically bind to an ETBR epitope consisting of amino acid numbers 64 to 101 of SEQ ID NO: 10. Alternatively, the anti-ETBR antibody has three variable heavy chain CDRs and three variable transchain CDRs, where VH CDR1 is SEQ ID NO: 1, VH CDR2 is SEQ ID NO: 2, and VH CDR3 is SEQ ID NO: 3, and VL CDR1 is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, and VL CDR3 is intended to be SEQ ID NO: 6. A further option contemplated is that the anti-ETBR antibody has a variable heavy chain and a variable transchain, where the VH is SEQ ID NO: 7 or 9. In yet another option, the anti-ETBR antibody has a variable heavy chain and a variable transchain, wherein the 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 drug selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes. And the cytotoxin is a toxin. In another embodiment, the cytotoxin is a toxin, wherein the toxin is selected from the group consisting of maytansinoids, calicheamicins and auristatins. In one embodiment, the toxin is a maytansinoid.

In one embodiment of the article of manufacture described above, it is also contemplated that the MAP kinase inhibitor is a BRAF inhibitor. In one embodiment of the article of manufacture described above, the BRAF inhibitor is propane-1-sulfonic acid {3- [5- (4-chlorophenyl) -1H-pyrrolo [2,3-b] pyridine-3-carbonyl]-. 2,4-difluoro-phenyl} -amide. In addition, BRAF inhibitors are intended to have the following chemical structure:

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

  One aspect of the present invention is intended to be the use of anti-ETBR antibody drug conjugates and MAP kinase inhibitors in the preparation of a medicament for TGI of melanoma. The anti-ETBR antibody is further intended to specifically bind to an ETBR epitope consisting of amino acid numbers 64 to 101 of SEQ ID NO: 10. Alternatively, the anti-ETBR antibody has three variable heavy chain CDRs and three variable transchain CDRs, where VH CDR1 is SEQ ID NO: 1, VH CDR2 is SEQ ID NO: 2, and VH CDR3 is SEQ ID NO: 3, and VL CDR1 is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, and VL CDR3 is intended to be SEQ ID NO: 6. A further option contemplated is that the anti-ETBR antibody has a variable heavy chain and a variable transchain, where the VH is SEQ ID NO: 7 or 9. In yet another option, the anti-ETBR antibody has a variable heavy chain and a variable transchain, wherein the 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 drug selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes. And the cytotoxin is a toxin. In another embodiment, the cytotoxin is a toxin, wherein the toxin is selected from the group consisting of maytansinoids, calicheamicins and auristatins. In one embodiment, the toxin is a maytansinoid.

In one embodiment of the pharmaceutical use described above, it is also contemplated that the MAP kinase inhibitor is a BRAF inhibitor. In one embodiment of the pharmaceutical use described above, the BRAF inhibitor is propane-1-sulfonic acid {3- [5- (4-chlorophenyl) -1H-pyrrolo [2,3-b] pyridine-3-carbonyl]. -2,4-difluoro-phenyl} -amide. In addition, BRAF inhibitors are intended to have the following chemical structure:

In another aspect of the pharmaceutical use described above, the MAP kinase inhibitor is a MEK inhibitor. In yet another aspect of the pharmaceutical use described above, the MEK inhibitor is (S)-(3,4-difluoro-2-((2-fluoro-4-iodophenyl) amino) phenyl) (3-hydroxy. -3- (piperidin-2-yl) azetidin-1-yl) methanone. In yet another aspect of the pharmaceutical use described above, the MEK inhibitor has the following chemical structure:

  One aspect of the present invention is intended to be the use of an article of manufacture comprising an anti-ETBR antibody drug conjugate and a MAP kinase inhibitor in the preparation of a medicament for TGI of melanoma. The anti-ETBR antibody is further intended to specifically bind to an ETBR epitope consisting of amino acid numbers 64 to 101 of SEQ ID NO: 10. Alternatively, the anti-ETBR antibody has three variable heavy chain CDRs and three variable transchain CDRs, where VH CDR1 is SEQ ID NO: 1, VH CDR2 is SEQ ID NO: 2, and VH CDR3 is SEQ ID NO: 3, and VL CDR1 is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, and VL CDR3 is intended to be SEQ ID NO: 6. A further option contemplated is that the anti-ETBR antibody has a variable heavy chain and a variable transchain, where the VH is SEQ ID NO: 7 or 9. In yet another option, the anti-ETBR antibody has a variable heavy chain and a variable transchain, wherein the VH is SEQ ID NO: 7 or 9, and VL is SEQ ID NO: 8. In another embodiment of the use of the article of manufacture, the anti-ETBR antibody is conjugated to a cytotoxin, wherein the cytotoxin is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes. Is a sex drug and the cytotoxin is a toxin. In another embodiment, the cytotoxin is a toxin, wherein the toxin is selected from the group consisting of maytansinoids, calicheamicins and auristatins. In one embodiment, the toxin is a maytansinoid.

In one embodiment of the use of the article of manufacture described above, it is also contemplated that the MAP kinase inhibitor is a BRAF inhibitor. In one embodiment of the use of the article of manufacture described above, the BRAF inhibitor is propane-1-sulfonic acid {3- [5- (4-chlorophenyl) -1H-pyrrolo [2,3-b] pyridine-3-carbonyl. ] -2,4-Difluoro-phenyl} -amide. In addition, BRAF inhibitors are intended to have the following chemical structure:

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

FIG. 1 is a schematic of the MAP kinase pathway. FIG. 2 demonstrates the relationship of receptor level to ADC cell death in vitro. The indicated number of receptor copies / cell was estimated by Scatchard analysis. Panel A, melanoma cell lines UACC-257X2.2, and Panel B shows cell killing by _anti-ET B R ADC titration of melanoma cell lines A2058. Anti _ET B R ADC of the specified concentration (Hu5E9v1-vc-MMAE), control IgG-vc-MMAE, or an equivalent amount of PBS vehicle control was incubated for 5 days with cells, relative cell viability (y-axis) Were evaluated using CellTiter-Glo. FIG. 3 shows the in vivo efficacy of anti-ETBR ADCs in a xenograft mouse model. Subcutaneous tumors were established in mice seeded with UACC-257X2.2 (panel A) or A2058 (panel B) cells. When the tumor volume reached approximately 200 mm ³ (day 0), the animals received a single vein at a dose that showed either a control ADC (control-VC-MMAE) or an anti-ETBR ADC (Hu5E9v1-vc-MMAE). It was injected internally. Mean tumor volume with standard deviation was determined from 10 animals per group (shown on the graph). Figure 4 shows the ET _B R expression in UACC-257X2.2 melanoma cells treated for 24 hours with BRAFi-945 at various concentrations. Panel A shows the ETBR transcript normalized to the RPL19 transcript. Panel B shows the expression of total ETBR and GAPDH (control) proteins in 50 μg whole cell lysate. Panel C shows surface ETBR protein expression in living cells as seen by flow cytometry, where the first peak shows cells treated against the secondary detection reagent alone and the middle peak shows the BRAF inhibitor Shows cells that have not been treated with the last peak, cells that have been treated with BRAF inhibitors. 5, against UACC-257X2.2 melanoma xenograft mouse model, indicating the effectiveness of the combination of an _{anti-ET B R ADC (Hu5E9v1-vc} -MMAE) and in vivo BRAFi-945 at various doses . Subcutaneous tumors were established in mice seeded with the UACC-257X2.2 cell line. When the tumor volume reached approximately 200 mm ³ (day 0), the animals were administered BRAFi-945 or vehicle control orally once daily for 21 days. On day 1 (after administering the BRAFi-945 2 times), in animals, a single intravenous injection at a dose shown to either vehicle or _anti-ET B R ADC was performed. Mean tumor volume with standard deviation was determined from 10 animals per group (shown on the graph). Information of the drug and the dose is as shown: Panel A shows a combination of _anti-ET B R-ADC of BRAFi-945 and 1 mpk of 1mpk (Hu5E9v1-vc-MMAE) ; Panel B, the 1 mpk exhibit a combination of BRAFi-945 and 3mpk anti _ET B R-ADC of (Hu5E9v1-vc-MMAE); panel C, _anti-ET B R-ADC of BRAFi-945 and 1mpk of 6mpk of (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 20 mpk BRAFi-945 and 3 mpk anti-ET _B R- Combination of ADC (Hu5E9v1-vc-MMAE) Indicates. FIG. 6 shows ETBR expression in COLO829 melanoma cells treated with various concentrations of BRAF inhibitor RG7204 for 24 hours. Panel A shows the ETBR transcript normalized to the RPL19 transcript. Panel B shows the expression of total ETBR and GAPDH (control) proteins in 50 μg whole cell lysate. Panel C shows surface ETBR protein expression in live cells as seen by flow cytometry, where the first peak shows cells treated against the secondary detection reagent alone and the second peak shows BRAF inhibition. Cells not treated with the agent are shown, and the third peak represents cells treated with the BRAF inhibitor. FIG. 7 demonstrates the in vivo efficacy of the anti-ETBR ADC and BRAF inhibitor RG7204 against the COLO829 melanoma xenograft mouse model. Subcutaneous tumors were established in mice seeded with the COLO829 melanoma cell line. When the tumor volume reached approximately 200 mm ³ (Day 0), the animals received RG7204 orally once daily for 21 days. On day 1 (RG7204 after administration three times), the animals, a single intravenous injection with either the indicated dose of vehicle or _{anti-ET B R ADC (Hu5E9v1-vc} -MMAE) has been performed. Mean tumor volume with standard deviation was determined from 9 animals per group (shown on the graph). Drug and dose information is as shown: Panel A shows 3 mpk anti-ET _B R-ADC (Hu5E9v1-vc-MMAE) combined with 30 mpk RG7204; Panel B shows 30 mpk RG7204 Panel 1 shows 1 mpk anti-ET _B R-ADC (Hu5E9v1-vc-MMAE) combined; 10 mpk anti-ET _B R-ADC (Hu5E9v1-vc-MMAE) combined with 10 mpk RG7204; Shows 3 mpk anti-ET _B R-ADC (Hu5E9v1-vc-MMAE) in combination with 10 mpk RG7204. FIG. 8 shows ETBR expression in A2058 melanoma cells treated with various concentrations of BRAF inhibitor RG7204 for 24 hours. Panel A shows the ETBR transcript normalized to the RPL19 transcript; Panel B shows the expression of total ETBR and GAPDH (control) protein in 100 μg whole cell lysate; Panel C shows the flow Shown is the expression of surface ETBR protein in living cells as seen by cytometry. The first peak represents cells treated with the secondary detection reagent alone, the second peak represents cells not treated with BRAF inhibitor, and the third peak treated with BRAF inhibitor Cells are shown. Figure 9 demonstrates the efficacy of the combination of at A2058 against melanoma xenograft mouse model, _anti-ET B vivo R ADC (Hu5E9v1-vc-MMAE ) and BRAF inhibitors RG7204. Subcutaneous tumors were established in mice seeded with the A2058 melanoma cell line. When the tumor volume reached approximately 200 mm ³ (Day 0), the animals received RG7204 orally twice daily for 21 days. On day 1 (RG7204 after administration three times), the animals, a single intravenous injection with either the indicated dose of vehicle or _{anti-ET B R ADC (Hu5E9v1-vc} -MMAE) has been performed. Mean tumor volume with standard deviation was determined from 10 animals per group (shown on the graph). The drug and dose information shown in each graph is as follows: Panel A shows 6 mpk anti-ET _B R-ADC (Hu5E9v1-vc-MMAE) combined with 10 mpk RG7204; , Shows 6 mpk anti-ET _B R-ADC (Hu5E9v1-vc-MMAE) in combination with 30 mpk RG7204; Panel C shows 3 mpk anti-ET _B R-ADC in combination with 10 mpk RG7204 (Hu5E9v1-vc-MMAE) Panel D shows 3 mpk anti-ET _B R-ADC (Hu5E9v1-vc-MMAE) in combination with 30 mpk RG7204. FIG. 10 shows a Western blot experiment performed with BRAFi RG7204, expressing total ETBR, Perk and erk proteins and control proteins GAPDH and β-tubulin in total cell lysates from 25 μg to 100 μg from IPC-298 melanoma cells. Is shown. FIG. 11 shows the expression of surface ETBR protein in live IPC-298 cells as seen by flow cytometry after incubation with 0.1 μM, 1 μM and 10 μM BRAFi RG7204 (panels A, B and C, respectively). . The first peak represents cells treated with the secondary detection reagent alone, the second peak represents cells treated with BRAF inhibitor and the third peak treated with BRAF inhibitor Not showing cells. FIG. 12 shows a Western blot experiment performed with MEKi-623 (panel A) and MEKi-973 (panel B) at concentrations of 0 μM, 0.01 μM, 0.1 μM and 1 μM, showing total cells from COLO829 melanoma cells Shown is expression of total ETBR, Perk and erk proteins and control proteins GAPDH and β-tubulin in 50 μg of lysate. Figure 13 shows MEKi-623 (panels A, B and C, respectively) or MEKi-973 (respectively, 0.01 μM (panels A and D), 0.1 μM (panels B and E) and 1 μM (panels C and F), respectively). Shows the expression of surface ETBR protein in living COLO829 cells as seen by flow cytometry after incubation with panels D, E and F). The first peak represents cells treated with the secondary detection reagent alone, the second peak represents cells that were not treated with MEK inhibitor, and the third peak was treated with MEK inhibitor Cells are shown. FIG. 14 shows ETBR mRNA expression normalized to RPL19 transcript in A2058 melanoma cells treated with various concentrations of MEki-623 (Panel A) or MEKi-973 (Panel B) for 24 hours. . FIG. 15 shows a Western blot experiment performed with MEKi-623 (panel A) and MEKi-973 (panel B) at concentrations of 0 μM, 0.01 μM, 0.1 μM and 1 μM, showing total cells from A2058 melanoma cells. The expression of total ETBR, Perk and erk proteins and control proteins GAPDH and β-tubulin is shown in 50-100 μg of lysate. FIG. 16 shows 0.01 μM (panels A and D), 0.1 μM (panels B and E) and 1 μM (panels C and F) MEKi-623 (panels A, B and C, respectively) or MEKi-973 (respectively Shows the expression of surface ETBR protein in live A2058 cells as seen by flow cytometry after incubation with panels D, E and F). The first peak represents cells treated with the secondary detection reagent alone, the second peak represents cells not treated with MEK inhibitor, and the third peak treated with MEK inhibitor Cells are shown. Figure 17 demonstrates the efficacy of the combination of in vivo A2058 against melanoma xenograft mouse model, _{anti-ET B R ADC (Hu5E9v1-vc} -MMAE) and Meki-973. Subcutaneous tumors were established in mice seeded with the A2058 melanoma cell line. When the tumor volume reached approximately 200 mm ³ (0 day), animals were administered once a day Meki-973 for 21 days orally. On day 1 (after administering Meki-973 twice), animals, a single intravenous injection with either the indicated dose of vehicle or _{anti-ET B R ADC (Hu5E9v1-vc} -MMAE) is performed It was. Mean tumor volume with standard deviation was determined from 9 animals per group (shown on the graph). The drug and dose information shown in each graph is as follows: Panel A shows 7.5 mpk of anti-drug combined with 7.5 mpk of MEKi-973 compared to vehicle control and anti-gD ADC alone. Panel B shows 7.5 mpk MEKi-973 compared to vehicle control and 7.5 mpk MEKi-973 alone (GDC-0973) or 6 mpk anti-ET _B R-ADC alone. shows the _anti-ET B R-ADC of 6mpk in combination with (Hu5E9v1-vc-MMAE). 18, in Meki-623 (Panel A) or MEKi-973 SK23-MEL melanoma cells treated for 24 hours with various concentrations of (Panel B), normalized to RPL19 transcript, ET _B R transcription The expression of the product is shown. FIG. 19 shows a Western blot experiment performed with MEKi-623 (panel A) and MEKi-973 (panel B) at concentrations of 0 μM, 0.01 μM, 0.1 μM and 1 μM, from SK23-MEL melanoma cells. The expression of total ETBR, Perk and erk proteins and control proteins GAPDH and β-tubulin is shown in 50 μg of total cell lysate. FIG. 20 shows 0.01 μM (panels A and D), 0.1 μM (panels B and E) and 1 μM (panels C and F) MEKi-623 (panels A, B and C, respectively) or MEKi-973 (respectively Shows the expression of surface ETBR protein in live SK23-MEL cells as seen by flow cytometry after incubation with panels D, E and F). The first peak represents cells treated with the secondary detection reagent alone, the second peak represents cells not treated with MEK inhibitor, and the third peak treated with MEK inhibitor Cells are shown. Figure 21 demonstrates the efficacy of the combination of in vivo SK23-MEL against melanoma xenograft mouse model, _{anti-ET B R ADC (Hu5E9v1-vc} -MMAE) and Meki-973. The drug and dose information shown in each graph is as follows: Panel A shows 7.5 mpk MEKi compared to vehicle control, 7.5 mpk MEKi-973 and 6 mpk anti-gD ADC alone. 6mpk anti-gD ADC combined with -973 (control); panel B shows 7.5 mpk compared to vehicle control and 7.5 mpk MEKi-973 alone or 6 mpk anti-ET _B R-ADC alone. It is shown in combination with Meki-973 _anti-ET B R-ADC (the "combination") 6mpk (Hu5E9v1-vc-MMAE ). Panel C, vehicle control, as compared with the _{anti-ET B R-ADC (Hu5E9v1-} vc-MMAE) or Meki-973 of 3 mpk of 3 mpk, in combination with Meki-973 in 3 mpk ( "combination") 3 mpk anti ET _B shows the R-ADC (Hu5E9v1-vc- MMAE). Panel D, compared to anti _-ET B R-ADC alone Meki-973 alone or 3 mpk of vehicle control and 7.5Mpk, in combination with Meki-973 of 7.5Mpk ( "combination") 3 mpk anti ET of _B shows the R-ADC (Hu5E9v1-vc- MMAE). Panel E shows 6 mpk anti-ET _B R-ADC in combination with 3 mpk MEKi-973 (“combination”) compared to vehicle control and 3 mpk MEKi-973 alone or 6 mpk anti-ET _B R-ADC alone. (Hu5E9v1-vc-MMAE) is shown. FIG. 22 shows a Western blot experiment performed with MEKi-623 (panel A) and MEKi-973 (panel B) at concentrations of 0 μM, 0.01 μM, 0.1 μM and 1 μM, from IPC-298 melanoma cells. The expression of total ETBR, Perk and erk proteins and control proteins GAPDH and β-tubulin is shown in 25-100 μg of total cell lysate. FIG. 23 shows 0.01 μM (panels A and D), 0.1 μM (panels B and E) and 1 μM (panels C and F) MEKi-623 (panels A, B and C, respectively) or MEKi-973 (respectively Shows the expression of surface ETBR protein in live IPC-298 cells as seen by flow cytometry after incubation with panels D, E and F). The first peak represents cells treated with the secondary detection reagent alone, the second peak represents cells not treated with MEK inhibitor, and the third peak treated with MEK inhibitor Cells are shown. Figure 24, against IPC-298 melanoma xenograft mouse model, demonstrating the efficacy of the combination of in vivo _{anti-ET B R ADC (Hu5E9v1-vc} -MMAE) and Meki-623. The drug and dose information shown in each graph is as follows: Panel A is combined with 1 mpk MEKi-623 compared to vehicle control, 1 mpk MEKi-623 and 6 mpk anti-gD ADC alone. 6 mpk anti-gD ADC (control); Panel B combined with 1 mpk MEKi-623 compared to vehicle control and 1 mpk MEKi-623 alone or 6 mpk anti-ET _B R-ADC alone ("" combination ") shows the _anti-ET B R-ADC of 6mpk (Hu5E9v1-vc-MMAE) . Figure 25 shows the efficacy of the combination of in vivo IPC-298 against melanoma xenograft mouse model, _{anti-ET B R ADC (Hu5E9v1-vc} -MMAE) and Meki-973. The drug and dose information shown in each graph is as follows: Panel A shows 7.5 mpk MEKi compared to vehicle control, 7.5 mpk MEKi-973 and 6 mpk anti-gD ADC alone. 6mpk anti-gD ADC combined with -973 (control); panel B shows 7.5 mpk compared to vehicle control and 7.5 mpk MEKi-973 alone or 6 mpk anti-ET _B R-ADC alone. It is shown in combination with Meki-973 _anti-ET B R-ADC (the "combination") 6mpk (Hu5E9v1-vc-MMAE ). FIG. 26 shows phosphorylated ERK and total ERK protein expression in COLO829 tumors treated with either vehicle or 30 mpk BRAFi RG7204. FIG. 27 shows 3-day ETBR transcript expression in COLO829 tumors treated with BRAFi RG7204 (panel A) and A2058 tumors treated with MEKi-973 (panel B). Panel A shows ETBR transcripts normalized to control GAPDH in the COLO829 cell line in vehicle controls or COLO829 tumors treated with either 10 mpk or 30 mpk RG7204. Panel B shows ETBR transcripts normalized to the control Hprtl in A2058 tumors treated with vehicle control or either 5 mpk or 10 mpk MEKi-973.

Detailed Description of Embodiments of the Invention Definitions For purposes herein, an “acceptor human framework” refers to a light chain variable domain (VL) framework obtained from a human immunoglobulin framework or a human consensus framework, as defined below, or A framework containing the amino acid sequence of the heavy chain variable domain (VH) framework. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence, or it may comprise changes in the amino acid sequence. 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 human immunoglobulin framework sequence or human consensus framework sequence.

  “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, “binding affinity” is an intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, antibody and antigen). Point to. The affinity of molecule X for its partner Y can generally be expressed as a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific exemplary and exemplary embodiments for measuring binding affinity are described below.

  An “affinity matured” antibody is one or more in one or more hypervariable regions (HVRs) that cause an improvement in the affinity of the antibody for the antigen compared to the parent antibody without the modification. It has a modification of.

The terms “anti-ETBR antibody” and “antibody that binds to ETBR” refer to the endothelin B receptor (ETBR with sufficient affinity such that the antibody targets ETBR and is useful as a diagnostic and / or therapeutic agent. ). In one embodiment, the degree of binding of the anti-ETBR antibody to an irrelevant, non-ETBR protein is less than about 10% of the binding of the antibody to ETBR, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to ETBR has a dissociation constant (Kd) of ≦ 1 μM, ≦ 100 nM, ≦ 10 nM, ≦ 1 nM, ≦ 0.1 nM, ≦ 0.01 nM, or ≦ 0.001 nM (eg, Less than 10 ⁻⁸ M, such as 10 ⁻⁸ M to 10 ⁻¹³ M, eg 10 ⁻⁹ M to 10 ⁻¹³ M). In certain embodiments, the anti-ETBR antibody binds to an epitope of ETBR that is conserved among ETBRs from different species.

  The term “antibody” is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and desired antigen binding activity. As long as the antibody fragment is included.

“Antibody fragment” refers to a molecule other than an intact antibody, including a portion of an intact antibody that binds an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ′, Fab′-SH, F (ab ′) ₂ , diabody, linear antibody, single chain antibody molecule (eg, scFv), and antibody fragment A multispecific antibody formed from

  “Antibodies that bind to the same epitope” as a reference antibody refers to an antibody that blocks binding of a reference antibody to its antigen in a competition assay by 50% or more, and conversely, 50% or more in a competition assay to that antigen of that antibody. The reference antibody that blocks binding. A typical competition assay is provided herein.

  The term “BRAF” as used herein is a protein encoded by the BRAF gene in humans, serine, also known as proto-oncogene B-Raf or v-Raf murine sarcoma virus oncogene homolog B1. / Threonine protein kinase B-Raf. B-Raf protein is involved in signaling and cell proliferation within the cell.

  As used herein, the term “BRAF inhibitor” or “BRAFi” is any number of known that can inhibit or interrupt the B-Raf / MEK step on the B-Raf / MEK / ERK pathway. Of small molecule drug compounds. Suitable BRAFi include, but are not limited to, international patent application PCT / US2010 / 047007 filed on August 27, 2010, international patent application PCT / US2010 / 046975 filed August 27, 2010, August 8, 2010. International Patent Application PCT / US2010 / 046952 filed on May 27, International Patent Application PCT / US2010 / 046955 filed on August 27, 2010, and International Patent Application PCT filed on June 21, 2006 / US2006 / 024361 may be included. Another example includes, but is not limited to, 5- [2- [4- [2- (dimethylamino) ethoxy] phenyl] -5- (4-pyridinyl) -1H-imidazol-4-yl] -2,3 GSK 2118436, also known as -dihydro-1H-inden-1-one oxime and having CAS registry number 405554-55-4 may be mentioned.

  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 and / or light chain is from a different source or species.

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and some of these are further subclasses (isotypes), eg, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IGA ₁ , and it can be divided into IgA _2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term “cytotoxic drug” as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (eg, At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ^212, and Lu). Body): chemotherapeutic agents or drugs (eg, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents), growth inhibitors, enzymes and the like Fragments, for example antibiotics such as nucleases, toxins such as small molecule toxins, or enzyme-active toxins from bacteria, fungi, plants or animals (including fragments and / or variants thereof) and disclosed below Various antitumor agents or anticancer agents.

  “Effector function” refers to biological activity attributable to the Fc region of an antibody, which varies with the antibody's isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell mediated cytotoxicity (ADCC); phagocytosis, cell surface receptors (eg, B cell receptor) Body) down-regulation and B cell activation.

  An “effective amount” of an agent, eg, a pharmaceutical formulation, refers to an effective amount at the dose and for the duration necessary to achieve the desired therapeutic or prophylactic result.

  Unless otherwise indicated, the term “ETBR” as used herein refers to any native form from any vertebrate, including mammals such as primates (eg, humans) and rodents (eg, mice, rats). Refers to the endothelin B receptor (ETBR). The term includes “full length” untreated ETBR as well as any form of ETBR resulting from processing in a cell. The term also encompasses naturally occurring variants of ETBR, such as splice variants or allelic variants. An exemplary amino acid sequence of human ETBR is shown in SEQ ID NO: 10 (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 that is conjugated to a toxin. Such toxins include but are not limited to maytansinoids or specifically monomethyl auristatin (MMAE). Anti-ETBR antibody-ADC is intended as a kind of “anti-ETBR antibody of the present invention”.

  The term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least part of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends 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 stated herein, the numbering of amino acid residues within an Fc region or constant region is the Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda. , MD, 1991, according to the EU numbering system, also called EU index.

  “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR is generally composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in the following sequences of VH (or VL): 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 have a structure substantially similar to or native to the native antibody structure herein. Refers to an antibody having a heavy chain comprising the defined Fc region.

As used herein, the term “945” or “BRAFi-945” refers to 4-amino-N- (6-chloro-2-fluoro-3- (3-fluoropropylsulfonamido) phenyl) thieno [3, 2-d] pyrimidine-7-carboxamide refers to a B-Raf enzyme inhibitor, which was filed on Aug. 27, 2010, and is incorporated herein by reference in its entirety. International Patent Application PCT / US2010 / It has a structure having the following formula disclosed in Example 15 of 046955.

  The terms “host cell”, “host cell line” and “host cell culture” are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include “transformants” and “transformed cells”, including primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Variant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein.

  A “human antibody” is one that is produced by a human or human cell or has an amino acid sequence corresponding to that of an antibody derived from a non-human source utilizing a repertoire of human antibodies or sequences encoding other human antibodies. is there. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues.

  A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. In general, a subgroup of sequences is described by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), 1 Subgroup in ~ 3 volumes. In one embodiment, for VL, the subgroup is subgroup kappa I in Kabat et al., Supra. In one embodiment, for VH, the subgroup is subgroup III in Kabat et al., Supra.

  A “humanized” antibody refers to a chimeric antibody comprising amino acid residues derived from non-human HVR and amino acid residues derived from human FRs. In certain embodiments, the humanized antibody comprises substantially at least one, typically all of the two variable domains, and all or substantially all of the HVR (eg, CDR) is non-human antibody. All or substantially all of the FR corresponds to that of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized” form of an antibody, eg, a non-human antibody, refers to an antibody that has undergone humanization.

  The term “hypervariable region” or “HVR” as used herein is an antibody variable domain whose sequence is hypervariable and / or forms a structurally defined loop (“hypervariable loop”). Each area. In general, a native 4-chain antibody comprises 6 HVRs, 3 in VH (H1, H2, H3) and 3 in VL (L1, L2, L3). HVRs generally contain amino acid residues from hypervariable loops and / or from “complementarity determining regions” (CDRs), the latter being the highest sequence variability and / or involved in antigen recognition. Yes. Exemplary hypervariable loops are amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 ( H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are amino acid residues 24-34 of L1, 50-56 of L2, 89 of L3. -97, H1-31-35B, H2-50-65, and H3-95-102. Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991). With the exception of CDR1 of VH, CDRs generally contain amino acid residues that form a hypervariable loop. CDRs also include “specificity determining residues” or “SDRs” that are residues that contact antigen. SDRs are contained within a region of CDRs called abbreviated- CDRs or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are amino acid residues L1 31-34 of L2, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)). Unless otherwise noted, HVR residues and other residues within the variable domain (eg, FR residues) are numbered herein according to Kabat et al., Supra.

  An “immunoconjugate” is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic drugs.

  An “individual” or “subject” is a mammal. Mammals include, but are not limited to, livestock animals (eg, cows, sheep, cats, dogs, horses), primates (eg, non-human primates such as humans, monkeys), rabbits, rodents (eg, mice and Rat). In certain embodiments, the individual or subject is a human.

  An “isolated” antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is determined, for example, by electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reverse phase HPLC). Purified to a purity of 95% or more or 99%. For a review of antibody purity assessment methods, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

  An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes nucleic acid molecules contained in cells that usually contain the nucleic acid molecule, but the nucleic acid molecule is present at a chromosomal location that is extrachromosomal or different from its natural chromosomal location.

  “Isolated nucleic acid encoding an anti-ETBR antibody” refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, such as in a single vector or separate vectors. Nucleic acid molecules as well as such nucleic acid molecules present at one or more locations in the host cell.

  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 group. The mammalian ERK1 / 2 pathway 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) which is an important 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 occupies a central role in the MAPK pathway. Expression of a constitutively active form of MEK results in cell line transformation.

  The term “MEK inhibitor” as used herein refers to any number of known small molecule drug compounds that can inhibit or interrupt the MEK step on the MAP kinase pathway. Examples of suitable MEKi may include, but are not limited to, those described as MEKi-623, MEKi-973, or GSK11012012.

The term “MEKi-973” as used herein refers to the MEK inhibitor (S)-(3,4-difluoro-2-((2-fluoro-4-iodophenyl) amino) phenyl) (3- Refers to hydroxy-3- (piperidin-2-yl) azetidin-1-yl) methanone and has the following structure:

  The term “monoclonal antibody” as used herein means an antibody obtained from a substantially homogeneous population of antibodies, ie, contains, for example, naturally occurring mutations or occurs during the manufacture of a monoclonal antibody formulation. However, the individual antibodies that make up the population are identical and / or bind to the same epitope, except for potential variant antibodies, such as variants that are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies to different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is against a single determinant on the antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention include, but are not limited to, various methods including hybridoma methods, recombinant DNA methods, phage display methods, methods utilizing transgenic animals containing all or part of a human immunoglobulin locus. Such methods and other exemplary methods for making monoclonal antibodies made by various techniques are described herein.

  “Naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radioactive label. Naked antibodies may be present in pharmaceutical formulations.

  “Natural antibody” refers to immunoglobulin molecules of various structures that occur in nature. For example, a native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

  The term “package insert” is customarily used for commercial packages of therapeutic products, including information on efficacy, usage, dosage, administration, combination therapy, contraindications, and / or warnings regarding the use of such therapeutic products. Used to refer to included instructions.

  “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence refers to any conservative substitution of sequence identity after aligning the sequences and introducing gaps as necessary to obtain maximum percent sequence identity. Defined as the percentage of amino acid residues in the candidate sequence that are not considered part of, are identical to the amino acid residues of the reference polypeptide. Alignments for the purpose of determining percent amino acid sequence identity are publicly available in various ways within the skill of the artisan, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software This can be achieved by using computer software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein,% amino acid sequence identity values are generated by using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code is from the US Copyright Office, Washington D.C. C. , 20559 together with user documentation and registered under US copyright registration number TXU510087. ALIGN-2 is also publicly available from Genentech, South San Francisco, California, or can be compiled from its source code. The ALIGN-2 program should be compiled for use on the UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is used for amino acid sequence comparison, the given amino acid sequence A is% amino acid sequence identity with or relative to a given amino acid sequence B (or with a given amino acid sequence B, or On the other hand, a given amino acid sequence A having or containing a predetermined% amino acid sequence identity) is calculated as follows:
100 times the fraction X / Y, where X is the number of amino acid residues with the same and consistent score in the alignment of that program of A and B by the sequence alignment program ALIGN-2, and Y is the total amino acid of B The number of residues. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, then the% amino acid sequence identity of A to B is different from the% amino acid sequence identity of B to A. Unless otherwise noted, all% amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

  The term “pharmaceutical formulation” includes other ingredients that are in a form that permits the biological activity of the active ingredient contained therein and that is unacceptable to the subject to whom the formulation is administered. Refers to a preparation that is not.

“Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation that is non-toxic to a subject and that is not an active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, vehicles, stabilizers, or preservatives.

The term “RG7204” refers to a B-Raf enzyme inhibitor having the molecular formula of C ₂₃ H ₁₈ CIF ₂ N ₃ O ₃ S and the following structure:

  As used herein, “treatment” (and grammatical variations such as “treat” or “treating”) will alter the natural course of the individual being treated. Refers to a clinical intervention that attempts to be performed either for prevention or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, preventing metastasis, and the rate of progression of the disease Delaying, improving or alleviating disease state, and improving remission or prognosis. In some embodiments, the antibodies of the invention are used to delay disease onset or slow disease progression.

  The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable heavy and light chains (VH and VL, respectively) of natural antibodies have domains that each contain four conserved framework regions (FR) and three hypervariable regions (HVR), and are generally similar. It has a structure like that. (See, for example, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies that bind to a specific antigen should be isolated using VH or VL domains from antibodies that specifically bind to the antigen in order to screen the respective library of complementary VL or VH domains. Can do. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

  As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as a self-replicating nucleic acid structure as well as vectors integrated into the genome of the host cell into which it has been introduced. Certain vectors can direct the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as “expression vectors”.

II. Compositions and Methods In one aspect, the invention is based in part on antibodies that bind to ETBR. The antibodies of the present invention are useful, for example, for the treatment of melanoma.

Exemplary Anti-ETBR Antibodies In one aspect, the invention provides an isolated antibody that binds to ETBR. In one embodiment, the anti-ETBR antibody comprises (a) CDR-L1 (KSSQSLLDSDGGTYLN, SEQ ID NO: 7), (b) CDR-L2 (LVSKLDS, SEQ ID NO: 8), (c) CDR-L3 (WQGTHFYPY; SEQ ID NO: 9). ), (D) CDR-H1 (GYTFTSYWMQ; SEQ ID NO: 1), (e) CDR-H2 (TIYPGDGDTSYAQKFKG; SEQ ID NO: 2), and (f) CDR-H3 (WGYAYDIDN; SEQ ID NO: 3) , 2, 3, 4, 5 or 6 CDRs.

  In any of the above embodiments, the anti-ETBR antibody is humanized. In one embodiment, the anti-ETBR antibody comprises a CDR in any of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another aspect, the antibody provides an isolated anti-ETBR antibody having the VL amino acid sequence of SEQ ID NO: 8 and the VH amino acid sequence of SEQ ID NO: 7. In yet another aspect, the present invention provides an anti-ETBR antibody having the VL amino acid sequence of SEQ ID NO: 8 and the VH amino acid sequence of SEQ ID NO: 9.

  In another embodiment, the anti-ETBR antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, relative to the amino acid sequence of SEQ ID NO: 7 or 9. Contains heavy chain variable domain (VH) sequences with 99% or 100% sequence identity. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence An anti-ETBR antibody containing a substitution (eg, a conservative substitution), insertion, or deletion, but containing that sequence retains the ability to bind to ETBR. In certain embodiments, in SEQ ID NO: 7 or 9, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (ie, within FRs).

  In another embodiment, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amino acid sequence of SEQ ID NO: 8 An anti-ETBR antibody comprising a light chain variable domain (VL) having sequence identity is provided. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. An anti-ETBR antibody containing a substitution (eg, a conservative substitution), insertion, or deletion, but containing that sequence retains the ability to bind to ETBR. In certain embodiments, in SEQ ID NO: 8, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted. In certain embodiments, substitutions, insertions, or deletions occur in regions outside of HVR (ie, within FR).

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

  In a further aspect, the present invention provides an antibody that binds to the same epitope as an anti-ETBR antibody provided herein. For example, in one embodiment, an antibody is provided that binds to the same epitope as an anti-ETBR antibody comprising a VH sequence of SEQ ID NO: 7 or 9 and a VL sequence of SEQ ID NO: 8. In certain embodiments, an anti-ETBR antibody is provided that binds to an epitope within the N-terminal extracellular domain # 1 of ETBR consisting of amino acid numbers 64 to 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, eg, an Fv, Fab, Fab ′, scFv, diabody, or F (ab ′) ₂ fragment. In other embodiments, the antibody is a full-length antibody, eg, an intact IgG1 antibody, or other antibody class or isotype as defined herein.

  In a further aspect, as described in Sections 1-7 below, the anti-ETBR antibody according to any of the above embodiments can incorporate any feature, alone or in combination.

Antibody Affinity In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of ≦ 1 μM, ≦ 100 nM, ≦ 10 nM, ≦ 1 nM, ≦ 0.1 nM, ≦ 0.01 nM, or ≦ 0. 0.001 nM (eg, less than 10 ⁻⁸ M, eg, 10 ⁻⁸ M to 10 ⁻¹³ M, eg, 10 ⁻⁹ M to 10 ⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed using the Fab form of the antibody of interest and its antigen, as illustrated by the following assay. Solution binding affinity of Fab to antigen by equilibrating Fab with minimal concentration of ( ^125I ) -labeled antigen in the presence of titration series of unlabeled antigen and capturing antigen bound to anti-Fab antibody coated plate Sex is measured (see, eg, Chen, et al., J. Mol. Biol. 293: 865-881 (1999)). To determine the assay conditions, a MICROTITER® multiwell plate (Thermo Scientific) was coated overnight with 50 mM sodium carbonate (pH 9.6) containing 5 μg / ml capture anti-Fab antibody (Cappel Labs). Then, block with PBS containing 2% (w / v) bovine serum albumin at room temperature (approximately 23 ° C.) for 2-5 hours. A non-adsorbed plate (Nunc # 269620) is mixed with the desired Fab serially diluted with 100 pM or 26 pM [ ¹²⁵ I] antigen (eg, Presta et al. (1997) Cancer Res. 57: 4593-4599 anti-VEGF antibody , Consistent with Fab-12 evaluation). The target Fab is then incubated overnight; however, the incubation may take a long time (eg, approximately 65 hours) to confirm that equilibrium has been reached. The mixture is then transferred to a capture plate and incubated at room temperature (eg, 1 hour). The solution is then removed and the plate is washed 8 times with PBS containing 0.1% polysorbate 20 (Tween 20®). When the plate is dry, 150 μl / well of luminescent material (MICROSCINT-20 ™; Packard) is added and the plate is measured for 10 minutes on a TOPCOUNT ™ γ instrument (Packard). Fabs with a concentration of 20% or less of maximum binding are selected and used for competitive binding measurements, respectively.

According to another embodiment, BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25 ° C. using a fixed antigen CM5 chip of 10 reaction units (RU). Kd is measured using a surface plasmon resonance assay with. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) was prepared using N-ethyl-N ′-(3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N -Activated with hydroxysuccinimide (NHS). Antigen is diluted to 5 μg / ml (˜0.2 μM) with 10 mM sodium acetate (pH 4.8) and injected at a flow rate of 5 μl / min so that the reaction units (RU) of the bound protein is approximately 10. After the injection of antigen, 1M ethanolamine is injected to block the unreacted group. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were added at 25 ° C. at a flow rate of approximately 25 μl / min with 0.05% polysorbate 20 (TWEEN 20 ™) surfactant. Injection into PBS containing agent (PBST). Using a simple one-to-one Langmuir binding model (BIACORE® Evaluation software version 3.2) by fitting the association and dissociation sensorgrams simultaneously, the association rate (kon ) And dissociation rate (koff). The equilibrium dissociation constant (Kd) was calculated as the koff / kon ratio. See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999). If the binding rate by the above surface plasmon resonance assay is greater than 10 ⁶ M- ¹ s- ¹ , a spectrometer such as a stop-flow equipped spectrophometer (Aviv Instruments) or a stirred cuvette 20 nM anti-antigen antibody at 25 ° C. in PBS (pH 7.2) in the presence of increasing concentrations of antigen as measured with an 8000 series SLM-AMINCO ™ spectrophotometer (ThermoSpectronic) equipped with The binding rate can be measured using a fluorescence quenching technique that measures the increase or decrease in the fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass = 16 nm) of the Fab type).

Antibody Fragments In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab ′, Fab′-SH, F (ab ′) ₂ , Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); See also 93/16185; and US Pat. Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F (ab ′) ₂ fragments containing salvage receptor binding epitope residues and having increased in vivo half-life.

  Diabodies are antibody fragments that have 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. See USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

  Single domain antibodies are antibody fragments that contain all or part of an antibody heavy chain variable domain, or all or part of a light chain variable domain. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Pat. No. 6,248,516 B1).

  Antibody fragments can be generated by a variety of techniques, including, but not limited to, intact antibody degradation, as well as production by recombinant host cells (eg, E. coli or phage), as described herein.

Chimeric and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in US Pat. No. 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 (eg, a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a “class switch” antibody in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody comprises an antigen-binding fragment thereof.

  In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized while retaining the specificity and affinity of the parent non-human antibody to reduce immunogenicity to humans. In general, a humanized antibody comprises one or more variable domains in which the HVR, eg, CDR (or portion thereof) is derived from a non-human antibody and FR (or portion thereof) is derived from a human antibody sequence. A humanized antibody optionally also will comprise at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody correspond to those derived from a non-human antibody (eg, the antibody from which the HVR residue is derived) to restore or improve antibody specificity or affinity. Substituted with a residue.

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

  Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the “best fit” method (eg, Sims et al. J. Immunol. 151: 2296 (1993)); Framework regions from human antibody consensus sequences of specific subgroups of variable regions of the chain or heavy chain (eg Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al. J. Immunol., 151: 2623 (1993)); human maturation (somatic mutation) framework region or human germline framework region (eg, Almagro and Fransson, Front. Biosci. 13: 1619- 1633 (2008)); and framework regions from FR library screening (eg, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosek et al., J. Biol. Chem). 271: 22611-22618 (1996)).

Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various 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 prepared by administering an immunogen to an intact human antibody or a transgenic animal with a human variable region or modified to produce an intact antibody in response to an antigen challenge. . Such animals typically replace all or one of the human immunoglobulin loci that replace the endogenous immunoglobulin loci or are either extrachromosomal or randomly integrated into the animal's chromosome. Part. In such 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). Also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE (TM) technology; U.S. Patent No. 5,770, describing HuMab (R) technology. 429; U.S. Pat. No. 7,041,870 describing KM MOUSE.RTM. Technology and U.S. Patent Application Publication No. 2007/0061900 describing VelociMouse.RTM. Technology). reference. Intact antibody-derived human variable regions produced in such animals may be further modified, for example, by combining with different human constant regions.

  Human antibodies can be made by hybridoma-based methods. Human myeloma and mouse-human heterocell lines have been described for the production of human monoclonal antibodies. (See, eg, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pages 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al. , J. Immunol., 147: 86 (1991)). Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Further methods are described, for example, in US Pat. No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006). ) (Describes human-human hybridomas). 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 selected Fv clone variable domain sequences from human-derived phage display libraries. Such variable domain sequences may then be combined with the desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

Antibodies from libraries The antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening libraries for antibodies with the desired binding properties. Such methods are reviewed, for example, in Hoogenboom et al. In Methods in Molecular Biology 178: 1-37 (O'Brien et al., Ed., Human Press, Totowa, NJ, 2001) and, for example, McCafferty et al., Nature 348: 552-554; 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 given phage display method, repertoires of VH and VL genes are individually cloned by polymerase chain reaction (PCR) and recombined randomly into a phage library, after which Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994) can be screened for antigen-binding phages. Phages usually present antibody fragments as either single chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, a naïve repertoire can be cloned (eg from a human) and antibodies against a wide range of non-self and self antigens without any immunization as described in Griffiths et al., EMBO J, 12: 725-734 (1993) It is possible to provide a single source of Finally, the naive library also cloned non-rearranged V gene segments from stem cells as described in Hoogenboom and Winter, J. Mol. Biol. 227: 381-388 (1992). It can be made synthetically by encoding a highly variable CDR3 region using PCR primers containing random sequences and achieving in vitro rearrangement. Patent publications describing human antibody phage libraries include, for example, US Pat. No. 5,750,373 and US Patent Application Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007 / No. 0117126, No. 2007/0160598, No. 2007/0237764, No. 2007/0292936 and No. 2009/0002360.

  An antibody or antibody fragment isolated from a human antibody library is considered herein a human antibody or a fragment of a human antibody.

Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, eg, bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for ETBR and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of ETBR. Bispecific antibodies can also be used to localize cytotoxic drugs to cells expressing ETBR. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

  Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829 and Traunecker et al., EMBO J. 10: 3655 (1991)) and “knob-in-hole” engineering (see, eg, US Pat. No. 5,731, 168). Multispecific antibodies also manipulate the electrostatic steering effect to create Fc-heterodimeric molecules of antibodies (WO 2009 / 089004A1), cross-linking two or more antibodies or fragments ( See, eg, US Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)) using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al. al., J. Immunol., 148 (5): 1547-1553 (1992)), using “diabody” technology to generate bispecific antibody fragments (eg, Hollinger et al. , Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)), using single chain Fv (sFv) dimers (eg, Gruber et al., J. Immunol., 152: 5368). (1994)) and, for example, as described in Tutt et al. J. Immunol. 147: 60 (1991) It can be created by preparing specific antibodies.

  Modified antibodies having three or more functional antigen binding sites, including “Octopus antibodies” are also included herein (see, eg, US Patent Application Publication No. 2006/0025576 A1).

  The antibody or fragment herein also includes a “Dual Acting FAb” or “DAF” that includes an antigen binding site that binds ETBR as well as other different antigens (eg, US Patent Application Publication No. 2008/2008). / 0069820).

Antibody Variants 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 can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, residue deletions and / or insertions and / or substitutions within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties, eg, antigen binding.

Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites subject to substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading “Conservative substitutions”. More substantial changes are given under the heading “Typical substitutions” in Table 1 and are further described below with reference to the amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product screened for the desired activity, eg, retention / improvement of antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Amino acids can be grouped based on common side chain properties:
Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;
Acidity: Asp, Glu;
Basic: His, Lys, Arg;
Residues that affect chain orientation: Gly, Pro;
Aromatic: Trp, Tyr, Phe.

  Non-conservative substitutions will require exchanging one member of these classes for another.

  One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). In general, variants that can be selected for further study will have altered (eg, improved) specific biological properties (eg, increased affinity, immunogens) compared to the parent antibody. And / or substantially retain certain biological properties of the parent antibody. A typical substitutional variant is an affinity matured antibody, which can be conveniently generated using, for example, affinity display techniques based on phage display described herein. Briefly, one or more HVR residues are mutated and mutant antibodies are displayed on phage and screened for a specific biological activity (eg, binding affinity).

  Changes (eg, substitutions) can be made with HVRs, eg, to improve antibody affinity. Such changes occur at HVR “hot spots”, ie, residues encoded by codons that are frequently mutated during the process of somatic maturation (eg, Chowdhury, Methods Mol. Biol. 207: 179-196 ( 2008)), and / or SDR (a-CDR), and the resulting mutant VH or VL is tested for binding affinity. Affinity maturation by constructing and reselecting from a secondary library is described, for example, by Hoogenboom et al. In Methods in Molecular Biology 178: 1-37 (O'Brien et al., Ed., Human Press, Totowa, NJ, ( In some embodiments of affinity maturation, the diversity is any of a variety of methods (eg, variant PCR, strand shuffling, or oligonucleotide-targeted mutagenesis). Into a variable gene selected for maturation, and then a secondary library is created, which is then screened to identify antibody variants with the desired affinity. Another way of introducing diversity involves an HVR-oriented approach in which several HVR residues (eg, 4 to 6 residues at a time) are randomized. The group is Example, if alanine scanning mutagenesis, or by using modeling, specifically can be identified .CDR-H3 and CDR-L3 is often targeted.

  In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs as long as such modification does not substantially reduce the antibody's ability to bind antigen. For example, conservative modifications that do not substantially reduce binding affinity (eg, conservative substitutions provided herein) can be made in HVRs. Such modifications may be outside the HVR “hot spot” or SDR. In certain embodiments of the variant VH or VL sequences given above, each HVR is either unchanged or contains only one, two or three amino acid substitutions.

  A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is described in “Alanine,” as described by Cunningham and Wells (1989) Science, 244: 1081-1085. This is called “scanning mutagenesis”. In this method, residues or groups of target residues (eg, charged residues such as arg, asp, his, lys and glu) are identified to determine whether the antigen-antibody interaction is affected. To that end, it is replaced with a neutral or negatively charged amino acid (eg alanine or polyalanine). Additional substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the first substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex identifies the point of contact between the antibody and the antigen. Such contact residues and neighboring residues can be targeted or excluded as candidates for substitution. Variants can be screened to determine if they contain the desired property.

  Amino acid sequence insertions include amino and / or carboxyl terminal fusions over the length of the polypeptide containing from one residue to over a hundred residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionine residue. Other insertional variants of the antibody molecule include fusions to the N-terminus or C-terminus of the antibody to the enzyme (eg in the case of ADEPT), or to a polypeptide that increases the serum half-life of the antibody.

Glycosylation variants 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 glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or deleted.

  If the antibody contains an Fc region, the sugar attached to it can be changed. Natural-type antibodies produced by mammalian cells typically contain branched, bi-branched oligosaccharides that are commonly attached by N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15: 26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, and fucose linked to a “trunk” GlcNAc of a biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the invention can be made to create antibody variants with certain improved properties.

  In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the fucose amount of such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is the sum of all sugar structures attached to Asn297 as measured by MALDI-TOF mass spectrometry, eg as described in WO 2008/077546 (eg complex, hybrid And high mannose structure) by calculating the average amount of fucose in the sugar chain of Asn297. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues), but Asn297 is also approximately ± 3 amino acids upstream or downstream of position 297, ie, the antibody. Due to minor sequence variations, it can be placed between positions 294 and 300. Such fucosylated mutants may have improved ADCC function. For example, see US Patent Application Publication No. 2003/0157108 (Presta, L.); US Patent Application Publication No. 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to “unmodified fucose” or “fucose-deficient” antibody variants include US 2003/0157108, WO 2000/61739, WO 2001/29246, US Patent Application Publication No. 2003/0115614, United States Patent Application Publication No. 2002/0164328, United States Patent Application Publication No. 2004/0093621, United States Patent Application Publication No. 2004/0132140, United States Patent Application Publication No. 2004/0110704, United States Patent Application Publication No. 2004/0110282, U.S. Patent Application Publication No. 2004/0109865, International Publication No. 2003/085119, International Publication No. 2003/084570, International Publication No. 2005/035586, International Publication No. 2005/035778. International publication 2005/053742; WO 2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) It is. Examples of cell lines capable of producing unfucose-modified antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Patents) Published application 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., In particular Example 11), and knockout cell line, alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (eg, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94 (4): 680-688 (2006); and internationally published. No. 2003/085107).

  The antibody variant further comprises, for example, a bisected oligosaccharide in which a biantennary oligosaccharide bound to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may reduce fucosylation and / or improve ADCC function. Examples of such antibody variants are, for example, WO 2003/011878 (Jean-Mairett et al.); US Pat. No. 6,602,684 (Umana et al.); And US Patent Publication No. 2005/0123546. (Umana et al.). Antibody variants are also provided that have at least one galactose residue within the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are, for example, WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.). It is described in.

Fc region variants In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. A variant of the Fc region may comprise a sequence of a human Fc region (eg, an Fc region of human IgG1, IgG2, IgG3 or IgG4) that includes an amino acid modification (eg, substitution) at one or more amino acid positions.

  In certain embodiments, the invention makes it a desirable candidate for applications where the half-life of the antibody in vivo is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. Antibody variants with some but not all effector functions are contemplated. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC activity and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that an antibody lacks FcγR binding (and therefore probably lacks ADCC activity) but retains FcRn binding ability. Primary cells that mediate ADCC, NK cells express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. Expression of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of molecules of interest include US Pat. No. 5,500,362 (eg, 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 (eg, Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays can be used (eg, ACTI ™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, 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, or in addition, ADCC of the molecule of interest Activity can be assessed in vivo, for example in animal models, as disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998). The antibody also binds body C1q. Can be done to confirm that it is not possible, and therefore lacks CDC activity, eg See the C1q and C3c binding ELISAs of 2006/0298779 and WO 2005 / 100402.CDC assays can be performed to assess complement activation (see, eg, Gazzano-Santoro et al., J. Biol. (See 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)). , And in vivo clearance / half-life measurements can also be performed using methods known in the art (eg, 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 residues 238, 265, 269, 270, 297, 327, 329 of the Fc region (US Pat. No. 6,737,056). Such Fc variants have substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called “DANA” Fc variants with substitutions of residues 265 and 297 to alanine. Fc variants are included (US Pat. No. 7,332,581).

  Certain antibody variants have been described that have improved or decreased binding to FcR. (For example, US Pat. 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 one or more amino acid substitutions that improve ADCC, eg, substitutions at positions 298, 333, and / or 334 of the Fc region (EU residue numbering).

  In some embodiments, alterations in the Fc region are made that result in altered (ie, improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC), eg, US Pat. No. 6,194. 551, International Publication No. WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

  Antibodies (Guyer et al., J. Immunol. 117: 587 (1976) and Kim with improved binding to the neonatal Fc receptor (FcRn), which have an increased half-life and are responsible for the transfer of maternal IgG to the fetus et al., J. Immunol. 24: 249 (1994)) is described in US Patent Application Publication No. 2005/0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants are residues of the Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 with one or more substitutions, for example those with residue 434 in the Fc region (US Pat. No. 7,371,826).

  Duncan & Winter, Nature 322: 738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821; and other publications of Fc region variants. See also 94/29351.

Cysteine engineered antibody variants In certain embodiments, it may be desirable to create a cysteine engineered antibody, eg, “thioMAb (s)”, in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the substituted residue occurs at an accessible site of the antibody. By substituting those residues with cysteine, the reactive thiol group is thereby positioned at the accessible site of the antibody to create an immunoconjugate as further described herein. Can be used to conjugate the antibody to other moieties such as, for example, drug moieties or linker-drug moieties. In certain embodiments, one or more of the following residues may be substituted with cysteine: light chain V205 (Kabat numbering); heavy chain A118 (EU numbering); and heavy chain Fc region S400 (EU number). Date). Cysteine engineered antibodies can be generated, for example, as described in US Pat. No. 7,521,541.

Antibody Derivatives In certain embodiments, the antibodies provided herein can be further modified to include additional non-protein moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, Poly-1,3,6-trioxolane, ethylene / maleic anhydride copolymer, polyamino acid (either homopolymer or random copolymer) and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol alone Polymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization is not limited, but may include specific properties or functions of the antibody to be improved, whether the antibody derivative is used in therapy under limited conditions, etc. Can be determined based on considerations.

  In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, but includes but is not limited to a wavelength that does not harm normal cells but heats the non-protein portion to a temperature at which the proximal cells of the antibody-non-protein portion are killed. It is.

Recombinant Methods and Compositions Antibodies can be produced using recombinant methods and compositions, for example, as described in US Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-ETBR antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence that includes the VL of the antibody and / or an amino acid sequence that includes the VH of the antibody (eg, the light and / or heavy chain of the antibody). In further embodiments, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In one embodiment, the host cell comprises (eg, transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and an amino acid sequence comprising an antibody VH, or (2) A first vector containing a nucleic acid encoding an amino acid sequence containing an antibody VL, and a second vector containing a nucleic acid encoding an amino acid sequence containing an antibody VH. In one embodiment, the host cell is a eukaryote, such as a Chinese hamster ovary (CHO) cell, or a lymphoid cell (eg, Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-ETBR antibody is provided, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as described above, And optionally recovering the antibody from the host cell (or host cell culture medium).

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

  Suitable host cells for cloning or expression of the antibody-encoding vector include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly where glycosylation and Fc effector function are not required. See, eg, US Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 for expression of antibody fragments and polypeptides in bacteria. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pages 245-254, describing the expression of antibodies in E. coli.) Thereafter, the antibodies can be isolated from the bacterial cell paste in the soluble fraction and further purified.

  In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including fungi and yeast strains, whose glycosylation pathway is “Humanized”, resulting in the production of antibodies having a partial or complete human glycosylation pattern. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

  Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified and can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

  Plant cell culture can be used as a host. For example, US Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (antibody production in transgenic plants) See PLANTIBODIES ™ technology).

  Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 strain transformed with SV40 (COS-7), human embryonic kidney strain (Graham et al., J. Gen Virol. 36:59 (1977) 293 cells or 293 cells, baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells described in Mather, Biol. Reprod. 23: 243-251 (1980)), monkey kidney cells (CV1) ), African green monkey kidney cells (VERO-76), human cervical cancer cells (HeLa), canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A), human lung cells (W138); human hepatocytes (HepG2), Mouse mammary tumors (MMT060562), such as TRI cells, MRC5 cells, and FS4 cells described in Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982) Other useful mammalian hosts Cell lines are Chinese hamster ovary (CHO) cells, including 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 a review of specific mammalian host cell lines suitable for antibody production, see, eg, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), page See 255-268 (2003).

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

Binding Assays and Other Assays In one embodiment, the antibodies of the invention are tested for their antigen binding activity by known methods such as, for example, ELISA, Western blotting.

  In other embodiments, competition assays are performed for binding to ETBR, eg, Hu5E9v. 1 or Hu5E9v. Can be used to identify antibodies that compete with 2. In certain embodiments, such competing antibodies are Hu5E9v. 1 or Hu5E9v. Bind to the same epitope bound by 2 (eg, linear or conformational epitope). A detailed exemplary method for mapping the epitope to which an antibody binds is described in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology 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 a typical competition assay, immobilized ETBR is tested for the ability to compete with a first labeled antibody that binds to ETBR (eg, Hu5E9v.1 or Hu5E9v.2) and the first antibody for binding to ETBR. Incubated in a solution containing the prepared second unlabeled antibody. The second antibody may be present in the hybridoma supernatant. As a control, immobilized ETBR is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to ETBR, excess unbound antibody is removed and the amount of label bound to immobilized ETBR is measured. If the amount of label bound to immobilized ETBR is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody competes with the first antibody for binding to ETBR. It shows that you are doing. See Harlow and Lane (1988) Antibodies: Laboratory Manual (A Laboratory Manual) ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Activity Assay In one aspect, an assay is provided to identify whether an anti-ETBR antibody and / or BRAFi compound has biological activity. Biological activity includes those described in the Examples, for example, in vitro melanoma cell survival assays, or in vivo xenograft models in which melanoma cell lines are transplanted into nude mice and tumor growth inhibition (TGI) is assessed. Can do.

Immunoconjugates The present invention also includes chemotherapeutic agents or drugs, growth inhibitory agents, toxins (eg, protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes, etc. Provided are immunoconjugates comprising an anti-ETBR antibody herein conjugated to one or more cytotoxic drugs.

  The invention also includes chemotherapeutic agents, drugs, growth inhibitors, toxins (eg, protein toxins, bacteria, filamentous fungi, enzymatically active toxins or fragments thereof from plants or animals), or radioisotopes (ie, radioconjugates). An immunoconjugate (referred to interchangeably with “antibody-drug conjugate” or “ADC”) is provided, comprising an antibody conjugated to one or more cytotoxic agents such as (gate).

  Immunoconjugates have been used for local delivery of cytotoxic drugs, ie, agents that kill or inhibit cell growth or proliferation in the treatment of cancer (Xie et al (2006) Expert. Opin Biol. Ther. 6 (3): 281-291; Kovtun et al (2006) Cancer Res. 66 (6): 3214-3121; Law et al (2006) Cancer Res. 66 (4): 2328-2337; Lambert, J. (2005) Curr. Opinion in Pharmacology 5: 543-549; Wu et al (2005) Nature Biotechnology 23 (9): 1137-1146; Payne, G. (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; US Pat. No. 4,975,278). Immunoconjugates allow targeted delivery of drug components to the tumor and intracellular accumulation there, where systemic administration of the unconjugated drug to normal cells as well as to tumor cells that need to be removed. Can cause unacceptable levels of toxicity (Baldwin et al., Lancet (Mar. 15, 1986) pp. 603-05; Thorpe (1985) “Antibody Carriers of Cytotoxic Drugs in Cancer Treatment: Summary” (“Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, ") in Monoclonal Antibodies '84: Biological And Clinical Applications (A. Pinchera et al., Ed.) Pp. 475-506. Polyclonal and monoclonal antibodies are useful in this strategy. (Rowland et al., (1986) Cancer Immunol. Immunother. 21: 183-87) Drugs used in this method include daunomycin, doxorvidin, methotrexate and vindezin (Rowland et al. (1986), supra) Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, geldanamycin (Mandler et al. (2000) Jour. Of the Nat. Cancer). Inst. 92 (19): 1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10: 1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13: 786-791), maytansinoids (EP 1391213 Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623), and calicheamicin (Lode et al., (1998) Cancer Res. 58: 2928; Hinman et al., (1993) Cancer Res. 53: 3336-3342), and efforts to design and improve ADCs include monoclonal antibody (mAbs) selectivity as well as drug mode of action, drug binding, drug / antibody ratio (loading). ), And focus on drug release properties (McDonagh (2006) Protein Eng. Design & Sel .; Doronina et al (2006) Bioconj. Chem. 17: 114-1 24; Erickson et al (2006) Cancer Res. 66 (8): 1-8; Sanderson et al (2005) Clin. Cancer Res. 11: 843-852; Jeffrey et al (2005) J. Med. Chem. 48 : 1344-1358; Hamblett et al (2004) Clin. Cancer Res. 10: 7063-7070). The toxins exert their cytotoxic effects by mechanisms including tubulin binding, DNA binding or topoisomerase inhibition. Certain cytotoxic agents tend to be less inactive or less active when conjugated to large antibodies or protein receptor ligands.

  Zevalin® (ZEVALIN) (ibritumomab tiuxetan, Biogen / Idec) is a mouse IgG1κ monoclonal antibody against the CD20 antigen found on the cell surface of normal and malignant B lymphocytes and 111In or 90Y radioactivity. It is an antibody-radioisotope conjugate in which an isotope is bound by a thiourea linker chelator (Wiseman et al. (2000) Eur. Jour. Nucl. Med. 27 (7): 766-77; Wiseman et al. (2002 ) Blood 99 (12): 4336-42; Witzig et al. (2002) J. Clin. Oncol. 20 (10): 2453-63; Witzig et al. (2002) J. Clin. Oncol. 20 (15): 3262 -69). Zevalin is active against B-cell non-Hodgkin lymphocytes (NHL), but administration causes severe and prolonged cytopenias in most patients. Myrotag ™ (MYLOTARG ™) (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate consisting of huCD33 antibody linked to calicheamicin, is used to treat acute myeloid leukemia Approved in 2000 (Drugs of the Future (2000) 25 (7): 686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762) No. 5739116; No. 5767285; No. 5773001). Antibody-drug conjugates consisting of the maytansinoid, DM1, linked to trastuzumab show potent antitumor activity in HER2 overexpressing trastuzumab-sensitive and resistant tumor cell lines and human cancer xenograft models. Trastuzumab-mcc-DM1 (T-DM1) is currently being evaluated in phase II clinical trials in patients whose disease is resistant to HER2-directed therapy (Beeram et al (2007) “Trastuzumab-MCC-DM1 ( T-DM1), Phase I study of trastuzumab-mcc-DM1 (T-DM1), a first-in-class HER2 antibody-drug conjugate (ADC) in patients with HER2 + metastatic breast cancer (BC) first-in-class HER2 antibody-drug conjugate (ADC), in patients (pts) with HER2 + metastatic breast cancer (BC)), 43rd American Society of Clinical Oncology June 1 (Abstract 1042; Krop et al., European Cancer Conference) ECCO, poster 2118, September 23-27, 2007, Barcelona; US Pat. No. 7,097,840; US Pat. No. 2005/0276812; US Pat. No. 2005/0166993. No.) Synthetic analogs of auristatin peptide, auristatin E (AE) and monomethyl auristatin (MMAE), dolastatin, and chimeric monoclonal antibody cBR96 (specific for Lewis Y on cancer cells) and It is conjugated to cAC10 (specific for CD30 on hematological malignancies) (Doronina et al. (2003) Nature Biotechnology 21 (7): 778-784) and is in the therapeutic development stage.

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

  In certain embodiments, immunoconjugates include antibodies and small molecule drugs and cytotoxic activity such as, but not limited to, calicheamicin, maytansinoids, dolastatin, auristatin, anthracyclines, taxanes, trichothecenes, and CC1065. One or more small molecule drug moieties (toxins) comprising derivatives of these drugs having Examples of these immunoconjugates are discussed in more detail below.

Exemplary Immunoconjugates The immunoconjugates (or “antibody-drug conjugates” (“ADC”)) of the present invention may be of the formula I below, wherein the antibody is either mono- or through any linker (L). Conjugated to multiple drug moieties (D).

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

A typical linker linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”). )), P-aminobenzyloxycarbonyl (“PAB”), N-succinimidyl 4 (2-pyridylthio) pentanoate (“SPP”), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1carboxylate (“ SMCC "), and N-succinimidyl (4-iodo-acetyl) aminobenzoate (" SIAB "). Various 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 into the cell. For example, acid labile linkers (eg, hydrazones), protease sensitive (eg, peptidase sensitive) linkers, photolabile linkers, dimethyl linkers or disulfide containing linkers can be used (Chari et al., Cancer Research, 52: 127- 131 (1992); US Pat. No. 5,208,020).

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

  Where A is a stretcher unit, 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 Ab; D and p are defined above with respect to Formula I. Exemplary embodiments of this linker are described in US Patent Application Publication No. 2005-0238649 A1, which is incorporated herein by reference.

In some embodiments, the linker component can include a “stretcher unit” that links the antibody to another linker component or to a drug moiety. A typical stretcher unit is shown below (where the wavy line indicates the site of covalent binding to the antibody):

  In some embodiments, the linker component can include amino acid units. In one such embodiment, the amino acid unit allows cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate when exposed to intracellular proteases such as lysosomal enzymes. 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. Typical dipeptides are valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); or N-methyl-valine-citrulline (Me-). val-cit). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid units can include naturally occurring amino acid residues, as well as trace amino acids and non-naturally occurring amino acid analogs such as citrulline. Amino acid units can be designed and optimized in particular for selectivity for enzymatic cleavage by enzymes such as tumor-associated proteases, cathepsins B, C and D or plasmin proteases.

  In some embodiments, the linker component can include a “spacer” unit that links the antibody to the drug moiety, either directly 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-sacrificing” spacer unit is one in which part or all of the spacer unit remains attached to the drug moiety by enzymatic (eg, proteolytic) cleavage of the ADC. Examples of non-self-sacrificing spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. Also contemplated are other combinations of peptidic spacers that are affected by sequence-specific enzymatic cleavage. For example, enzymatic cleavage of a ADC containing a glycine-glycine spacer unit by a tumor cell-associated protease will release the glycine-glycine-drug moiety from the remaining ADC. In such embodiments, the glycine-glycine-drug moiety is subjected to a different hydrolysis treatment of the tumor cells, thereby separating the glycine-glycine spacer unit from the drug moiety.

  A “self-sacrificing” spacer unit releases the drug moiety without a different hydrolysis treatment. In some embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In such embodiments, p-aminobenzyl alcohol is linked to the amino acid unit by an amide bond, and a carbamate, methyl carbamate or carbonate is made between the benzyl alcohol and the cytotoxic drug. 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, the phenylene moiety of the p-aminobenzyl unit is replaced with Qm, where Q is -C1-C8 alkyl, -O- (C1-C8 alkyl), -halogen, -nitro, or -cyano; And m is an integer in the range of 0-4. Examples of self-sacrificing spacer units include, but are not limited to, aromatic compounds that are electronically similar to p-aminobenzyl alcohol (see, eg, US Patent Publication No. 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 are included. Spacers that are cyclized by amide bond hydrolysis, eg substituted or unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223); appropriately substituted bicyclo [2 2.1] and bicyclo [2.2.2] ring systems (Storm et al., J. Amer. Chem. Soc., 1972, 94, 5815); and 2-aminophenylpropionic acid amides (Amsberry et al., J. Org. Chem., 1990, 55, 5867) 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-sacrificing spacer useful for ADCs.

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

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

  In other embodiments, the linker L may be a dendritic linker for covalent attachment of one or more drug components to the antibody by a branched, multifunctional linker component (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12: 2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11: 1761-1768). Dendritic linkers can increase, or load, the molar ratio of drug to antibody, which is related to the potency of the ADC. Thus, if a cysteine engineered antibody has only one reactive cysteine thiol group, many drug components can be attached via a dendritic linker.

Exemplary linker components and combinations thereof are shown below in connection with the ADC of Formula II:

  A linker component containing a stretcher, a spacer and an amino acid unit can be synthesized by methods known in the art, such as those described in US Patent Application Publication No. 2005-0238649 A1, for example.

Typical drug moieties maytansine and maytansinoids

  In some embodiments, the immunoconjugate comprises an antibody conjugated with one or more maytansinoid molecules. Maytansinoids are mitotic inhibitors that act to inhibit tubulin polymerization. Maytansine was originally isolated from the East African shrub Maytenus serrata (US Pat. No. 3,896,111). It was subsequently discovered that certain microorganisms produce maytansinoids, such as maytansinol and C-3 maytansinol esters (US Pat. No. 4,115,042). Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,776; 4330928; 4331529; 4317821; 43232348; 43331598; 43361650; 43364866; 44424219; 4436263; 43621663; and 43671533 It is disclosed.

  The maytansinoid drug moiety is an attractive drug moiety in antibody-drug conjugates. Because they are: (i) relatively available for preparation by fermentation or chemical modification, derivatization of fermentation products, (ii) suitable for conjugation to antibodies through non-disulfide linkers Suitable for derivatization with functional groups present, (iii) stable in plasma, and (iv) effective against various tumor cell lines.

  Maytansine compounds suitable for use as maytansinoid drug moieties are well known in the art and can be isolated from natural sources according to known methods or can be produced using genetic engineering techniques. Maytansinol and maytansinol analogs can also be prepared synthetically according to known methods.

  Exemplary maytansinoid drug moieties include those having a modified aromatic ring, such as C-19-dechloro (US Pat. No. 4,424,219) (prepared by lithium aluminum hydride reduction of ansamitocin P2 ); C-20-hydroxy (or C-20-dimethyl) +/- C-19 dechloro (US Pat. Nos. 4,361,650 and 4,307,016) (demethylation using Streptomyces or actinomycetes or using LAH) C-20-dimethoxy, C-20-acyloxy (OCOR), +/- dechloro (US Pat. No. 4,294,757) (prepared by acylation with acyl chloride) and others Those having a modification at the position of are included.

Typical maytansinoid drug moieties include those having a modified, for example, be prepared by the reaction of C-9-SH (U.S. Pat. No. 4424219) _{(H 2} S or a maytansinol having _P 2 _{S 5} C-14-alkoxymethyl (demethoxy / CH ₂ OR) (US Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH ₂ OH or CH ₂ OAc) (US Pat. No. 4,450,254) C-15-hydroxy / acyloxy (US Pat. No. 4,364,866) (prepared by conversion of maytansinol by Streptomyces); C-15-methoxy (US Pat. Nos. 4,313,946 and 4,315,929) ( Isolated from Trewia nudlflora); C-18-N-demethyl (US patent) 4362663 and 4322348) (prepared by demethylation of maytansinol by the genus Streptomyces); and 4,5-deoxy (US Pat. No. 4,371,533) (maytansinol titanium trichloride / Prepared by LAH reduction).

  Depending on the type of binding, many positions on the maytansine compound are known to be useful as binding positions. For example, to form an ester bond, the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group are all suitable. .

Maytansinoid drug components include those having the following structure.

Here, the wavy line indicates the covalent bond of the sulfur atom of the maytansinoid drug component to the linker of the ADC. R may independently be H or C ₁ -C ₆ alkyl. The alkylene chain with the amide group attached to the sulfur atom may be methanyl, ethanyl or propyl, ie, m is 1, 2 or 3 (US Pat. No. 6,334,410; US Pat. No. 5,208,020; US) Patent No. 7276497; Chari et al (1992) Cancer Res. 52: 127-131; Liu et al (1996) Proc. Natl. Acad. Sci USA 93: 8618-8623).

All stereoisomers of maytansinoid drug components are contemplated for the compounds of the present invention, ie any combination of R and S coordination at the chiral carbon of D (US Pat. No. 7,276,497; US Pat. No. 6,931,748). U.S. Pat. No. 6,441,163; U.S. Pat. No. 6,334,410 (RE39151); U.S. Pat. No. 5,208,020; Widdison et al (2006) J. Med. Chem. 49: 4392-4408, which are incorporated by reference in their entirety. )). In one embodiment, the maytansinoid drug component will have the following stereochemistry.

Exemplary embodiments of maytansinoid drug components include DM1; DM3; and DM4 having the following structures:

  Here, the wavy line indicates the covalent bond 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 Patent Application Publication No. 2005/0276812; US Patent Application Publication No. 2005/016993).

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

A typical antibody-drug conjugate in which DM1 is linked to the antibody thiol group by a BMPEO linker has the following structure and abbreviations:
Here, Ab is an antibody, n is 0, 1 or 2, and p is 1, 2, 3 or 4.

Immunoconjugates containing maytansinoids, methods for their preparation and their therapeutic uses are disclosed, for example, in US Pat. Nos. 5,208,020 and 5,541,064, US Patent Application Publication No. 2005/0276812 A1, and EP 0425235 B1. The disclosure of which is incorporated herein by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93: 8618-8623 (1996) describes an immunoconjugate containing a maytansinoid designated DM1, which binds to the monoclonal antibody C242 against human colorectal cancer. Has been. The conjugate has been found to be highly cytotoxic to cultured colon cancer cells and exhibits antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research, 52: 127-131 (1992), describes a mouse antibody A7 in which a maytansinoid binds to an antigen of a human colon cancer cell line via a disulfide bond, or a HER-2 / neu oncogene. Other mouse monoclonal antibodies that bind to TA. An immunoconjugate bound to 1 is described. TA. The cytotoxicity of 1-maytansinoid conjugates was tested in vitro on a human breast cancer cell line SK-BR-3 that expresses 3 × 10 ⁵ HER-2 surface antigen per cell. Drug conjugates achieve a degree of cytotoxicity similar to that of the free maytansinoid agent, which is increased by increasing the number of maytansinoid molecules per antibody molecule. A7-maytansinoid conjugates showed low systemic cytotoxicity in mice.

  Antibody-maytansinoid conjugates are prepared by chemically coupling an antibody to a maytansinoid molecule with little reduction in the biological activity of either the antibody or the maytansinoid molecule. See, for example, US Pat. No. 5,208,020, the disclosure of which is expressly incorporated by reference. A single molecule of toxin / antibody is expected to increase cytotoxicity in the use of naked antibodies, but an average of 3-4 maytansinoid molecules bound per antibody molecule is the function or lysis of the antibody. It exhibits the effect of improving cytotoxicity against target cells without adversely affecting sex. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in US Pat. No. 5,208,020 and other patents and publications that are not the aforementioned patents. Preferred maytansinoids are maytansinol analogs, such as maytansinol, and various maytansinol esters modified at the aromatic ring or other positions of the maytansinol molecule.

  For example, US Pat. No. 5,208,020 or European Patent No. 0425235 B1, Chari et al., Cancer Research, 52: 127-131 (1992), and US Patent Application Publication No. 2005/016993 A1 (the disclosures of which are clearly identified by reference) There are many linking groups known in the art for making antibody-maytansinoid conjugates, including those disclosed in US Pat. Antibody-maytansinoid conjugates comprising the linker component SMCC are prepared as disclosed in “Antibody-drug conjugates and methods” of US Patent Publication No. 2005/0276812 A1. Can be done. Linkers include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups as disclosed in the aforementioned patents. Additional linkers are described and exemplified herein.

  Conjugates of antibodies and maytansinoids have various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane -1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (eg dimethyl adipimidate HCL), active esters (eg disuccinimidyl suberate), aldehydes (eg , Glutaraldehyde), bisazide compounds (eg bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (eg bis- (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (eg toluene-2,6- Isocyanate), and bis-active fluorine compounds (e.g., can be made using 1,5-difluoro-2,4-dinitrobenzene). In one embodiment, N-succinimidyl-4- (2-pyridylthio) pentanoate (SPP) or N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173: 723-737 (1978)).

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

Auristatins and dolastatins In some embodiments, the immunoconjugate is conjugated to dolastatin or a drostatin peptidic analog or derivative such as auristatin (US Pat. Nos. 5,635,483; 5,780,588). Antibody. Dolastatin and auristatin prevent microtubule dynamics, GTP hydrolysis and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45 (12): 3580-3584), anticancer activity (US Patent No. 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42: 2961-2965). The dolastatin or auristatin drug component may be attached to the antibody by the N (amino) terminus or the C (carboxyl) terminus of the peptidyl drug molecule (WO 02/088172).

  Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF (US Pat. No. 7,498,298).

The peptidic drug moiety may be selected from the following formulas D _E and D _F.

Here, the wavy lines D _E and D _F independently represent the site of covalent binding to the antibody or antibody-linker component at each position.

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

R ³ is 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 Selected from _3- C ₈ heterocycle and C ₁ -C ₈ alkyl- (C ₃ -C ₈ heterocycle);

R ⁴ is 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 Selected from _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 together form a cyclic carbon, and Ra and Rb are independently selected from H, C ₁ -C ₈ alkyl and C ₃ -C ₈ carbocycle. Having the formula — (CR ^a R ^b ) _n —, where 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 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 Selected from _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 ^{R 12} is located in the ^{NR 12} is _C 1 -C ₈ alkyl;

R ¹¹ is from 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 ) _2. Selected;

  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;

Each occurrence of R ¹⁵ is independently H, COOH, — (CH ₂ ) _n —N (R ¹⁶ ) ₂ , — (CH ₂ ) n —SO ₃ H, or — (CH ₂ ) _n —SO _3. It is -C ₁ -C ₈ alkyl;

Each occurrence of R ¹⁶ 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 selected from carbocyclic); n is an integer from 0 to 6.

In one ^embodiment, R 3, ^{R 4} and ^{R 7} are independently isopropyl or sec- butyl, 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 yet another embodiment, R ² and R ⁶ are each methyl and R ⁹ is —H. In still other embodiments, each occurrence of R ₈ is —OCH ₃ . In an exemplary embodiment, 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 each R ⁸ Occurrence of —OCH ₃ and R ⁹ is —H. In one embodiment, Z is —O— or —NH—. In one embodiment, R ¹⁰ is aryl.
In one 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 —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 formula _DE is MMAE, where the wavy line indicates the covalent attachment of the antibody-drug conjugate to the linker (L).

Embodiments of the exemplary auristatin of formula D _F is MMAF, wherein the wavy line antibody - indicates a covalent bond to the drug conjugate of the linker (L) (U.S. Pat. No. 7,498,298 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124)).

  Other exemplary embodiments include a monomethylvaline compound having a phenylalanine carboxy modification at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008848) and a phenylalanine side at the C-terminus of the pentapeptide auristatin drug moiety. Monomethyl valine compounds having chain modifications (WO 2007/008603) are included.

Other drug moieties include the following MMAF derivatives, where the wavy line indicates the covalent attachment of the antibody-drug conjugate to the linker (L).

In one aspect, hydrophilic groups including, but not limited to, triethylene glycol ester (TEG) shown above can be attached to the drug moiety at R ¹¹ . Without being bound by any particular theory, hydrophilic groups are involved 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 Pat. No. 7,498,298 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124, which is clearly referenced Specifically incorporated herein by reference. An exemplary embodiment of an ADC of formula I comprising MMAE or MMAF and various linker components has the following structure and abbreviations (where “Ab” is an antibody and p is from 1 to about 8: "Val-Cit" is a valine-citrulline dipeptide and "S" is a sulfur atom.

  Exemplary embodiments of ADCs of formula I comprising MMAF and various linker components further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Interestingly, an immunoconjugate comprising MMAF attached to the antibody by a linker that does not undergo proteolytic cleavage is comparable to an immunoconjugate comprising MMAF attached to the antibody by a proteolytically cleavable linker. It has been shown to have See, for example, Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. In such cases, drug release appears to be affected by intracellular antibody degradation. Same as above.

  Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods well known in the field of peptide chemistry (E. Schroder and K. Lubke, “The Peptides” ”, Volume 1, page 76 -136, 1965, Academic Press). The auristatin / dolastatin drug moiety is described in US Patent Application Publication No. 2005-0238649 A1; US Pat. No. 5,635,483; US Pat. No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc. 111: 5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13: 243-277; Pettit, GR, et al. Synthesis, 1996, 719-725; and Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5: 859-863 And Doronina (2003) Nat. Biotechnol. 21 (7): 778-784.

In particular, auristatin / dolastatin drug moieties of formula D _F, for example MMAF and derivatives thereof, U.S. Patent No. 7498298 and Doronina etc. (2006) Bioconjugate Chem 17:. Can be prepared using methods described in 114-124. The auristatin / dolastatin drug moiety of formula _DE , such as MMAE and its derivatives, can be prepared using the method described in Doronina et al. (2003) Nat. Biotech. 21: 778-784. The drug-linker moieties MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE are described in, for example, Doronina et al. (2003) Nat. Biotech. 21: 778-784, and the United States. It may be conveniently synthesized by conventional methods as described in patent 7498298 and then conjugated to the antibody of interest.

Calicheamicin In other embodiments, the immunoconjugate comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics can produce double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, U.S. Pat. Company). The calicheamicin structural analogs that can be used include, but are not limited to, γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-acetyl-γ 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 aforementioned American Cyanamid US patent). Another anti-tumor agent to which the antibody can bind is QFA, which is an antifolate. Both calicheamicin and QFA have a site of action in the cell and do not easily cross the plasma membrane. Thus, the uptake of these drugs into cells by antibody-mediated internalization greatly improves the cytotoxic effect.

Other cytotoxic drugs Other anti-tumor agents that can be conjugated to antibodies are described in BCNU, streptozocin, vincristine and 5-fluorouracil, US Pat. Nos. 5,053,394, 5,770,710 and collectively The family of drugs known as the LL-E33288 complex, as well as esperamicin (US Pat. No. 5,877,296) are included.

  Usable enzyme active toxins and fragments thereof include diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin ) A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPIII and PAP-S), momordica momordica charantia inhibitor, curcin, crotine, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and trichothecens (tr icothecenes). See, for example, International Publication No. 93/21232 published October 28, 1993.

  The present invention further contemplates immunoconjugates formed between an antibody and a compound having nucleolytic activity (eg, ribonuclease or DNA endonuclease, eg, deoxyribonuclease; DNase).

In certain embodiments, the immunoconjugate may contain atoms with high radioactivity. A variety of radioactive isotopes are available for generating radioconjugated antibodies. Examples include radioactive isotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , Zr ⁸⁹ and Lu. If the immunoconjugate is used for detection, it may be a radioactive atom for scintigraphic studies, such as tc ^99m or I ¹²³ , or a spin label for nuclear magnetic resonance (NMR) imaging (magnetic resonance imaging, also known as mri), For example, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron may be contained.

Radio- or other labels can be introduced into the immunoconjugate in a known manner. For example, peptides are biosynthesized or synthesized by chemical amino acid synthesis using a suitable amino acid precursor containing fluorine-19 instead of hydrogen. Labels such as tc ^99m or I ¹²³ , Re ¹⁸⁶ , Re ¹⁸⁸ , Zr ⁸⁹ and In ¹¹¹ can be attached via the cysteine residue of the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to introduce iodine-123. Details of other methods are described in “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989).

  In certain embodiments, an immunoconjugate comprises an antibody conjugated to a prodrug activating enzyme that converts a prodrug (eg, a peptidyl chemotherapeutic agent, see WO81 / 01145) to an active agent such as an anticancer agent. May be included. Such immunoconjugates are useful for antibody-dependent enzyme-mediated prodrug therapy (“ADEPT”). Enzymes that can be conjugated to antibodies include, but are not limited to, alkaline phosphatases useful for converting phosphate-containing prodrugs to free drugs; useful for converting sulfate-containing prodrugs to free drugs. Arylsulfatase; cytosine deaminase useful for converting non-toxic 5-fluorocytosine to anticancer agent 5-fluorouracil; proteases such as serratia protease, thermolysin, subtilisin, carboxypeptidase and cathepsins (eg cathepsins B and L), peptide-containing Useful for converting prodrugs to free drugs; D-alanyl carboxypeptidases useful for converting prodrugs containing D-amino acid substituents; carbohydrate cleaving enzymes such as glycosylated prodrugs Neuraminidase and β-galactosidase useful for converting glucoside to free drug; β-lactamase useful for converting drug derivatized with β-lactam to free drug; and penicillin amidase such as phenoxyacetyl or Penicillin V amidase or penicillin G amidase useful for converting drugs derivatized at their aminic nitrogen with phenylacetyl groups into free drugs is included. The enzyme may be covalently attached to the antibody 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 represented by p, which is the average number of drug moieties per antibody in the molecule of formula I. Drug loading may range from 1 to 20 drug moieties (D) per antibody. The ADC of formula I includes a collection of antibodies conjugated in the range of 1 to 20 drug moieties. The average number of drug moieties per antibody in the preparation of ADC from the conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assay and HPLC. The quantitative distribution of ADC in terms of p can also be determined. In some cases, separation, purification and characterization of homogeneous ADCs where p is a constant value for ADCs with other drug loadings can be achieved by means such as reverse phase HPLC or electrophoresis.

  For some antibody-drug conjugates, p may be limited by the number of additional sites on the antibody. For example, as in the exemplary embodiment above, if the linkage is a cysteine thiol, the antibody may have only one or more cysteine thiol groups, or only one or more to which a linker may be attached. It may have a sufficiently reactive thiol group. In certain embodiments, high drug loading, such as p> 5, can cause aggregation, poor solubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the drug loading of the ADC of the invention ranges from 1 to about 8, from about 2 to about 6, or from about 3 to about 5. In fact, for some ADCs, a suitable ratio of drug moieties per antibody may be less than 8, or may be from about 2 to about 5. See U.S. Patent Application Publication No. 2005-0238649 A1.

  In some embodiments, less than the theoretical maximum drug moiety is conjugated to the antibody during the conjugation reaction. An antibody can include, 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 reactive cysteine thiol groups that can bind to the drug moiety, and in fact, most cysteine thiol residues of antibodies exist as disulfide bridges. In certain embodiments, the antibody is reacted with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partially or fully reducing conditions to produce a reactive cysteine thiol group. Can be reduced. In certain embodiments, antibodies exhibit reactive nucleophilic groups such as lysine and cysteine when exposed to denaturing conditions.

  ADC loading (drug / antibody ratio), for example, (i) limits the molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limits the reaction time or temperature of the conjugate, And (iii) may be controlled in different ways, such as by partial or limited reduction conditions for cysteine thiol modification.

  When one or more nucleophilic groups react with a linker reagent or drug-linker intermediate followed by a drug moiety reagent, the resulting product is a mixture of ADC compounds having a distribution of one or more drug moieties bound to the antibody. It is thought that. The average number of drugs per antibody can be calculated from the mixture by a dual ELISA assay that is antibody specific and drug specific. Individual ADC molecules can be identified in the mixture by mass spectrometry and separated by HPLC, eg hydrophobic interaction chromatography (for example McDonagh et al. (2006) Prot. Engr. Design & Selection 19 (7): 299-307 Hamblett et al (2004) Clin. Cancer Res. 10: 7063-7070; Hamblett, KJ, et al. "Effect of drug loading on pharmacology, pharmacokinetics and toxicity of anti-CD30 antibody-drug conjugates" "Drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate,") Abstract 624, American Association for Cancer Research, 2004 Annual Meeting, March 2004 27-31 May, AACR Proceedings of the AACR, 45, March 2004; Alley, SC, et al. "Controlling the location of the drug in the antibody-drug conjugate." drug attachment in antibody-drug conjugates, ")" Abstract # 627, USA Society, 2004 Annual General Meeting, March 27-31, 2004, AACR Proceedings, Vol. 45, referring to the March 2004). In certain embodiments, a homogeneous ADC with a single loading value may be isolated from the conjugate mixture by electrophoresis or chromatography.

Certain Preparation Methods for Immunoconjugates The ADCs of Formula I can be prepared by a variety of means using organic chemical reactions, conditions and reagents known to those skilled in the art, such as (1) to form Ab-L by covalent bonding. Reaction of the divalent linker with the nucleophilic group of the antibody followed by reaction with the drug moiety D; and (2) a divalent linker to form DL by covalent bonding and the nucleophilic group of the drug moiety. And the subsequent reaction with the nucleophilic group of the antibody. An exemplary method for preparing Formula I ADCs by the latter route is described in US Patent Publication No. 2005-0238649 A1, which is expressly incorporated herein by reference.

  Antibody nucleophilic groups include, but are not limited to, (i) N-terminal amine groups, (ii) side chain amine groups such as lysine, (iii) side chain thiol groups such as cysteine, and (iv) antibodies Includes a sugar hydroxyl group or an amino group to be glycosylated. Amines, thiols and hydroxyl groups are nucleophilic and can react to form a covalent bond with an electrophilic group on the linker moiety and linker reagent. The linker moiety and linker reagent include (i) active esters such as NHS esters, HOBt esters, haloformic acids and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes , Ketones, carboxyl and maleimide groups. Certain antibodies have reducible interchain disulfides, ie cysteine bridges. An antibody may be conjugated with a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) so that the antibody is completely or partially reduced. Good. Thus, each cysteine bridge theoretically forms two reactive thiol nucleophiles. By modification of the lysine residue, additional nucleophilic groups can be introduced into the antibody, for example, by reacting lysine with 2-iminothiolane (a trout reagent) that converts an amine to a thiol. A reactive thiol group introduces 1, 2, 3, 4 or more cysteine residues (eg, preparing a variant antibody comprising one or more unnatural cysteine amino acid residues). May be introduced into the antibody.

  The antibody-drug conjugate of the present invention may be produced by a reaction between an electrophilic group on the antibody such as an aldehyde or ketone carbonyl group and a nucleophilic group on a linker reagent or drug. Useful nucleophilic groups on the linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide. In one embodiment, the antibody is modified to introduce an electrophilic moiety that can be reacted with a nucleophilic substituent on the linker reagent or drug. In other embodiments, the glycosylated antibody carbohydrate is oxidized, eg, using a periodate oxidant, to form an aldehyde or ketone group that can react with an amine group of a linker reagent or drug moiety. Also good. The resulting imine Schiff base group may form a stable bond or may be reduced, for example, with a borohydride reagent that forms a stable amine bond. In one embodiment, reaction of a carbohydrate moiety of a glycosylated antibody with either galactose oxidase or sodium metaperiodate, the antibody carbonyl (which can react with an appropriate group on a drug (Hermanson, Bioconjugate Techniques) ( Aldehyde and ketone) groups can be formed. In another embodiment, an antibody containing an N-terminal serine or threonine residue reacts with sodium metaperiodate to produce an aldehyde instead of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3: 138-146; U.S. Pat. No. 5,362,852). Such aldehydes can react with drug moieties or linker nucleophilic groups.

  Nucleophilic groups on the drug moiety include, but are not limited to amines, thiols, hydroxyls, hydrazides, oximes, which can react and covalently bond with electrophilic groups on the linker moiety and linker reagent. Hydrazine, thiosemicarbazone, hydrazine carboxylic acid ester and aryl hydrazide groups are included. The linker moiety and linker reagent include (i) active esters such as NHS esters, HOBt esters, haloformic acids and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes , Ketones, carboxyl and maleimide groups.

  The compounds of the present invention include, but are not limited to, the following crosslinkers: commercially available (eg, from Pierce Biotechnology, Inc., Rockford, IL., USA) BMPS, EMCS, GMBS, HBVS, LC-SMCC , MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl) Particularly contemplated are ADCs prepared with-(4-vinylsulfone) benzoate).

  Immunoconjugates comprising antibodies and cytotoxic drugs can be obtained from various bifunctional protein coupling agents such as N-succinimidyl 3- (2-pyridylthio) propionate (SPDP), succinimidyl 4- (N-maleimidomethyl) cyclohexane. -1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (eg dimethyl adipimidate HCl), active esters (eg disuccinimidylsbelate), aldehydes (eg glutaraldehyde), Bis-azide compounds (such as bis (p-azidobenzoyl) hexanediamine), bisdiazonium derivatives (such as bis (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) It may be created using such fine bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionucleotide to an antibody, WO 94/11026. reference.

  Alternatively, a fusion protein containing the antibody and cytotoxic drug is made, for example, by recombinant techniques or peptide synthesis. The recombinant DNA molecule can include regions encoding the antibody and cytotoxic portions of the conjugate that are separated by regions that encode linker peptides that do not destroy the desired properties of the conjugate, or that are adjacent to each other.

  In yet another embodiment, the antibody is conjugated to a “receptor” (eg, streptavidin) for use in tumor pre-targeting, wherein the antibody-receptor conjugate is administered to the patient followed by a clarifier. It may be used to remove unbound conjugate from the circulation and administer a “ligand” (eg, avidin) that is conjugated to a cytotoxic agent (eg, a radionucleotide).

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

  A therapeutic formulation containing the antibody or immunoconjugate of the present invention can be obtained by mixing an antibody or immunoconjugate with the desired purity and optionally a pharmaceutically acceptable carrier, vehicle or stabilizer ( Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), lyophilized or prepared in the form of an aqueous solution and stored. Acceptable carriers, vehicles or stabilizers are nontoxic to recipients at the dosages and concentrations used, and buffers such as acetate, tris, phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine Preservatives (eg octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin, or immunoglobulin; hydrophilic poly, such as polyvinylpyrrolidone -Amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride Sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes); and / or TWEEN®, PLURONICS® or polyethylene glycol ( PEG) and other nonionic surfactants. Pharmaceutical formulations used for in vivo administration are generally sterile. This is easily accomplished by sterilization with a sterile filtration membrane.

  The active ingredient can also be incorporated into microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly- (methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g. Albumin microspheres, microemulsions, nano-particles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

  Sustained release formulations may be prepared. Suitable examples of sustained release formulations include a semi-permeable matrix of a hydrophobic solid polymer comprising the antibody or immunoconjugate of the present invention, which matrix is in the form of a molding, such as a film or microcapsule. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactides (US Pat. No. 3,773,919), L-glutamic acid and γ ethyl-L- Copolymers of glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-)-3-hydroxybutyric acid is included. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for over 100 days, certain hydrogels release proteins in a shorter time. When encapsulated antibodies or immunoconjugates remain in the body for a long time, they denature or aggregate upon exposure to moisture at 37 ° C., resulting in reduced biological activity and possible immunogenicity Bring about changes. Reasonable methods can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular S—S bond formation through thio-disulfide exchange, stabilization may include modification of sulfhydryl residues, lyophilization from acidic solutions, control of water content, appropriate Addition of various additives and the development of specific polymer matrix compositions.

  The antibody may be prepared in any suitable manner for delivery to the target cell / tissue. For example, the antibody may be prepared as an immunoliposome. “Liposomes” are small vesicles composed of various types of lipids, phospholipids and / or surfactants that are useful for the delivery of drugs to mammals. The components of the liposome are generally arranged in a bilayer, similar to the lipid arrangement of biological membranes. Liposomes containing antibodies are described in Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); No. 4544545; and WO 97/38731 published Oct. 23, 1997, and are prepared by methods known in the art. Liposomes with increased circulation time are disclosed in US Pat. No. 5,013,556.

  Particularly useful liposomes can be made by the reverse phase evaporation method with a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter with a defined pore size to obtain liposomes having a desired diameter. Fab ′ fragments of antibodies of the invention can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982). . In some cases, the chemotherapeutic agent is included within the liposome. See Gabizon et al., J. National Cancer Inst. 81 (19) 1484 (1989).

  In other embodiments, immunoconjugates include, but are not limited to, antibodies described herein conjugated to enzymatically active toxins or fragments thereof, diphtheria A chain, non-binding active fragment of diphtheria toxin. , Exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, phytraca americana (Phytolaca americana) protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria oficinalis inhibitor, gelonin , Mitogellin, les Rikutoshin (restrictocin), phenol mycin (phenomycin), include Enomaishin (enomycin) and trichothecenes (tricothecenes).

In other embodiments, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include radioactive isotopes of At ⁸⁹ , I ²¹¹ , I ¹³¹ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu. When using a conjugate for detection, it is for a radioactive atom for scintigraphic testing, such as tc99m or I123, or nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging mri). Spin labels such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

  Conjugates of antibodies and cytotoxic drugs are available in various bifunctional protein coupling agents such as N-succinimidyl 3- (2-pyridylthio) propionate (SPDP), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 Carboxylates (SMCC), iminothiolanes (IT), bifunctional derivatives of imide esters (eg dimethyl adipimidate HCl), active esters (eg disuccinimidylsbelate), aldehydes (eg glutaraldehyde), bis- Azide compounds (eg bis (p-azidobenzoyl) hexanediamine), bisdiazonium derivatives (eg bis (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (eg toluene 2,6-diisocyanate) and bisactive Fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) using such can be created. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionucleotide to an antibody, WO 94/11026. reference. The linker may be a “cleavable linker” that facilitates release of the cytotoxic drug into the cell. For example, acid labile linkers, peptidase sensitive linkers, photosensitive linkers, dimethyl linkers or disulfide containing linkers (Chari et al., Cancer Res. 52: 127-131 (1992); US Pat. No. 5,208,020 ) Can be used.

  As used herein, immunoconjugates or ADCs include, but are not limited to, commercially available (eg, from Pierce Biotechnology, Inc., Rockford, IL., USA), BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS MPBH. , SBAP, SIA, SIAB, SMCC, SMPB, SMPH, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-MBS, Sulfo-SIAB, Sulfo-SMCC, and Sulfo-SMPB, SVSB (succinimidyl (4-vinylsulfone Conjugates prepared with a crosslinker reagent comprising)) benzoate) are explicitly contemplated.

Pharmaceutical Formulations Pharmaceutical formulations of the anti-ETBR antibodies described herein can be prepared by combining the antibody with any desired degree of purity and any pharmaceutically acceptable carrier (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed .: Williams and Wilkins PA, USA (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are not toxic to recipients at the dosages and concentrations used and are not limited to buffers such as phosphates, citrates and other organic acids. Antioxidants including ascorbic acid and methionine; preservatives (eg octadecyl dimethylthiol ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or Propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymer; example Polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; mannosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as Nonionic surfactants such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium, metal complexes (eg Zn-protein complexes); and / or polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein include intervening drug dispersants such as water soluble neutral active hyaluronidase glycoprotein (sHASEGP) such as rHuPH20 (HYLENEX®, Baxter International, Inc.) and other human soluble PH-20 hyaluronidase glycoproteins. Certain exemplary sHASEGPs and uses include rHuPH20 and are disclosed in US Patent Application Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glucosaminoglycans such as chondroitinase.

  A typical lyophilized antibody formulation is described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006/044908, the latter formulation comprising a histidine-acetate buffer.

  The formulations herein may also contain one or more active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide a BRAF inhibitor, MEK inhibitor or anti-CTLA-4 antibody, ipilimumab. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

  The active ingredients can be used in colloidal drug delivery systems (eg liposomes, albumin microcapsules), for example by coacervation techniques or interfacial polymerization methods, eg hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively. Spheres, microemulsions, nanoparticles and nanocapsules) or microcapsules prepared with macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

  Sustained release formulations may be prepared. Suitable examples of sustained release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing antibodies, which matrix is in the form of a molded article, such as a film or microcapsule.

  Formulations used for in vivo administration are generally sterile. Sterility can be achieved, 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 for use as a medicament is provided. In another aspect, the method provides an anti-ETBR antibody in combination with a BRAF inhibitor that is useful as a medicament. In further embodiments, the combination is useful for the treatment of melanoma and / or metastatic melanoma. In certain embodiments, an anti-ETBR antibody in combination with a BRAF inhibitor for use in a method of treatment is provided. In certain embodiments, the invention is used in a method of treating an individual having melanoma and / or metastatic melanoma comprising administering to the individual an effective amount of an anti-ETBR antibody and an effective amount of a BRAF inhibitor. An anti-ETBR antibody for is provided. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent for the described combination, eg, as described below. 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 present invention is for use in a method of inhibiting tumor growth in a subject comprising administering to the subject an effective amount of an anti-ETBR antibody in combination with a BRAF inhibitor that inhibits tumor growth. Anti-ETBR antibodies in combination with other BRAF inhibitors are provided. The “subject” according to any of the above embodiments is preferably a human.

  In a further aspect, the present invention provides the use of an anti-ETBR antibody in combination with a BRAF inhibitor in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of melanoma or metastatic melanoma. In a further embodiment, the medicament is for use in a method of treating melanoma or metastatic melanoma comprising administering to an individual having melanoma or metastatic melanoma an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, eg, as described below. In a further embodiment, the medicament is for tumor growth inhibition. In a further embodiment, the medicament is for use in a method of inhibiting tumor growth in an individual comprising administering to the individual an effective amount of the medicament to inhibit tumor growth. An “individual” according to any of the above embodiments can be a human.

  In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-ETBR antibodies provided herein, eg, in combination with a BRAF inhibitor, for use in any of the above therapies. . In one embodiment, the pharmaceutical formulation comprises any of the anti-ETBR antibodies provided herein in combination with a BRAF inhibitor and a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical formulation comprises any of the anti-ETBR antibodies provided herein in combination with a BRAF inhibitor and at least one additional therapeutic agent, eg, as described below.

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

  Such combination therapies described above include combination administration (two or more therapeutic agents are contained in the same or separate formulations), and administration of the antibodies of the invention to administration of additional therapeutic agents and / or adjuvants. Including separate administration that can occur before, simultaneously, and / or thereafter. The antibodies of the present invention can also be used in combination with radiation therapy.

  The antibodies of the invention (and any additional therapeutic agent, such as a BRAF inhibitor) can be administered by any suitable means, and oral, pulmonary, and intranasal, and topical treatment is desired, Intralesional administration is included. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing depends in part on whether the administration is short-term or long-term and can be performed by any suitable route, for example, injection such as intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including but not limited to single or multiple doses, bolus doses, pulse infusions over various time points. Alternatively, the BRAF inhibitor may be administered orally either in tablet or capsule or liquid form.

  The antibodies of the invention are formulated, administered and dosed in a manner consistent with good medical practice. Factors to consider in this regard include the specific disorder to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration and others known to the health care professional Including the factors. The antibody is optionally, but not necessarily, formulated with one or more drugs currently in use for the prevention or treatment of the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally at the same dose and route of administration as described herein, or from 1% to 99% of the dose described herein, or empirically / clinically relevant. Used by any dose and any route determined to be.

  For the prevention or treatment of a disease, an appropriate dose of an antibody of the invention (when used alone or in combination with one or more additional therapeutic agents) is the type of disease to be treated, It depends on the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatment, patient clinical history and responsiveness to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg of antibody (eg, 0.1 mg / kg to 10 mg / kg), whether by one or more separate doses or by continuous infusion Can be an initial candidate dose for administration to a patient. One typical daily dose ranges from about 1 μg / kg to 100 mg / kg or more, depending on the factors described above. For repeated administrations over several days or longer, depending on the condition, the treatment will continue until the desired suppression of disease symptoms occurs. One exemplary antibody dose ranges from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses (or any combination thereof) of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (eg, so that the patient receives about 2 to about 20, eg, about 6 doses of antibody). One or more low doses may be administered after the initial high load dose. However, other dosage regimens may be beneficial. The progress of this therapy is easily monitored by conventional techniques and assays.

Articles of Manufacture In another embodiment of the invention, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the disorders described above is provided. The article of manufacture includes a container and a label or a package insert that is on or attached to the container. Suitable containers include, by way of example, bottles, vials, syringes, IV infusion bags and the like. The container may be formed from a variety of materials such as glass or plastic. The container may contain a compound that is effective in treating, preventing, and / or diagnosing a disease, either as such or in combination with other compositions, and may have a sterile access port (eg, the container is subcutaneous It may be an intravenous solution bag or vial with a pierced stopper by a syringe needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used for the treatment of a specific condition. Further, the article of manufacture comprises (a) a first container that includes a composition that includes an antibody of the invention; and (b) a composition that includes additional cytotoxic or other therapeutic agents. A second container containing the object may be included. The article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a particular disease. Alternatively or in addition, the article of manufacture comprises a second (or third) pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. The container may further be included. This may further include other materials desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

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

Example 1: In vitro evaluation of specific cell killing by anti-ETBR ADCs The anti-ETBR antibody-ADC candidate Hu5E9v1-ADC is ETBR cell line A2058 (obtained from American Type Culture Collection). In the case of the relatively low copy number, or in the case of the cell line UACC-257X2.2, it was evaluated in vitro in a melanoma cell line that expresses a high copy number of ETBR. The UACC-257X2.2 cell line is a derivative of the parent UACC-257 cell line optimized for growth in vivo. Parental UACC-257 was injected subcutaneously into the right flank of female NCr nude mice, and one tumor was harvested and isolated and expanded in vitro to obtain the UACC-257X1.2 cell line. The UACC-257X1.2 strain was again subcutaneously injected into the right flank of female NCr nude mice for the purpose of improving cell line growth. Tumors from this study were collected and again adapted for growth in vitro to generate the UACC-257X2.2 cell line. As determined by flow cytometry, the cell line expresses ET B _R at high levels. The relationship of receptor level to Hu5E9v1-vc-MMAE cell killing in these cell lines was evaluated as follows.

Melanoma cell lines A2058 and UACC-257X2.2 were grown in appropriate media at 37 ° C., 5% CO ₂ . To assess the effect of Hu5E9v1-ADC on cell viability, cells were seeded at 1,500 per well in 50 μL of normal growth medium in 96 well clear bottom black plates. After 24 hours, an additional 50 μL of culture medium containing a serial dilution of Hu5E9v1-ADC was added to triplicate wells. After 5 days, cell viability was determined with an EnVision 2101 multilabel reader using CellTiter-Glo luminescent cell viability reagent (G7572, Promega).

  Determination of antibody binding sites per cell (Scatchard analysis): The affinity constant and the number of cell surface binding sites in each antibody fixed the concentration of 125I-labeled Hu5E9v1-ADC and unlabeled Hu5E9v1-ADC. A combination of increasing concentrations was assessed by incubating melanoma cells on ice for 4 hours. Data were analyzed by non-linear curve fitting using an analysis program method.

  As shown in FIGS. 2A and 2B, Scatchard analysis estimated the number of Hu5E9v1-ADC binding sites on A2058 and UACC-257X2.2 as 1,582 and 33939 sites per cell, respectively. Titration of these cell lines with anti-ETBR ADC candidates showed specific cell killing compared to control ADC, which was generally proportional to the level of ETBR expression.

Example 2: In vitro evaluation of specific tumor killing by anti-ETBR ADCs Based on the studies described in Example 1 above, the melanoma cell lines A2058 and UACC-257X2.2 were transformed in vivo to represent a broad range of ETBR expression. Was selected as a suitable model for the study of antitumor activity. The UACC-257X2.2 melanoma cell line is a derivative of the parent UACC-257 melanoma cell line (National Cancer Institute (NCI)) optimized for in vivo growth. Specifically, the parent UACC-257 was injected subcutaneously into the right flank of female NCr nude mice, and one tumor was collected and expanded in vitro to obtain the UACC-257X1.2 cell line. The UACC-257X1.2 strain was again subcutaneously injected into the right flank of female NCr nude mice for the purpose of improving cell line growth. Tumors from this study were collected and again adapted for growth in vitro to generate the UACC-257X2.2 cell line. This cell line and tumors derived from this line express ETBR comparable to the parent cell line UACC-257 (data not shown).

Next, efficacy studies were performed using the melanoma cell line in the xenograft mouse model described above. All studies were conducted according to guidelines for laboratory animal management and use (Reference: Institute of Laboratory Animal Resources (NIH publication no. 85-23), Washington, DC: National Academies Press; 1996). 10-14 week old female CRL Nu / Nu or NCr nude mice from Charles River Laboratories are 5x10 ⁶ UACC-257X2.2 cells or Matrigel in HBSS containing Matrigel on the dorsal right flank. Either 5 × 10 ⁶ A2058 cells in HBSS containing were inoculated subcutaneously. When the tumor volume reached approximately 200 mm ³ (Day 0), the animals were randomly divided into groups of 10 each.

  For single drug efficacy studies, anti-ETBR ADC candidate Hu5E9v1-ADC was administered as a single intravenous (IV) injection on day 0 at 1 mpk, 3 mpk or 6 mpk (mg / kg) . A control ADC antibody and vehicle control were also administered. Mean tumor volume with standard deviation was determined from 10 animals per group (shown on the graph). Tumor volume was measured twice a week until the end of the study.

  The results are shown in FIG. 3A for the high ETBR copy number UACC-257X2.2 cell line and in FIG. 3B for the low ETBR copy number cell line A2058. Consistent with the in vitro cell killing experiment described in Example 1, UACC-257X2.2 xenograft tumors were responsive to Hu5E9v1-ADC. Efficacy was not evident in the dosing group at 1 mg / kg, but sustained tumor regression was observed in response to single doses of Hu5E9v1-ADC at 3 mg / kg and 6 mg / kg (FIG. 3A). .

  3 and 6 mg / kg Hu5E9v1-ADC, 6 mg / kg control ADC or vehicle control were administered to animals with low ETBR copy number A2058 tumors. A partial decrease in tumor volume was observed at a high dose of 6 mpk of Hu5E9v1-ADC compared to a matching dose of control ADC or vehicle. Efficacy was not apparent in the group dosed with 3 mg / kg of Hu5E9v1-ADC. Thus, the reduction in tumor burden in the A2058 xenograft model, which represents the lower spectrum of ETBR expression in human melanoma (Asundi et al, 2011), in tumors corresponding to the full range of expression of ETBR encountered in human melanoma, It suggests that efficacy can be achieved using the candidate Hu5E9v1-ADC as a single agent.

Example 3: Effect of BRAF inhibitory drug on expression level of ETBR The effect of a BRAF inhibitory drug on the expression level of ETBR transcripts and proteins (total protein and cell surface protein) is, for example, a variant of BRAF (V600E), BRAF Various melanoma cells representing various genetic backgrounds of melanoma, such as wild type and RAS (Q61L) mutants, were evaluated.

  Melanoma cell lines UACC-257X2.2, A2058, COLO829, IPC-298 (ATCC) are BRAF inhibitory drugs (“BRAFi”), specifically, RG7204 is cultured on 4 well dishes for 24 hours. Different concentrations were treated by adding the appropriate drug amount to the cells inside.

  To determine the effect of BRAFi on the transcript levels of ETBR and control ribosomal protein L19 (RPL19), the following experiment was performed. Cells treated with RG7204 for 24 hours were harvested by scraping from the plate and processed for total RNA using Qiasredder and RNeasy mini kits (79654, 74104, Qiagen, Valencia, Calif.). The Taqman assay was prepared using reagents from Applied Biosystems (ABI, Foster City, Calif.) And assayed using a 7500 real-time PCR instrument and software from ABI. The primer-probe set was designed taking into account the primers adjacent to the fluorogenic probe doubly labeled with the reporter dye FAM and the quencher dye TAMRA.

  The primer-probe set of RPL19 is as follows:

  Forward primer—5′AGC GGA TTC TCA TGG AAC A (SEQ ID NO: 11); reverse primer—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 of ETBR is as follows:

  Forward primer-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 UACC-257X2.2 are shown in FIG. 4A, the results for A2058 are shown in FIG. 8A, and the results for COLO829 are shown in FIG. 6A. These results show that treatment with BRAFi RG7204 for 24 hours appears to increase ETBR transcripts in all cell lines tested compared to control cells without added BRAFi RG7204. .

  To test whether the increase in ETBR transcripts due to BRAFi treatment also resulted in any change in ETBR total protein levels, Western blot experiments were performed on the same cell line treated with BRAFi RG7204 as described above. . The following reagents were used for Western blotting: for protein detection: anti-ETBR in-house generated monoclonal antibody 1H 1.8.5, anti-phospho p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) Antibody (9101, Cell Signaling Technology), anti-p44 / 42 MAPK (ERK1 / 2) antibody (9102, Cell Signaling Technology) and, as a control, rabbit polyclonal anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody (PA1) -987; Affinity Bioreagents) and mouse monoclonal anti-β-tubulin antibody (556321, BD Pharmingen). The results for UACC-257X2.2 are shown in FIG. 4B, the results for A2058 are shown in FIG. 8B, the results for COLO829 are shown in FIG. 6B, and the results for IPC-298 are shown in FIG. . These results show that treatment with BRAFi RG7204 for 24 hours increased total ETBR protein in all tested UACC-257X2.2, A2058, and COLO 829 cell lines compared to control cells without added BRAFi. Shows that it seems to be. However, for cell line IPC-298, which is wild type for BRAF and mutant for RAS (Q61L), BRAFi does not appear to increase the level of ETBR compared to various BRAFi dosage levels, rather As shown in FIG. 10, it appears to activate the level of phosphorylated ERK.

Since the increase observed in total protein levels of ET B _R according BRAFi treatment also determines whether results in increased ET B _R surface protein levels, was performed fluorescence activated cell sorting (FACS) analysis. Cells were collected in PBS containing 2.5 mmol / L EDTA and washed in PBS buffer containing 1% FBS. All subsequent steps were performed at 40 ° C. Cells were incubated with 3 μg / mL anti-ETBR antibody, Hu5E9v1, for 1 hour, followed by anti-human IgG fluorescence detection reagent (A11013; Invitrogen). Cells were then analyzed on a FACS Calibur flow cytometer (BD Biosciences). The results for UACC-257X2.2 are shown in FIG. 4C, the results for A2058 are shown in FIG. 8C, and the results for COLO829 are shown in FIG. 6C. These results, the treatment with BRAFi RG7204 of 24 hours, compared to control cells without the addition of BRAFi, seem to increase the surface level of ET _B R protein expressed in all cell lines tested Is shown. However, for cell line IPC-298, BRAFi RG7204 appears to reduce the level of ETBR at all dosage levels tested, as shown in FIGS.

Example 4: Effect of BRAF inhibitory drug on in vivo efficacy of anti-ETBR ADC In view of the results shown in Example 3 above, the effect of BRAF inhibitory drug on in vivo efficacy of anti-ETBR ADC was tested. In order to do this, the in vivo efficacy for various combinations of Hu5E9v1-ADC and BRAFi-945 was evaluated against the UACC-257X2.2 melanoma model described above. Tumors were grown to an average size of about 200 mm ³ on which the animals were randomly divided into groups of 10 animals. Appropriate vehicle control (Klucel LF) or BRAFi-945 was administered orally at a dose of 1 mpk, 6 mpk, or 20 mpk once daily for 21 days beginning on test day 0. A single 1 mpk or 3 mpk dose of Hu5E9v1-ADC or control, histidine buffer # 8, was administered intravenously via the tail vein (after two doses of 945) on day 1 of the study.

Average tumor volume was determined from 10 animals per group. Tumor volume was measured twice a week until the end of the study. Tumor volume was measured in two dimensions (length and width) using an UltraCal IV caliper (Model 54 10 111, Fred V. Fowler Company; Newton, Mass.). To calculate the tumor volume, the following equation Excel version 12.2.8; used in (Microsoft Redmond, WA): tumor volume (mm3) = (length × width ²⁾ × 0.5

A mixed modeling linear mixed effects (LME) approach was used to analyze repeated measurements of tumor volume from the same animal over time (Pinheiro et al. 2009). This approach can address both repeated measures and non-treatment related discontinuation rates in animals prior to the end of the study. A 3D regression spline was used to fit a non-linear profile over time for log2 tumor volume at each dosage level. These non-linear profiles were then related to dose within the mixed model. Tumor growth inhibition (TGI) as a percentage of vehicle was calculated as the area percentage under the approximate curve per day (AUC) for the vehicle using the following formula:

  Using this formula, a TGI value of 100% indicates tumor stasis, not less than <100%,> 1% indicates tumor growth delay, and> 100% indicates tumor regression. To obtain the% TGI uncertainty interval, an approximate curve and an approximate covariance matrix were used to generate randomized samples as an approximation of the distribution of% TGI. The randomized sample consists of 1000 simulated realizations of the approximate mixed model, where% TGI is recalculated for each realization. Here, there is a time value of 95% in the reported UI, and assuming an approximate model, the recalculated value of% TGI settles in this region. The 2.5 and 97.5 percentiles of the simulated distribution were used as the upper and lower UI.

  The results are shown in FIGS. 5A, 5B, 5C, 5D and 5E. All combinations of Hu5E9v1-ADC and BRAFi-945 showed better efficacy than any drug of the drug as a single drug alone. The two drugs combined at the lowest level were tested to obtain a combination efficacy that was almost indistinguishable from the combination efficacy achieved at the highest dosage level tested.

Example 5: In vivo dose test of combination of anti-ETBR ADC and BRAFi in COLO829 xenografts The test described in the above example is based on the BRAF inhibitor drug RG7204. In combination with Hu5E9v1-ADC allows refinement of in vivo efficacy assessment. Lack of antagonism between drugs is expected and therefore the effectiveness of the drug combination was tested at low doses. The COLO829 xenograft model was selected as a representative of moderate levels of ETBR expression that further increases the stringency of the combination study. Tumors were grown to an average size of about 200 mm ³ on which the animals were randomly divided into groups of 9 animals. Appropriate vehicle control (Klucel LF) or G00044364.1-12 (RG7204) was administered orally at a dose of 10 mpk or 30 mpk once daily for 21 days starting on test day 0. A single dose of either 1 or 3 mpk of Hu5E9v1-ADC or control histidine buffer # 8 was administered intravenously on day 1. The results are shown in FIGS. 7A, 7B, 7C, and 7D.

  Medium doses of both drugs (30 mg / kg RG7204 and 3 mg / kg Hu5E9v1-ADC) were mixed well together to obtain greater combined efficacy than either drug alone. The combination of the two drugs at other low doses showed a similar trend with the sole exception of the lowest dose tested in combination.

Example 6: In vivo dose testing of anti-ETBR ADC and BRAFi combination in A2058 xenografts The efficacy of Hu5E9v1-ADC and RG7204 in an A2058 xenograft model was tested. This model is particularly important because of the high level of stringency it exhibits. The A2058 xenograft model shows the lower limit of the expression spectrum of ETBR found in melanoma patients, thereby making it a challenging model for realizing anti-ETBR ADCs. Furthermore, despite its BRAF V600E mutation status, this model shows that the in vitro lethal effect has> 20 μM and is non-responsive to RG7204.

Tumors were grown to an average size of about 200 mm ³ on which the animals were randomly divided into groups of 10 animals. Appropriate vehicle control (Klucel LF) or RG7204 was administered orally at a dose of 10 mpk or 30 mpk once daily for 21 days starting on test day 0. A single dose of Hu5E9v1-ADC, either 3 mpk or 6 mpk, or control histidine buffer # 8 was administered intravenously into the 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 showed greater efficacy than any single agent alone. A 10 mg / kg dose of RG7204 alone did not show the effectiveness of a single agent against the A2058 model (see FIGS. 9A and 9C). However, when combined with 6 mg / kg Hu5E9v1-ADC, better efficacy was achieved than with either drug alone. The combination of 10 mg / kg RG7204 and 6 mg / kg Hu5E9v1-ADC was the combined dose achieved with the highest dose level tested, ie 30 mpk RG7204 and 6 mpk Hu5E9v1-ADC as shown in FIG. 9B The effectiveness of the combination is almost indistinguishable.

  Table 3 shows the percent difference in combined use of BRAF inhibitor and anti-ETBR ADC when compared to either the percent TGI of anti-ETBR ADC as a single agent or the percent of TGI of BRAF inhibitor as a single agent. To demonstrate the combined efficacy represented as (last column), we summarize the three melanoma xenograft models tested at various doses as described above. The percent TGI was calculated using a linear mixed effects (LME) modeling approach as described above.

Example 7: Effect of MEK inhibitory drug on expression level of ETBR The effect of MEK inhibitory drug on the expression level of ETBR transcripts and proteins (total protein and cell surface protein) was determined to be BRAF wild type or mutant and / or RAS wild. type or mutant forms of various melanoma cells which is ^{either: COLO829 (BRAF V600E), A2058} (BRAF V600E), evaluated at ^{SK23-MEL (BRAF WT / RAS} WT), or ^{IPC-298 (BRAF WT / RAS} C61L) It was done.

  Melanoma cell lines A2058, COLO 829, SK23-MEL and IPC-298 (ATCC) are MEK inhibitor drugs (“MEKi-973” or “MEKi”) at various concentrations (0 μM, 0.01 μM, 0.1 μM or 1 μM). -623 ") was treated by adding the appropriate amount of drug to cells in culture for 24 hours on 4-well dishes.

  To determine the effect of MEKi on the ETBR transcript, the following experiment was performed as described in Example 3. Cells treated with one MEKIi-973 for 24 hours were harvested by scraping from the plate and processed for total RNA using Qiasredder and RNeasy mini kit (79654, 74104, Qiagen, Valencia, Calif.). The Taqman assay was prepared using reagents from Applied Biosystems (ABI, Foster City, Calif.) And assayed using a 7500 real-time PCR instrument and software from ABI. The primer-probe set was designed taking into account the primers adjacent to the fluorogenic probe doubly labeled with the reporter dye FAM and the quencher dye TAMRA.

  The primer-probe set of RPL19 is as follows:

  Forward primer—5′AGC GGA TTC TCA TGG AAC A (SEQ ID NO: 11); reverse primer—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 of ETBR is as follows:

  Forward primer-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 treated with MEKi-623 at the specified dose are shown in FIG. 14A and the results for A2058 treated with MEKi-973 are shown in FIG. 14B. These results indicate that treatment with a MEK inhibitor for 24 hours appears to increase ETBR transcripts compared to control cells without the addition of MEK inhibitor.

To test whether the increase in ETBR transcripts resulting from treatment with MEKi also resulted in changes in ETBR total protein levels, as described above, Western blot experiments were performed using cell lines COLO829 (BRAF ^V600E ), A2058 (BRAF ^V600E ), SK23-MEL (BRAF ^WT / RAS ^WT ), or IPC-298 (BRAF ^WT / RAS ^C61L ). The following reagents were used for Western blotting: for protein detection: anti-ETBR in-house generated monoclonal antibody 1H 1.8.5, anti-phospho p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) Antibody (9101, Cell Signaling Technology), anti-p44 / 42 MAPK (ERK1 / 2) antibody (9102, Cell Signaling Technology) and, as a control, rabbit polyclonal anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody (PA1) -987; Affinity Bioreagents) and mouse monoclonal anti-β-tubulin antibody (556321, BD Pharmingen). The results for A2058 treated with MEKi-623 are in FIG. 15A, the results for A2058 treated with MEKi-973 are in FIG. 15B, the results for COLO829 treated with MEKi-623 are in FIG. 12A, and MEKi− The results for COLO829 treated with 973 are in FIG. 12B, the results for SK23-MEL treated with MEKi-623 are in FIG. 19A, and the results for SK23-MEL treated with MEKi-973 are in FIG. 19B. The results for IPC-298 treated with MEKi-623 are shown in FIG. 22A and the results for IPC-298 treated with MEKi-973 are shown in FIG. 22B. These results indicate that treatment with MEKi-623 or MEKi-973 for 24 hours appears to increase total ETBR protein in all cell lines tested compared to control cells without the addition of MEKi. Is shown.

To determine whether the increase was observed in total protein levels of ET B _R according MEKi treatment also results in an increase in the ET B _R surface protein level, performed fluorescence activated cell sorting (FACS) analysis as described above did. Cells were collected in PBS containing 2.5 mmol / L EDTA and washed in PBS buffer containing 1% FBS. All subsequent steps were performed at 40 ° C. Cells were incubated with 3 μg / mL anti-ETBR antibody, Hu5E9v1, for 1 hour, followed by anti-human IgG fluorescence detection reagent (A11013; Invitrogen). Cells were then analyzed on a FACS Calibur flow cytometer (BD Biosciences). Results for A2058 are shown in FIGS. 16A-F, results for COLO829 are shown in FIGS. 13A-F, results for SK23-MEL are shown in FIGS. 20A-F, and results for IPC-298 are shown in FIGS. 23A-F. Show. These results, the treatment with Meki-623 or Meki-973 for 24 hours, an increase compared to control cells without the addition of Meki, the level of the surface of all Come ET _B R protein on the cell lines tested Shows that it seems to be.

Example 8: Considering the results shown in Example 7 on the effects of MEK inhibitory drug for in vivo efficacy of anti ETBR ADC, MEK inhibitors described herein for in vivo efficacy of _anti-ET B R ADC The effect of the agent was tested. To do this, the in vivo efficacy for various combinations of Hu5E9v1-ADC and MEKi-623 and / or MEKi-973 was compared to A2058 and SK-MEL-23 and IPC-298 in an in vivo model. The evaluation was carried out as described in FIG. Appropriate methylcellulose tween vehicle control (0.5% methylcellulose, 0.2% Tween-80 (MCT) or MEK inhibitor is administered once daily for 21 days starting on test day 0, doses of 1 mpk, 3 mpk A single 3 mpk or 6 mpk dose of Hu5E9v1-ADC or control, histidine buffer # 8 was administered via the tail vein on day 1 of the study (MEK inhibitor 2 It was administered intravenously (after a single dose).

  The results are shown in FIGS. 17A-B, FIG. 21, FIG. 24 and FIG. Surprisingly, all of the tested Hu5E9v1-ADC inhibitor and MEK inhibitor combinations showed greater efficacy than the additive efficacy of one drug as a single agent alone.

Example 9: PD study of A2058 and COLO829 melanoma xenografts Tumors collected at the end of the study shown in FIG. This may be due to the fact that the timing of tumor collection was well past the washout period of the administered BRAFi drug. Does the in vitro action of BRAFi / MEKi (ie, increase ETBR and decrease Perk) occur in vivo in cell lines and therefore allow higher efficacy in the combination of anti-ETBR ADC and BRAFi / MEKi? In order to evaluate whether or not, the following experiment was conducted.

A2058 or COLO829 tumors were grown to an average size of about 200 mm ³ on which the animals were randomly divided into groups of 5-6. In the BRAFi PD study, the appropriate vehicle control (Klucel LF) or RG7204 was orally administered once daily for 3 days at a dose of 10 mpk or 30 mpk (FIG. 27A). In the MEKi PD study, the appropriate vehicle control or MEKi-973 was orally administered once daily for 3 days at a dose of 5 mpk or 10 mpk (FIG. 27B). Rapid frozen tumors collected at the end of the study were homogenized and processed for RNA and / or protein. The Taqman assay was prepared using reagents from Applied Biosystems (ABI, Foster City, Calif.) And assayed using a 7500 real-time PCR instrument and software from ABI. The primer-probe set was designed taking into account the primers adjacent to the fluorogenic probe doubly labeled with the reporter dye FAM and the quencher dye TAMRA. The level of ETBR transcripts can be determined using primer and probe pairs specific for human homologues of these genes, such as Hprt1 (hypoxanthine phosphoribosyltransferase 1) or GAPDH (glyceraldehyde 3-phosphate dehydrogenase), etc. Normalized to the transcript level of the reference gene.

  The primer and probe pairs of the reference gene Hprt1 (hypoxanthine phosphoribosyltransferase 1) are as follows:

  Forward primer-5 'CAC ATC AAA GAC AGC ATC 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 of ETBR is as follows:

  Forward primer-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 combination of the reference gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is as follows:

  Forward primer-5′GAA GAT GGT 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 (SEQ ID NO: 22).

  FIG. 27A shows that BRAFi induces ETBR mRNA in vivo compared to the control vehicle. FIG. 27B shows that MEKi-973 induces ETBR mRNA in vivo compared to the control vehicle.

Phosphorylated erk and total erk protein levels were assessed in tumors by Western blot using the following reagents: anti-phospho p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) antibody (9101, Cell Signaling Technology) Anti-p44 / 42 MAPK (Erk1 / 2) antibody (9102, Cell Signaling Technology), and mouse monoclonal anti-β-tubulin antibody (556321, BD Pharmingen) as a control (FIG. 26). Here, BRAFi appears to inhibit Perk in vivo compared to the control.

  Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are incorporated by reference in their entirety.

Claims (73)

An agent for inhibiting tumor growth in a subject suffering from melanoma by administration in combination with an effective amount of a MAP kinase inhibitor, comprising an effective amount of an anti-endothelin B receptor (ETBR) antibody,
The anti-ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs, VH CDR1 is SEQ ID NO: 1, VH CDR2 is SEQ ID NO: 2, VH CDR3 is SEQ ID NO: 3, and VL CDRl is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, VL CDR3 is Ri SEQ ID NO: 6 der,
The anti-ETBR antibody is conjugated to a cytotoxin, and the MAP kinase inhibitor is a BRAF inhibitor or a MEK inhibitor . An agent for inhibiting tumor growth in a subject suffering from melanoma by administration in combination with an effective amount of an anti-endothelin B receptor (ETBR) antibody, comprising an effective amount of a MAP kinase inhibitor,
The anti-ETBR antibody has three variable heavy chain CDRs and three variable light chain CDRs, VH CDR1 is SEQ ID NO: 1, VH CDR2 is SEQ ID NO: 2, VH CDR3 is SEQ ID NO: 3, and VL CDRl is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, VL CDR3 is Ri SEQ ID NO: 6 der,
The anti-ETBR antibody is conjugated to a cytotoxin, and the MAP kinase inhibitor is a BRAF inhibitor or a MEK inhibitor .   3. The agent according to claim 1 or 2, wherein the combination is synergistic.   4. The agent according to any one of claims 1 to 3, wherein tumor growth inhibition (TGI) observed in the combination is greater than TGI observed using anti-ETBR antibody alone.   The agent according to any one of claims 1 to 4, wherein the TGI observed in the combination is greater than the TGI observed using a MAP kinase inhibitor alone.   The TGI observed with the combination is greater than about 10%, or greater than about 15%, or greater than about 20%, or greater than about 25%, or greater than the use of anti-ETBR antibody alone. More than 30%, or more than about 35%, or more than about 40%, or more than about 45%, or more than about 50%, or more than about 55%, or about 60% 5. The agent of claim 4, wherein the agent is greater than or greater than about 65% or greater than about 70%.   The TGI observed with the combination is greater than about 10%, or greater than about 15%, or greater than about 20%, or greater than about 25% than the use of a MAP kinase inhibitor alone, or Greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, or about 60 6. The agent of claim 5, wherein the agent is greater than%, greater than about 65%, or greater than about 70%.   The drug according to any one of claims 1 to 7, wherein the anti-ETBR antibody specifically binds to an ETBR epitope consisting of amino acid numbers 64 to 101 of SEQ ID NO: 10.   The drug according to any one of claims 1 to 8, wherein the anti-ETBR antibody has a variable heavy chain and a variable light chain, and the VH is SEQ ID NO: 7 or 9.   10. The agent according to claim 9, wherein the VL is SEQ ID NO: 8. The drug according to any one of claims 1 to 10 , wherein the cytotoxin is a cytotoxic drug selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes. 12. The agent of claim 11 , wherein the cytotoxin is a toxin. 13. A medicament according to claim 12 , wherein the toxin is selected from the group consisting of maytansinoids, calicheamicins and auristatins. 14. The agent of claim 13 , wherein the toxin is a maytansinoid. The agent according to any one of claims 1 to 14 , wherein the MAP kinase inhibitor is a BRAF inhibitor. The BRAF inhibitor is propane-1-sulfonic acid {3- [5- (4-chlorophenyl) -1H-pyrrolo [2,3-b] pyridine-3-carbonyl] -2,4-difluoro-phenyl}-. 16. The agent according to claim 15 , which is an amide. 16. The agent of claim 15 , wherein the BRAF inhibitor has the following chemical structure:
The agent according to any one of claims 1 to 14 , wherein the MAP kinase inhibitor is a MEK inhibitor. The MEK inhibitor is (S)-(3,4-difluoro-2-((2-fluoro-4-iodophenyl) amino) phenyl) (3-hydroxy-3- (piperidin-2-yl) azetidine- The agent according to claim 18 , which is 1-yl) methanone. 19. The agent of claim 18 , wherein the MEK inhibitor has the following chemical structure:
A medicament for treating melanoma in a subject by administration in combination with a MAP kinase inhibitor, comprising a therapeutically effective amount of an anti-endothelin B receptor (ETBR) antibody, the anti-ETBR antibody comprising three variable heavy chains It has a CDR and three variable light chain CDRs, VH CDR1 is SEQ ID NO: 1, VH CDR2 is SEQ ID NO: 2, VH CDR3 is SEQ ID NO: 3, and VL CDR1 is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, VL CDR3 is Ri SEQ ID NO: 6 der,
The medicament, wherein the anti-ETBR antibody is conjugated to a cytotoxin, and the MAP kinase inhibitor is a BRAF inhibitor or a MEK inhibitor . A medicament for treating melanoma in a subject by administration in combination with an anti-endothelin B receptor (ETBR) antibody, comprising a therapeutically effective amount of a MAP kinase inhibitor, the anti-ETBR antibody comprising three variable heavy chains It has a CDR and three variable light chain CDRs, VH CDR1 is SEQ ID NO: 1, VH CDR2 is SEQ ID NO: 2, VH CDR3 is SEQ ID NO: 3, and VL CDR1 is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, VL CDR3 is Ri SEQ ID NO: 6 der,
The medicament, wherein the anti-ETBR antibody is conjugated to a cytotoxin, and the MAP kinase inhibitor is a BRAF inhibitor or a MEK inhibitor . 23. A medicament according to claim 21 or 22 , wherein the combination is synergistic. 24. The medicament according to any one of claims 21 to 23 , wherein the tumor growth inhibition (TGI) observed in the combination is greater than the TGI observed using anti-ETBR antibody alone. 25. The medicament according to any one of claims 21 to 24 , wherein the TGI observed in the combination is greater than the TGI observed using a MAP kinase inhibitor alone. The TGI observed with the combination is greater than about 10%, or greater than about 15%, or greater than about 20%, or greater than about 25%, or greater than the use of anti-ETBR antibody alone. More than 30%, or more than about 35%, or more than about 40%, or more than about 45%, or more than about 50%, or more than about 55%, or about 60% 25. The medicament of claim 24 , greater than or greater than about 65% or greater than about 70%. The TGI observed with the combination is greater than about 10%, or greater than about 15%, or greater than about 20%, or greater than about 25% than the use of a MAP kinase inhibitor alone, or Greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, or about 60 26. The medicament of claim 25 , greater than%, greater than about 65%, or greater than about 70%. 28. The medicament according to any one of claims 21 to 27 , wherein the anti-ETBR antibody specifically binds to an ETBR epitope consisting of amino acid numbers 64 to 101 of SEQ ID NO: 10. 29. The medicament according to any one of claims 21 to 28 , wherein the anti-ETBR antibody has a variable heavy chain and a variable light chain, and the VH is SEQ ID NO: 7 or 9. 30. The medicament of claim 29 , wherein the VL is SEQ ID NO: 8. The medicament according to any one of claims 21 to 30 , wherein the cytotoxin is a cytotoxic drug selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes. 32. The medicament of claim 31 , wherein the cytotoxin is a toxin. 33. The medicament of claim 32 , wherein the toxin is selected from the group consisting of maytansinoids, calicheamicins and auristatins. 34. The medicament of claim 33 , wherein the toxin is a maytansinoid. The medicament according to any one of claims 21 to 34 , wherein the MAP kinase inhibitor is a BRAF inhibitor. The BRAF inhibitor is propane-1-sulfonic acid {3- [5- (4-chlorophenyl) -1H-pyrrolo [2,3-b] pyridine-3-carbonyl] -2,4-difluoro-phenyl}-. 36. The medicament according to claim 35 , which is an amide. 36. The medicament of claim 35 , wherein the BRAF inhibitor has the following chemical structure:
35. The medicament according to any one of claims 21 to 34 , wherein the MAP kinase inhibitor is a MEK inhibitor. The MEK inhibitor is (S)-(3,4-difluoro-2-((2-fluoro-4-iodophenyl) amino) phenyl) (3-hydroxy-3- (piperidin-2-yl) azetidine- 39. The medicament according to claim 38 , which is 1-yl) methanone. 39. The medicament of claim 38 , wherein the MEK inhibitor has the following chemical structure:
41. The medicament according to any one of claims 21 to 40 , wherein the melanoma is ETBR positive. 42. The medicament according to any one of claims 21 to 41 , wherein the melanoma is metastatic. 43. The medicament according to any one of claims 21 to 42 , wherein the subject has not received prior treatment with a MAP kinase inhibitor. 44. The medicament according to any one of claims 21 to 43 , wherein the subject has a V600E BRAF gene mutation. 45. The medicament according to any one of claims 21 to 44 , wherein the subject is V600E wild type. 46. The medicament according to any one of claims 21 to 45 , wherein the MAP kinase inhibitor is first administered to a subject in need thereof. 47. The medicament according to any one of claims 21 to 46 , wherein the anti-ETBR antibody is administered after administration of the MAP kinase inhibitor. 44. The medicament according to any one of claims 21 to 43 , wherein the anti-ETBR antibody and the MAP kinase inhibitor are administered simultaneously. 44. The medicament according to any one of claims 21 to 43 , wherein the anti-ETBR antibody and the MAP kinase inhibitor are administered sequentially. 50. The medicament of claim 49 , wherein the anti-ETBR antibody is first administered to the subject. 51. The medicament of claim 50 , wherein the MAP kinase inhibitor is administered to the subject after administration of the anti-ETBR antibody. 50. The medicament of claim 49 , wherein the MAP kinase inhibitor is first administered to the subject. 53. The medicament of claim 52 , wherein the anti-ETBR antibody is administered to a subject after administration of a MAP kinase inhibitor. 54. The medicament according to any one of claims 21 to 53 , wherein the anti-ETBR antibody is administered intravenously. The anti-ETBR antibody is about 0.1 mpk, or about 0.2 mpk, or about 0.3 mpk, or about 0.5 mpk, or about 1 mpk, or about 5 mpk, or about 10 mpk, or about 15 mpk, or about 20 mpk, or 55. The medicament according to any one of claims 21 to 54 , wherein the medicament is administered at about 25 mpk, or about 30 mpk. 56. The medicament according to any one of claims 21 to 55 , wherein the MAP kinase inhibitor is administered orally. The MAP kinase inhibitor is about 1 mpk, or about 2 mpk, or about 3 mpk, or about 4 mpk, or about 5 mpk, or about 6 mpk, or about 7 mpk, or about 8 mpk, or about 9 mpk, or about 10 mpk, or about 11 mpk, or 57. The medicament according to any one of claims 21 to 56 , wherein the medicament is administered at about 12 mpk, or about 15 mpk, or about 20 mpk or about 30 mpk. An article of manufacture for inhibiting tumor growth in a subject suffering from melanoma comprising a package comprising an anti-ETBR antibody composition and a MAP kinase inhibitor composition comprising:
The anti-ETBR antibody composition comprises an anti-ETBR antibody having three variable heavy chain CDRs and three variable light chain CDRs, wherein VH CDR1 is SEQ ID NO: 1, VH CDR2 is SEQ ID NO: 2, and VH CDR3 is a sequence a number 3, and VL CDRl is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, VL CDR3 is Ri SEQ ID NO: 6 der,
An article of manufacture wherein the anti-ETBR antibody is conjugated to a cytotoxin and the MAP kinase inhibitor is a BRAF inhibitor or MEK inhibitor . 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 comprising:
The anti-ETBR antibody composition comprises an anti-ETBR antibody having three variable heavy chain CDRs and three variable light chain CDRs, wherein VH CDR1 is SEQ ID NO: 1, VH CDR2 is SEQ ID NO: 2, and VH CDR3 is a sequence a number 3, and VL CDRl is SEQ ID NO: 4, VL CDR2 is SEQ ID NO: 5, VL CDR3 is Ri SEQ ID NO: 6 der,
An article of manufacture wherein the anti-ETBR antibody is conjugated to a cytotoxin and the MAP kinase inhibitor is a BRAF inhibitor or MEK inhibitor . 60. The product of claim 58 or 59 , wherein the anti-ETBR antibody specifically binds to an ETBR epitope consisting of amino acid numbers 64 to 101 of SEQ ID NO: 10. 60. The article of manufacture of claim 58 or 59 , wherein the anti-ETBR antibody has a variable heavy chain and a variable light chain, and the VH is SEQ ID NO: 7 or 9. 62. The article of manufacture of claim 61 , wherein the VL is SEQ ID NO: 8. 63. The article of manufacture according to any one of claims 58 to 62 , wherein the cytotoxin is a cytotoxic drug selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes. 64. The article of manufacture of claim 63 , wherein said cytotoxin is a toxin. 65. The article of manufacture of claim 64 , wherein the toxin is selected from the group consisting of maytansinoids, calicheamicins and auristatins. 66. The article of manufacture of claim 65 , wherein the toxin is a maytansinoid. 60. The article of manufacture of claim 58 or 59 , wherein the MAP kinase inhibitor is a BRAF inhibitor. The BRAF inhibitor is propane-1-sulfonic acid {3- [5- (4-chlorophenyl) -1H-pyrrolo [2,3-b] pyridine-3-carbonyl] -2,4-difluoro-phenyl}-. 68. The article of manufacture of claim 67 , which is an amide. 68. The article of manufacture of claim 67 , wherein the BRAF inhibitor has the following chemical structure:
60. The article of manufacture of claim 58 or 59 , wherein the MAP kinase inhibitor is a MEK inhibitor. The MEK inhibitor is (S)-(3,4-difluoro-2-((2-fluoro-4-iodophenyl) amino) phenyl) (3-hydroxy-3- (piperidin-2-yl) azetidine- 71. The article of manufacture of claim 70 , which is 1-yl) methanone. 71. The article of manufacture of claim 70 , wherein the MEK inhibitor has the following chemical structure:
60. Use of an article of manufacture according to claim 58 or 59 in the preparation of a medicament for TGI of melanoma.