[go: up one dir, main page]

HK1141586B - Predicting response to a her dimerisation inhibitor based on low her3 expression - Google Patents

Predicting response to a her dimerisation inhibitor based on low her3 expression Download PDF

Info

Publication number
HK1141586B
HK1141586B HK10107891.3A HK10107891A HK1141586B HK 1141586 B HK1141586 B HK 1141586B HK 10107891 A HK10107891 A HK 10107891A HK 1141586 B HK1141586 B HK 1141586B
Authority
HK
Hong Kong
Prior art keywords
cancer
antibody
her3
antibodies
her2
Prior art date
Application number
HK10107891.3A
Other languages
Chinese (zh)
Other versions
HK1141586A1 (en
Inventor
Lukas C. Amler
Merrill Birkner
Chin-Yu Lin
Joachim Moecks
Andreas Strauss
Original Assignee
健泰科生物技术公司
霍夫曼-拉罗奇有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 健泰科生物技术公司, 霍夫曼-拉罗奇有限公司 filed Critical 健泰科生物技术公司
Priority claimed from PCT/US2008/055502 external-priority patent/WO2008109440A2/en
Publication of HK1141586A1 publication Critical patent/HK1141586A1/en
Publication of HK1141586B publication Critical patent/HK1141586B/en

Links

Description

Predicting response to HER dimerization inhibitors based on low HER3 expression
Technical Field
The present invention concerns the use of low HER3 as a selection criterion for treating cancer patients, such as ovarian cancer patients, with a HER inhibitor, such as Pertuzumab.
Furthermore, the invention relates to the use of a high HER 2: HER3 ratio as selection criteria for treating cancer patients, such as ovarian cancer patients, with a HER inhibitor, such as Pertuzumab.
In addition, the invention relates to the use of high HER3 as a selection criterion for treating cancer patients with chemotherapeutic agents, such as gemcitabine.
Background
HER receptor and antibody against the same
The HER family of receptor tyrosine kinases is an important mediator of cell growth, differentiation and survival. The receptor family includes four distinct members, including epidermal growth factor receptor (EGFR, ErbB1 or HER1), HER2(ErbB2 or p 185)neu) HER3(ErbB3) and HER4(ErbB4 or tyro 2).
EGFR encoded by the erbB1 gene has been causally linked to human malignancies. In particular, increased EGFR expression is observed in breast, bladder, lung, head, neck and stomach cancers as well as glioblastomas. Increased EGFR receptor expression is often associated with increased production of EGFR ligand transforming growth factor alpha (TGF- α) by the same tumor cell, leading to receptor activation through an autocrine stimulatory pathway. Baselga and Mendelsohn, pharmac. ther.64: 127-154(1994). Monoclonal antibodies against EGFR, or its ligands TGF-alpha and EGF, have been evaluated as therapeutic agents in the treatment of such malignancies. See, e.g., Baselga and Mendelsohn, supra; masui et al, cancer research 44: 1002-1007 (1984); wu et al, j.clin.invest.95: 1897-1905(1995).
Second member of the HER family, p185neuThe product of the transforming gene was originally identified as being derived from neuroblastoma in chemically treated rats. The activated form of the neu proto-oncogene is derived from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the human homolog of neu was observed in breast and ovarian cancers and was associated with a poor prognosis (Slamon et al, Science 235: 177-. To date, no point mutations similar to those in the neu protooncogene have been reported for human tumors. Overexpression of HER2 has also been observed in other cancers (frequently but heterogeneously due to gene amplification), including cancers of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder. See King et al, Science 229: 974 (1985); yokota et al, Lancet 1: 765-; fukushige et al, mol.cell biol.6: 955- "958 (1986); guerin et al, Oncogene Res.3: 21-31 (1988); cohen et al, Oncogene 4: 81-88 (1989); yonemura et al, Cancer Res.51: (1034 1991); borst et al, gynecol. oncol.38: 364 (1990); weiner et al, Cancer Res.50: 421-; kern et al, Cancer Res.50: 5184 (1990); park et al, Cancer Res.49: 6605 (1989); zhau et al, mol. carminog.3: 254-257(1 990) (ii) a Aasland et al, br.j. cancer 57: 358-363 (1988); williams et al, Pathiology 59: 46-52 (1991); McCann et al, Cancer 65: 88-92(1990), and so forth. HER2 can be overexpressed in prostate Cancer (Gu et al, Cancer Lett.99: 185-9 (1996); Ross et al, hum. Pathol.28: 827-33 (1997); Ross et al, Cancer 79: 2162-70 (1997); Sadasavan et al, J.Urol.150: 126-31 (1993)).
For rat p185neuAnd the human HER2 protein product have been described.
Drebin and colleagues prepared p185 gene product for rat neuneuThe antibody of (1). See, e.g., Drebin et al, Cell 41: 695-706 (1985); myers et al, meth.enzym.198: 277-290 (1991); WO 94/22478. Drebin et al, Oncogene 2: 273-277(1988) reported the correlation with p185neuThe mixture of antibodies reactive with the two different regions produced a synergistic antitumor effect on neu-transformed NIH-3T3 cells implanted in nude mice. See also U.S. patent No. 5,824,311 issued on 10/20/1998.
Hudziak et al, mol.cell.biol.9 (3): 1165-1172(1989) described the generation of a panel of antibodies to HER2 and characterized using the human breast tumor cell line SK-BR-3. Relative cell proliferation was determined by crystal violet staining of cell monolayers 72 hours after exposure of SK-BR-3 cells to the antibody. Using this assay, maximum inhibition was obtained with an antibody designated 4D5, which inhibited cell proliferation by 56%. The other antibodies of the panel reduced cell proliferation to a lesser extent in this assay. Antibody 4D5 was also found to sensitize breast tumor cell lines overexpressing HER2 to the cytotoxic effects of TNF- α. See also U.S. patent No. 5,677,171 issued 10/14 in 1997. The HER2 antibody discussed in the Hudziak et al article was further characterized in the following references: fendly et al, Cancer Research 50: 1550 and 1558 (1990); kotts et al, In Vitro 26 (3): 59A (1990); sarup et al, growth regulation 1: 72-82 (1991); shepard et al, j.clin.immunol.11 (3): 117-127 (1991); kumar et al, mol.cell.biol.11 (2): 979-; lewis et al, Cancer immunol.immunoher.37: 255-; pietras et al, Oncogene 9: 1829-1838 (1994); vitetta et al, Cancer Research 54: 5301-5309 (1994); sliwkowski et al, j.biol.chem.269 (20): 14661-14665 (1994); scott et al, j.biol.chem.266: 14300-5 (1991); d' souza et al, proc.natl.acad.sci.91: 7202-; lewis et al, cancer research 56: 1457-1465 (1996); schaefer et al, Oncogene 15: 1385-1394(1997).
Recombinant humanized versions of murine HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, trastuzumab orU.S. Pat. No. 5,821,337) clinically works on patients with metastatic breast cancer that overexpresses HER2 and who have received a large number of existing anti-cancer therapies (Baselga et al, j.clin.oncol.14: 737-744(1996)). Trastuzumab received marketing approval from the U.S. food and drug administration at 9/25 of 1998 and was used to treat metastatic breast cancer patients whose tumors overexpress HER2 protein.
Other HER2 antibodies with various properties are described in the following documents: tagliabue et al, int.j. cancer 47: 933 937 (1991); McKenzie et al, Oncogene 4: 543 and 548 (1989); maier et al, Cancer res.51: 5361-5369 (1991); bacillus et al, Molecular Carcinogenesis 3: 350-362 (1990); stancovski et al, PNAS (USA) 88: 8691 and 8695 (1991); bacus et al, Cancer Research 52: 2580 + 2589 (1992); xu et al, int.j.cancer 53: 401-408 (1993); WO 94/00136; kasprzyk et al, Cancer Research 52: 2771-2776 (1992); hancock et al, Cancer Res.51: 4575-4580 (1991); shawver et al, Cancer Res.54: 1367-; aretag et al, Cancer Res.54: 3758-3765 (1994); harwerth et al, J.biol.chem.267: 15160- > 15167 (1992); U.S. Pat. nos. 5,783,186; klaper et al, Oncogene 14: 2099-2109(1997).
Homology screening allows two other members of the HER receptor family to be identified: HER3 (U.S. Pat. Nos. 5,183,884 and 5,480,968 and Kraus et al, PNAS (USA) 86: 9193-. Both receptors exhibit increased expression in at least some breast cancer cell lines.
HER receptors are commonly found in various combinations in cells, and heterodimerization is thought to increase the diversity of cellular responses to various HER ligands (ear et al, Breast Cancer Research and treatment 35: 115-132 (1995)). EGFR is bound by 6 different ligands: epidermal Growth Factor (EGF), transforming Growth factor alpha (TGF-. alpha.), amphiregulin, heparin-binding epidermal Growth factor (HB-EGF), betacytokinin, and epithelial regulatory protein (Groenen et al, Growth Factors 11: 235-257 (1994)). One family of heregulin proteins that results from alternative splicing of a single gene are ligands of HER3 and HER 4. The heregulin family includes the alpha, beta and gamma heregulins (Holmes et al, Science 256: 1205-1210 (1992); U.S. Pat. No. 5,641,869; Schaefer et al, Oncogene 15: 1385-1394 (1997)); neu Differentiation Factor (NDF); glial Growth Factor (GGF); acetylcholine Receptor Inducing Activity (ARIA); and sensory and motor neuron derived factor (SMDF). For a review see Groenen et al, Growth Factors 11: 235-257 (1994); lemke, g., mol. & cell. neurosci.7: 247- "262" (1996); lee et al, pharm. rev.47: 51-85(1995). Three additional HER ligands have recently been identified: neuregulin-2 (NRG-2), which has been reported to bind HER3 or HER4(Chang et al, Nature 387: 509-; neuregulin-3, which binds HER4(Zhang et al, PNAS (USA)94 (18): 9562-7 (1997)); and neuregulin-4, which binds HER4(Harari et al, Oncogene 18: 2681-89 (1999)). HB-EGF, betacytokinin and epithelial regulatory protein also bind HER 4.
Although EGF and TGF α do not bind HER2, EGF stimulates EGFR and HER2 to form a heterodimer, which activates EGFR and leads to transphosphorylation of HER2 in the heterodimer. Dimerization and/or transphosphorylation appear to activate HER2 tyrosine kinase. See Earp et al, supra. Similarly, an active signal complex is formed when HER3 is co-expressed with HER2, and antibodies to HER2 are capable of disrupting this complex (Sliwkowski et al, J.biol. chem.269 (20): 14661-14665 (1994)). In addition, HER3 increased affinity for regulatory proteins (HRG) to a higher affinity state when co-expressed with HER 2. For the HER2-HER3 protein complex see also Levi et al, Journal of Neuroscience 15: 1329-; morrissey et al, proc.natl.acad.sci.usa 92: 1431-1435 (1995); lewis et al, Cancer Res.56: 1457-1465(1996). HER4, like HER3, forms an active signaling complex with HER2 (Carraway and Cantley, Cell 78: 5-8 (1994)).
Patent publications relating to HER antibodies include: US5,677,171; US5,720,937; US5,720,954; US5,725,856; US5,770,195; US5,772,997; US6,165,464; US6,387,371; US6,399,063; US 2002/0192211a 1; US6,015,567; US6,333,169; US 4,968,603; US5,821,337; US6,054,297; US6,407,213; US6,719,971; US6,800,738; US 2004/0236078a 1; US5,648,237; US6,267,958; US6,685,940; US6,821,515; WO 98/17797; US6,127,526; US6,333,398; US6,797,814; US6,339,142; US6,417,335; US6,489,447; WO 99/31140; US2003/0147884A 1; US 2003/0170234a 1; US 2005/0002928a 1; US6,573,043; US 2003/0152987a 1; WO 99/48527; US 2002/0141993a 1; WO 01/00245; US 2003/0086924; US 2004/0013667a 1; WO 00/69460; WO 01/00238; WO 01/15730; US6,627,196B 1; US6,632,979B 1; WO 01/00244; US 2002/0090662a 1; WO 01/89566; US 2002/0064785; US 2003/0134344; WO 04/24866; US 2004/0082047; US 2003/0175845a 1; WO 03/087131; US 2003/0228663; WO 2004/008099a 2; US 2004/0106161; WO 2004/048525; US 2004/0258685a 1; US5,985,553; US5,747,261; US 4,935,341; US5,401,638; US5,604,107; WO 87/07646; WO 89/10412; WO 91/05264; EP 412,116B 1; EP 494,135B 1; US5,824,311; EP 444,181B 1; EP 1,006,194a 2; US 2002/0155527a 1; WO 91/02062; US5,571,894; US5,939,531; EP 502,812B 1; WO 93/03741; EP554,441B1, respectively; EP 656,367a 1; US5,288,477; US5,514,554; US5,587,458; WO 93/12220; WO 93/16185; US5,877,305; WO 93/21319; WO 93/21232; US5,856,089; WO 94/22478; US5,910,486; US6,028,059; WO 96/07321; US5,804,396; US5,846,749; EP 711,565; WO 96/16673; US5,783,404; US5,977,322; US6,512,097; WO 97/00271; US6,270,765; US6,395,272; US5,837,243; WO 96/40789; US5,783,186; US6,458,356; WO 97/20858; WO 97/38731; US6,214,388; US5,925,519; WO 98/02463; US5,922,845; WO 98/18489; WO 98/33914; US5,994,071; WO 98/45479; US6,358,682B 1; US 2003/0059790; WO 99/55367; WO 01/20033; US 2002/0076695a 1; WO 00/78347; WO 01/09187; WO 01/21192; WO 01/32155; WO 01/53354; WO 01/56604; WO 01/76630; WO 02/05791; WO 02/11677; US6,582,919; US2002/0192652A 1; US 2003/0211530a 1; WO 02/44413; US 2002/0142328; US6,602,670B 2; WO 02/45653; WO 02/055106; US 2003/0152572; US 2003/0165840; WO 02/087619; WO 03/006509; WO 03/012072; WO 03/028638; US 2003/0068318; WO 03/041736; EP 1,357,132; US 2003/0202973; US 2004/0138160; US5,705,157; US6,123,939; EP 616,812B 1; US 2003/0103973; US 2003/0108545; US6,403,630B 1; WO 00/61145; WO 00/61185; US6,333,348B 1; WO 01/05425; WO 01/64246; US 2003/0022918; US 2002/0051785a 1; US6,767,541; WO 01/76586; US 2003/0144252; WO 01/87336; US 2002/0031515a 1; WO 01/87334; WO 02/05791; WO 02/09754; US 2003/0157097; US 2002/0076408; WO 02/055106; WO 02/070008; WO 02/089842; and WO 03/86467.
Diagnostic agent
Patients treated with HER2 antibody trastuzumab were selected for treatment based on HER2 overexpression/amplification. See, e.g., WO99/31140(Paton et al), US2003/0170234A1(Hellmann, S.), and US2003/0147884(Paton et al); and WO01/89566, US2002/0064785 and US2003/0134344(Mass et al). For Immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH) to detect overexpression and amplification of HER2 see also US2003/0152987(Cohen et al).
WO2004/053497 and US2004/024815A1(Bacus et al), and US2003/0190689(Crosby and Smith) are directed to determining or predicting response to trastuzumab therapy. US2004/013297A1(Bacus et al) is directed to determining or predicting response to ABX0303 EGFR antibody therapy. WO2004/000094(Bacus et al) is concerned with determining the response to GW572016, a small molecule EGFR-HER2 tyrosine kinase inhibitor. WO2004/063709(Amler et al) relates to biomarkers and methods for determining sensitivity to EGFR inhibitors, erlotinib hydrochloride. US2004/0209290(Cobleigh et al) relates to gene expression markers for the prognosis of breast cancer.
Patients treated with Pertuzumab can be selected to receive treatment based on HER activation or dimerization. Patent publications relating to the selection of Pertuzumab and patients treated therewith include: WO01/00245(Adams et al); US2003/0086924(Sliwkowski, M.); US2004/0013667A1 (Sliwkowski, M.); and WO2004/008099A2 and US2004/0106161(Bossenmai et al).
Cronin et al, am.j.path.164 (1): 35-42(2004) describe the measurement of gene expression in paraffin-embedded tissues retained. Ma, et al, Cancer Cell 5: 607 (2004) describes gene profiling by gene oligonucleotide microarrays using isolated RNA from tumor tissue sections of retained primary biopsies.
Pertuzumab (also known as recombinant human monoclonal antibody 2C 4; OMNITARG)TMGenetech, Inc, South San Francisco) was the first of a new class of agents known as HER Dimerization Inhibitors (HDI) to emerge, which function to inhibit the ability of HER2 to form active heterodimers with other HER receptors such as EGFR/HER1, HER3 and HER4, and which are active regardless of HER2 expression levels. See, e.g., Harari and Yarden, Oncogene 19: 6102-14 (2000); yarden and Sliwkowski, Nat Rev Mol Cell Biol 2: 127-37 (2001); sliwkowski, Nat StructBiol 10: 158-9 (2003); cho et al, Nature 421: 756-60 (2003); malik et al, Pro Am Soccancer Res 44: 176-7(2003).
It has been shown that blockade of HER2-HER3 heterodimer formation by Pertuzumab inhibits critical Cell signaling in tumor cells, resulting in tumor expansion and decreased survival (Agus et al, Cancer Cell 2: 127-37 (2002)).
Pertuzumab has been clinically tested as a single agent with phase Ia trials in patients with advanced cancer and phase II trials in patients with ovarian and breast cancer as well as lung and prostate cancer. In the phase I study, patients with incurable, locally advanced, recurrent or metastatic solid tumors that developed during or after standard treatment were treated with Pertuzumab intravenously every three weeks. Tolerance to Pertuzumab is generally better. Tumor regression was achieved in 3 of 20 patients evaluable in response. Two patients demonstrated partial responses. Stable disease was observed for more than 2.5 months in 6 of 21 patients (Agus et al, Pro Am Soc Clin Oncol 22: 192 (2003)). At doses of 2.0-15mg/kg, the pharmacokinetics of Pertuzumab is linear, with a mean clearance in the range of 2.69-3.74 mL/day/kg and a mean terminal elimination half-life in the range of 15.3-27.6 days. No antibody was detected against Pertuzumab (Allison et al, Pro Am Soc Clin Oncol 22: 197 (2003)).
Sergina et al reported that the biological marker used to assess the efficacy of HER Tyrosine Kinase Inhibitors (TKIs) should be transphosphorylation rather than autophosphorylation of HER3 (Sergina et al, Nature 445 (7126): 437-441 (2007)).
Jazaeri et al assessed gene expression profiles associated with response to chemotherapeutic agents in epithelial ovarian cancer (Jazaeri et al, Clin. cancer Res.11 (17): 6300-6310 (2005)).
Tanner et al reported that HER3 predicts survival in ovarian cancer (Tanner et al, j.clin.oncol.24 (26): 4317-.
Summary of The Invention
The present application is directed, at least in part, to the unexpected observation that cancer patients whose cancer expresses HER3 at low levels (e.g., ovarian cancer patients) respond better to HER dimerization inhibitors in human clinical trials than those patients whose cancer expresses HER3 at high levels. In general, such patients have a high HER 2: HER3 ratio (due to low levels of HER 3), so assessing the relative levels of both HER2 and HER3 provides an additional or alternative means to select patients for HER dimerization inhibitor therapy.
Thus, in a first aspect, the invention herein concerns a method for treating a patient having a type of cancer which is capable of responding to a HER inhibitor comprising administering to the patient a therapeutically effective amount of a HER inhibitor, wherein the patient's cancer expresses HER3 at a level less than the median level for HER3 expression in the cancer type. Examples of contemplated HER inhibitors include HER antibodies or small molecule inhibitors; a HER2 antibody or small molecule inhibitor; tyrosine kinase inhibitors, including but not limited to lapatinib, Tykerb; and the like. More preferably, the HER inhibitor is a HER dimerization inhibitor. Thus, the invention provides a method for treating a patient having a type of cancer which is capable of responding to a HER dimerization inhibitor, comprising administering to the patient a therapeutically effective amount of a HER dimerization inhibitor, wherein the patient's cancer expresses HER3 at a level less than the median level for HER3 expression in the cancer type.
According to this embodiment, preferably the patient's cancer expresses HER3 at a level less than the 25 th percentile for HER3 expression in that cancer type. Optionally, the cancer of such patients expresses HER 2: HER3 at a level greater than the 25 th percentile, preferably greater than the median level, most preferably greater than the 75 th percentile of HER 2: HER3 expression in that cancer type. Preferred assays for measuring HER3 (and HER2) expression include Polymerase Chain Reaction (PCR), most preferably quantitative real-time polymerase chain reaction (qRT-PCR).
Preferably, the HER dimerization inhibitor is an antibody, most preferably, a HER2 antibody, such as Pertuzumab.
Preferably, the type of cancer to be treated or diagnosed herein is selected from ovarian cancer, peritoneal cancer, fallopian tube cancer, Metastatic Breast Cancer (MBC), non-small cell lung cancer (NSCLC), prostate cancer, and colorectal cancer. Most preferably, the type of cancer to be treated or diagnosed herein is ovarian, peritoneal, or fallopian tube cancer. The cancer type may be chemotherapy-resistant, platinum-resistant, advanced, refractory, and/or recurrent. The method can prolong survival in the patient, including Progression Free Survival (PFS) and Overall Survival (OS).
The HER inhibitor may be administered as a single anti-tumor agent, or may be combined with one or more other therapies. In one embodiment, the HER inhibitor is administered with one or more chemotherapeutic agents, such as gemcitabine (gemcitabine), carboplatin (carboplatin), paclitaxel (paclitaxel), docetaxel (docetaxel), topotecan (topotecan), and liposomal doxorubicin (lipomal doxorubicin), preferably an antimetabolite, such as gemcitabine. The HER inhibitor may also be combined with trastuzumab, erlotinib, or bevacizumab.
In another aspect, the invention pertains to a method for treating a patient having ovarian, peritoneal, or fallopian tube cancer comprising administering a therapeutically effective amount of Pertuzumab to the patient, wherein the patient's cancer expresses HER3 at a level less than the median level for HER3 expression in ovarian, peritoneal, or fallopian tube cancer.
The invention herein further concerns a method for selecting a therapy for a patient with a type of cancer that is capable of responding to a HER inhibitor (e.g., a HER dimerization inhibitor), comprising determining HER3 expression in a cancer sample from the patient, and selecting a HER inhibitor (e.g., a HER dimerization inhibitor) as the therapy if the cancer sample expresses HER3 at a level less than the median level for HER3 expression in the cancer type.
In addition, the invention provides an article of manufacture comprising, packaged together, a pharmaceutical composition comprising a HER dimerization inhibitor in a pharmaceutically acceptable carrier and a label stating that the inhibitor or pharmaceutical composition is indicated for use in treating a patient with a type of cancer that is capable of responding to the HER dimerization inhibitor, wherein the patient's cancer expresses HER3 at a level less than the median level for HER3 expression in the type of cancer.
In another aspect, the invention pertains to a method for the manufacture of a HER dimerization inhibitor or a pharmaceutical composition thereof, comprising combining in a package said inhibitor or pharmaceutical composition and a label stating that said inhibitor or pharmaceutical composition is indicated for use in treating a patient with a type of cancer that is capable of responding to a HER dimerization inhibitor, wherein said patient's cancer expresses HER3 at a level less than the median level for HER3 expression in the type of cancer.
In yet another embodiment, the invention provides a method of advertising a HER dimerization inhibitor or a pharmaceutically acceptable composition thereof, comprising promoting to an audience of interest (audience) the use of a HER dimerization inhibitor or a pharmaceutical composition thereof for treating a patient population having a cancer type, wherein the patient's cancer expresses HER3 at a level less than the median level for HER3 expression in the cancer type.
In addition to the above invention, the human clinical data provided herein demonstrate that cancer patients whose cancer expresses HER3 at high levels (e.g., ovarian cancer patients) have a better clinical response to chemotherapeutic agents, such as gemcitabine, than those whose cancer expresses HER3 at low levels.
With respect to this aspect of the invention, the invention provides a method of selecting a therapy for a patient with a type of cancer that is likely to respond to a chemotherapeutic agent, comprising determining HER3 expression in a cancer sample from the patient, and selecting a chemotherapeutic agent as the therapy if the cancer sample expresses HER3 at a level greater than the median level for HER3 expression in the type of cancer. Preferably, the cancer type is ovarian, peritoneal, or fallopian tube cancer, including platinum-resistant ovarian, peritoneal, or fallopian tube cancer, as well as advanced, refractory, or recurrent ovarian cancer. Preferably, the chemotherapeutic agent of choice is an antimetabolite, such as gemcitabine.
The invention also concerns a method for treating a patient having a type of cancer capable of responding to a chemotherapeutic agent comprising administering to the patient a therapeutically effective amount of a chemotherapeutic agent, wherein the patient's cancer expresses HER3 at a level greater than the median level for HER3 expression in the cancer type. Preferably, the patient's cancer expresses HER3 at a level greater than the 25 th percentile for HER3 expression in that cancer type. Preferred assays for measuring HER3 expression include Polymerase Chain Reaction (PCR), most preferably quantitative real-time polymerase chain reaction (qRT-PCR).
Preferably, the chemotherapeutic agent is an antimetabolite, most preferably gemcitabine.
Preferably, the type of cancer to be treated or diagnosed in accordance with this aspect of the invention is ovarian, peritoneal, or fallopian tube cancer. The cancer type may be chemotherapy-resistant (chemotherapeutic-resistant), platinum-resistant (platinum-resistant), advanced, refractory (refractory), and/or recurrent (recurrents). The method can prolong survival in the patient, including Progression Free Survival (PFS) and Overall Survival (OS).
In another aspect, the invention pertains to a method for treating a patient having ovarian, peritoneal, or fallopian tube cancer comprising administering to said patient a therapeutically effective amount of gemcitabine, wherein the patient's cancer expresses HER3 at a level greater than the median level for HER3 expression in ovarian, peritoneal, or fallopian tube cancer.
The invention also provides an article of manufacture comprising a pharmaceutical composition comprising a chemotherapeutic agent (such as gemcitabine) in a pharmaceutically acceptable carrier and a label packaged together stating that the chemotherapeutic agent or pharmaceutical composition is indicated for use in treating a patient with a type of cancer, wherein the patient's cancer expresses HER3 at a level greater than the median level for HER3 expression in the type of cancer.
In yet another aspect, the invention concerns a method for the manufacture of a chemotherapeutic agent (such as gemcitabine) or a pharmaceutical composition thereof, comprising combining in a package said chemotherapeutic agent or pharmaceutical composition and a label stating that said chemotherapeutic agent or pharmaceutical composition is indicated for the treatment of a patient suffering from one cancer type, wherein said patient's cancer expresses HER3 at a level greater than the median level for HER3 expression in that cancer type.
In yet another embodiment, the invention provides a method of advertising a chemotherapeutic agent or a pharmaceutically acceptable composition thereof, comprising promoting to a target audience the use of a chemotherapeutic agent or a pharmaceutical composition thereof for treating a population of patients having a cancer type, wherein the patients' cancer expresses HER3 at a level greater than the median level for HER3 expression in the cancer type.
The present invention provides human clinical data demonstrating that patients with high HER 2: HER3 expression respond more favorably to HER inhibitors such as Pertuzumab. Thus, in another aspect, the invention provides a means for selecting patients by assessing HER2 and HER3 expression levels and excluding from therapy those patients whose cancers express HER 2: HER3 at low levels.
Thus, the invention also concerns a method for treating a patient with a type of cancer which is capable of responding to a HER inhibitor comprising administering to the patient a therapeutically effective amount of a HER inhibitor, wherein the patient's cancer expresses HER 2: HER3 at a level greater than the 25 th percentile for HER 2: HER3 expression in the cancer type. Preferably, the patient's cancer expresses HER 2: HER3 at a level greater than the median HER 2: HER3 expression in that cancer type, most preferably greater than the 75 th percentile.
Additionally, a method for treating a patient having ovarian, peritoneal, or fallopian tube cancer comprising administering a therapeutically effective amount of Pertuzumab to the patient, wherein the patient's cancer expresses HER 2: HER3 at a level greater than the 25 th percentile for HER 2: HER3 expression in ovarian, peritoneal, or fallopian tube cancer is provided.
In another aspect, the invention concerns a method for selecting a therapy for a patient with a type of cancer that is capable of responding to a HER inhibitor, comprising determining HER2 and HER3 expression in a cancer sample from the patient, and selecting a HER inhibitor as the therapy if the cancer sample expresses HER 2: HER3 at a level greater than the 25 th percentile for HER 2: HER3 expression in the cancer type.
Furthermore, the invention relates to an article of manufacture comprising, packaged together, a pharmaceutical composition comprising a HER inhibitor in a pharmaceutically acceptable carrier and a label stating that the inhibitor or pharmaceutical composition is indicated for use in treating a patient with a type of cancer which is capable of responding to the HER inhibitor, wherein the patient's cancer expresses HER 2: HER3 at a level greater than the 25 th percentile for HER 2: HER3 expression in the cancer type.
Furthermore, the invention provides a method for the manufacture of a HER inhibitor or a pharmaceutical composition thereof, comprising combining in a package said inhibitor or pharmaceutical composition and a label stating that said inhibitor or pharmaceutical composition is indicated for the treatment of a patient with a type of cancer capable of responding to a HER inhibitor, wherein the patient's cancer expresses HER 2: HER3 at a level greater than the 25 th percentile for HER 2: HER3 expression in the cancer type.
In addition, the invention relates to a method of advertising a HER inhibitor or a pharmaceutically acceptable composition thereof, comprising promoting to a target audience the use of a HER inhibitor or a pharmaceutical composition thereof for treating a patient population suffering from a cancer type, wherein the patient's cancer expresses HER 2: HER3 at a level greater than the 25 th percentile for HER 2: HER3 expression in the cancer type.
Brief Description of Drawings
FIG. 1 provides a schematic representation of the structure of the HER2 protein, and the amino acid sequences of domains I-IV of its extracellular domain (SEQ ID Nos. 19-22, respectively).
FIGS. 2A and 2B depict the light chain variable region (V) of murine monoclonal antibody 2C4L) (FIG. 2A) and heavy chain variable region (V)H) (FIG. 2B) (SEQ ID Nos. 1 and 2, respectively); v of variant 574/PertuzumabLAnd VHDomains (SEQ ID Nos. 3 and 4, respectively); and a person VLAnd VHAlignment of the amino acid sequences of the consensus frameworks (hum. kappa.1, light chain kappa subgroup I; hum III, heavy chain subgroup III) (SEQ ID Nos. 5 and 6, respectively). Asterisks indicate the differences between the Pertuzumab variable domain and murine monoclonal antibody 2C4 or between the Pertuzumab variable domain and human framework. The Complementarity Determining Regions (CDRs) are enclosed in parentheses.
FIGS. 3A and 3B show the amino acid sequences of the Pertuzumab light (FIG. 3A; SEQ ID No.13) and heavy (FIG. 3B; SEQ ID No.14) chains. CDRs are shown in bold. The calculated molecular weights of the light and heavy chains were 23,526.22Da and 49,216.56Da (cysteine in reduced form). The carbohydrate module (motif) is attached to Asn299 of the heavy chain.
Figure 4 graphically depicts the binding of 2C4 at the heterodimer binding site of HER2, thereby preventing heterodimerization with activated EGFR or HER 3.
FIG. 5 depicts the conjugation of HER2/HER3 to MAPK and Akt pathways.
Figure 6 compares the various activities of Trastuzumab and Pertuzumab.
FIGS. 7A and 7B show the amino acid sequences of the Trastuzumab light chain (FIG. 7A; SEQ ID No.15) and heavy chain (FIG. 7B; SEQ ID No.16), respectively.
FIGS. 8A and 8B depict the variant Pertuzumab light chain sequence (FIG. 8A; SEQ ID No.17) and the variant Pertuzumab heavy chain sequence (FIG. 8B; SEQ ID No.18), respectively.
Figure 9 depicts the study design/protocol of the clinical trial in example 1 involving patients with platinum-resistant ovarian, primary peritoneal, or fallopian tube cancer treated with gemcitabine and placebo or gemcitabine and Pertuzumab.
Figure 10A depicts Progression Free Survival (PFS) from all patients studied in example 1.
Fig. 10B is an updated version of fig. 10A. PFS has been estimated by randomized stratification factors (ECOG PS, number of prior treatment regimens (regimens) for platinum resistant disease, and disease testability) using a stratified Cox model and a stratified longrank test.
Figure 11A presents PFS obtained by predicting pHER2 status.
FIG. 11B is an updated version of FIG. 11A.
Figure 12A presents PFS by qRT-PCR EGFR (HER1) retention.
Figure 12B is another representation of PFS by wRT-PCR EGFR (HER1) cutoff, also indicating the number of subjects in HER1 (high) and HER1 (low) groups at various EGFR cutoff values.
Figure 13A presents PFS by qRT-PCR HER2 retention.
Figure 13B is another presentation of PFS by qRT-PCR HER2 cutoff, also indicating the number of subjects in HER1 (high) and HER1 (low) groups at various HER2 cutoff values.
Figure 14A presents PFS by qRT-PCR HER3 retention.
Figure 14B is another presentation of PFS by qRT-PCR HER3 cutoff, also indicating the number of subjects in HER3 (high) and HER3 (low) groups at various HER3 cutoff values.
Figure 15A shows PFS by HER3 subgroup. Pertuzumab activity is greatest in patients with low HER3 expressing tumors and tends to increase with decreased levels of HER3 gene expression.
Figure 15B is another presentation of PFS by qRT-PCR HER3 levels.
Figure 16A shows PFS by HER3 subgroup. The data show that there may be a negative interaction between Pertuzumab and gemcitabine in patients with tumors with high expression of HER 3.
Figure 16B is another presentation of PFS by qRT-PCR HER3 levels. The data further demonstrate that there may be a negative interaction between Pertuzumab and gemcitabine in patients with tumors with high expression of HER 3.
Figure 17A summarizes PFS by HER3 subgroup: a high HER3 expression subgroup and a low HER3 expression subgroup.
Figure 17B is an updated version of the PFS shown in figure 17B obtained by qRT-PCR HER3 levels.
Figure 18A further demonstrates PFS by HER3 subgroup.
Figure 18B is an updated version of the PFS analysis shown in figure 18A by HER3 expression quartile.
Figure 19A shows PFS, 50/50 resolution by HER3 qRT-PCR; low HER3 expression is in less than the 50 th percentile, while high HER3 expression is in greater than or equal to the 50 th percentile.
FIG. 19B is a newer version of the separation of the PFS, 50/50 by HER3 qRT-PCR shown in FIG. 19A
Figure 20A shows PFS, 25/75 resolution by HER3 qRT-PCR; low HER3 expression is in less than the 25 th percentile, while high HER3 expression is in greater than or equal to the 25 th percentile.
Figure 20B is a newer version of the separation of PFS, 25/75 by HER3 qRT-PCR shown in figure 20A.
Fig. 21A shows preliminary data for Overall Survival (OS) in all patients. The data is based on 46/130 events.
Fig. 21B is an updated graph of OS data, a hierarchical Cox model and a hierarchical logrank-test estimated by randomized stratification factors (ECOG PS, number of prior treatment regimens for platinum-resistant disease, and disease testability).
Fig. 22A illustrates OS preliminary data obtained by HER3 in qRT-PCR. The data is based on 43/119 events.
Figure 22B is a newer graph of OS by HER3 in qRT-PCR, split 50/50, low HER3 expression in less than the 50 th percentile and high HER3 expression in greater than or equal to the 25 th percentile.
Figure 23A shows PFS by HER3 qRT-PCR comparing high to low Hazard Ratio (HR).
Figure 23B is a more recent plot of PFS by HER3 qRT-PCR, higher ratio of high to low Hazard (HR).
Figure 24A shows a full set of data for Pertuzumab platinum-resistant ovarian cancer in example 1, and PFS by qRT-PCR HER 3. Note that: the HR and Log-rank p values were not adjusted for multiple comparisons.
Figure 24B is another set of data for Pertuzumab platinum-resistant ovarian cancer, and PFS by qRT-PCR HER 3. As in FIG. 24A, the HR and Log-rank p values were not adjusted for multiple comparisons.
Figure 25 shows PFS and OS obtained by HER3 qRT-PCR for patients treated with Pertuzumab monotherapy as in example 2. High HER3 is in greater than and equal to percentage 75 patients; patients in low HER3 were those in less than the 75 th percentile. Median survival for low expressing patients was 3.31 years (95% CI, 1.93-4.69); the median survival for patients with high HER3 expression was 1.80 (95% CI, 0.83-2.78).
Figure 26A shows HER3 calibration normalized ratio (normalized ratio); the expression range is about 20-80 fold. The CP of most samples was between about 23 and 30.
Figure 26B is another graph showing HER3 calibration normalization ratios; the expression range is about 20-80 fold. The CP of most samples was between about 23 and 30.
FIG. 27 shows LIGHTCYCLER in the In Vitro Diagnostic (IVD) assay workflow2.0Pertuzumab qRT-PCR。
Figure 28 shows the Pertuzumab IVD assay workflow and analysis with one marker and reference.
Figure 29A provides PFS by HER 2: HER3 percentiles for patients treated in example 1.
Figure 29B is another graph showing PFS obtained by HER 2: HER3 percentiles for patients treated in example 1. Note that: the HR and log-rank p values were not adjusted for multiple comparisons.
Figure 30A PFS by HER 2: HER3 ratio was evaluated for example 1 using a Kaplan Meyer diagram, specifically for patients with HER2 to HER3 ratios above the median, or above the 75 th percentile.
Figure 30B is a graph showing the renewal of PFS by HER 2: HER3 ratio for example 1 using a Kaplan Meyer plot, particularly for patients with HER2 to HER3 ratios above the median, or above the 75 th percentile.
Figure 31A evaluates PFS obtained by HER 2: HER3 ratio quartile subgroup, also from example 1.
Figure 31B is another summary of PFS analysis by HER2/HER3 quartile recurrent ovarian cancer.
Figure 32 shows a Kaplan-Meier plot of PFS for subjects with ovarian cancer having HER3 levels less than or equal to or greater than the median, respectively, treated as described in example 3.
Figure 33 shows a PFS Kaplan-Meier plot for subjects treated with chemotherapy or Pertuzumab for ovarian cancer with HER3 levels less than median and equal to or greater than median in a patient group.
Figure 34 shows a PFS Kaplan-Meier plot for subjects treated with Pertuzumab and chemotherapy or with Pertuzumab alone for ovarian cancer with HER2/HER3 ratios below median and equal to or greater than median.
Figure 35 shows a PFS Kaplan-Meier plot for subjects treated with chemotherapy or Pertuzumab with HER2/HER3 ratios below and equal to or greater than median for ovarian cancer.
Detailed Description
I. Definition of
The "HER receptor" is a receptor protein tyrosine kinase belonging to the HER receptor family, including the EGFR, HER2, HER3 and HER4 receptors. A HER receptor will typically comprise an extracellular domain which can bind a HER ligand and/or dimerize with another HER receptor molecule; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxy-terminal signal domain containing several phosphorylatable tyrosine residues. The HER receptor may be a "native sequence" HER receptor or an "amino acid sequence variant" thereof. Preferably, the HER receptor is a native sequence human HER receptor.
The terms "ErbB 1", "HER 1", "epidermal growth factor receptor", and "EGFR" are used interchangeably herein and refer to, for example, Carpenter et al, ann.rev.biochem.56: EGFR disclosed in 881-914(1987), including naturally occurring mutant forms thereof (e.g., deletion mutant EGFR in Humphrey et al, PNAS (USA) 87: 4207-4211 (1990)). erbB1 refers to the gene encoding the EGFR protein product.
The expressions "ErbB 2" and "HER 2" are used interchangeably herein and refer to, for example, Semba et al, pnas (usa) 82: 6497-: 230-234(1986), and human HER2 protein (Genebank number X03363). The term "erbB 2" refers to the gene encoding human ErbB2, while "neu" refers to the gene encoding rat p185neuThe gene of (1). Preferred HER2 is native sequence human HER 2.
As used herein, "HER 2 extracellular domain" or "HER 2 ECD" refers to the extracellular domain of HER2, either anchored to the cell membrane or in the circulation, including fragments thereof. In one embodiment, the extracellular domain of HER2 may comprise 4 domains: "Domain I" (about amino acid residues 1 to 195; SEQ ID NO: 19), "Domain II" (about amino acid residues 196 and 319; SEQ ID NO: 20), "Domain III" (about amino acid residues 320 and 488; SEQ ID NO: 21) and "Domain IV" (about amino acid residues 489 and 630; SEQ ID NO: 22) (the residue numbering does not include a signal peptide). See Garrett et al, mol.cell.11: 495-505 (2003); cho et al, Nature 421: 756 and 760 (2003); franklin et al, Cancer Cell 5: 317-; plowman et al, Proc. Natl. Acad. Sci.90: 1746, 1750(1993), and FIG. 1 herein.
"ErbB 3" and "HER 3" refer, for example, to U.S. Pat. Nos. 5,183,884 and 5,480,968 and Kraus et al, PNAS (USA) 86: 9193-9197 (1989).
The terms "ErbB 4" and "HER 4" refer to, for example, european patent application No. 599,274; plowman et al, proc.natl.acad.sci USA 90: 1746 — 1750 (1993); plowman et al, Nature 366: 473, 475(1993), including isoforms thereof, such as disclosed in WO99/19488 published 4/22 in 1999.
"HER ligand" refers to a polypeptide that binds to and/or activates a HER receptor. HER ligands of particular interest herein are natural sequence human HER ligands such as Epidermal Growth Factor (EGF) (Savage et al, J.biol.chem.247: 7612-7621 (1972)); transforming growth factor alpha (TGF-. alpha.) (Marquardt et al, Science 223: 1079-1082 (1984)); amphiregulin (ampheirulin), also known as Schwannomoma or keratinocyte autocrine growth factor (Shoyab et al, Science 243: 1074-1076 (1989); Kimura et al, Nature 348: 257-260 (1990); Cook et al, mol.cell.biol.11: 2547-2557 (1991)); beta cell regulator (betacellulin) (Shing et al, Science 259: 1604-1607 (1993); Sasada et al, biochem. Biophys. Res. Commun.190: 1173 (1993)); heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al, Science 251: 936-939 (1991)); epithelial regulatory protein (epiregulin) (Toyoda et al, J.biol.chem.270: 7495-7500 (1995); Komurasaki et al, Oncogene 15: 2841-2848 (1997)); alpha regulin (heregulin) (see below); neuregulin-2 (NRG-2) (Carraway et al, Nature 387: 512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al, Proc. Natl. Acad. Sci.94: 9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari et al, Oncogene 18: 2681-89 (1999)); and cripto (CR-1) (Kannan et al, J.biol.chem.272 (6): 3330-3335 (1997)). HER ligands that bind EGFR include EGF, TGF- α, amphiregulin, betacellulin, HB-EGF and epiregulin. HER ligands that bind HER3 include heregulin. HER ligands capable of binding to HER4 include beta cell regulator, epithelial regulatory protein, HB-EGF, NRG-2, NRG-3, NRG-4 and heregulin.
"Modulator protein" (HRG) as used herein refers to a polypeptide encoded by a regulatory protein gene, such as that described in U.S. Pat. No. 5,641,869 or Marchionni et al, Nature 362: 312, 318 (1993). Examples of heregulins include heregulin-alpha, heregulin-beta 1, heregulin-beta 2 and heregulin-beta 3(Holmes et al Science 256: 1205-1210 (1992); U.S. Pat. No. 5,641,869); neu Differentiation Factor (NDF) (Peles et al, Cell 69: 205-216 (1992)); acetylcholine Receptor Inducing Activity (ARIA) (Falls et al, Cell 72: 801-815 (1993)); glial Growth Factor (GGF) (Marchionni et al, Nature 362: 312-318 (1993)); sensory and motor neuron derived factor (SMDF) (Ho et al, J.biol.chem.270: 14523-; gamma-heregulin (Schaefer et al Oncogene 15: 1385-1394 (1997)).
"HER dimer" refers herein to a non-covalently bound dimer comprising at least two HER receptors. Such complexes may form upon exposure of cells expressing two or more HER receptors to HER ligands, may be isolated by immunoprecipitation and analyzed by SDS-PAGE, as described, for example, by Sliwkowski et al, j.biol.chem.269 (20): 14661-. Other proteins, such as cytokine receptor subunits (e.g., gp130) can bind to the dimer. Preferably, the HER dimer comprises HER 2.
"HER heterodimer" herein refers to a non-covalently bound heterodimer comprising at least two different HER receptors, such as EGFR-HER2, HER2-HER3, or HER2-HER4 heterodimers.
"HER inhibitor" refers to an agent that interferes with HER activation or function. Examples of HER inhibitors include HER antibodies (e.g., EGFR, HER2, HER3, or HER4 antibodies); an EGFR-targeting agent; a small molecule HER antagonist; HER tyrosine kinase inhibitors; HER2 and EGFR dual tyrosine kinase inhibitors, such as lapatinib/GW 572016; antisense molecules (see, e.g., WO 2004/87207); and/or agents that bind to or interfere with the function of downstream signaling molecules such as MAPK or Akt (see figure 5). Preferably, the HER inhibitor is an antibody or small molecule that binds to a HER receptor.
"HER dimerization inhibitor" refers to an agent that inhibits HER dimer or HER heterodimer formation. Preferably, the HER dimerization inhibitor is a HER2 dimerization inhibitor and/or inhibits HER dimerization. Preferably, the HER dimerization inhibitor is an antibody, e.g. an antibody that binds HER2 at the heterodimeric binding site of HER 2. The most preferred HER dimerization inhibitor herein is Pertuzumab or MAb2C 4. The binding of 2C4 to the heterodimeric binding site of HER2 is shown in figure 4. Other examples of HER dimerization inhibitors include antibodies that bind EGFR and inhibit dimerization thereof with one or more other HER receptors (e.g., EGFR monoclonal antibody 806, MAb 806, which binds activated or "untethered" EGFR; see Johns et al, J.biol.chem.279 (29): 30375-30384 (2004)); an antibody that binds to HER3 and inhibits dimerization thereof with one or more other HER receptors; an antibody that binds to HER4 and inhibits dimerization thereof with one or more other HER receptors; peptide dimerization inhibitors (U.S. Pat. No. 6,417,168); an antisense dimerization inhibitor; and so on.
"HER 2 dimerization inhibitor" refers to an agent that inhibits the formation of dimers or heterodimers comprising HER 2.
"HER antibody" refers to an antibody that binds to a HER receptor. Optionally, the HER antibody also interferes with HER activation or function. Preferably, the HER antibody binds to the HER2 receptor. A HER2 antibody of particular interest herein is Pertuzumab. Another example of a HER2 antibody is trastuzumab. Examples of EGFR antibodies include cetuximab and ABX 0303.
"HER activation" refers to the activation or phosphorylation of any one or more HER receptors. Generally, HER activation results in signal transduction (e.g., caused by an intracellular kinase domain of a HER receptor that phosphorylates tyrosine residues in the HER receptor or substrate polypeptide). HER activation may be mediated by HER ligands that bind to HER dimers comprising the HER receptor of interest. HER ligands that bind to HER dimers may activate the kinase domain of one or more HER receptors in the dimer, thereby resulting in phosphorylation of tyrosine residues in one or more HER receptors and/or phosphorylation of tyrosine residues in other substrate polypeptides such as Akt or MAPK intracellular kinases. See, for example, fig. 5.
"phosphorylation" refers to the addition of one or more phosphate groups to a protein, such as a HER receptor, or a substrate thereof.
An antibody that "inhibits HER dimerization" refers to an antibody that inhibits or interferes with HER dimer formation. Preferably, such antibodies bind HER2 at the heterodimeric binding site of HER 2. The most preferred dimerization inhibiting antibody herein is Pertuzumab or MAb 2C 4. The binding of 2C4 to the heterodimeric binding site of HER2 is shown in figure 4. Other examples of antibodies that inhibit HER dimerization include antibodies that bind EGFR and inhibit dimerization thereof with one or more other HER receptors (e.g., EGFR monoclonal antibody 806, MAb 806, which binds activated or "untethered" EGFR; see Johns et al, J.biol. chem.279 (29): 30375-30384 (2004)); an antibody that binds to HER3 and inhibits dimerization thereof with one or more other HER receptors; and antibodies that bind to HER4 and inhibit dimerization thereof with one or more other HER receptors.
An antibody that "blocks HER receptor activation by a ligand more effectively than trastuzumab" refers to an antibody that reduces or eliminates HER receptor activation by a HER ligand more effectively (e.g., at least about a 2-fold increase in efficacy) than trastuzumab. Preferably, such antibodies block HER ligand activation of HER receptor at least about as effectively as the murine monoclonal antibody 2C4 or a Fab fragment thereof or as Pertuzumab or a Fab fragment thereof. The ability of an antibody to block activation of HER receptors by a ligand can be assessed by direct study of HER dimers, or by assessing HER activation or downstream signaling due to HER dimerization, and/or by assessing antibody-HER 2 binding site, among others. Assays for screening antibodies that inhibit ligand activation of HER receptor more effectively than trastuzumab are described in Agus et al, Cancer Cell 2: 127-137(2002) and WO01/00245(Adams et al). By way of example only, the following may be determined: inhibition of HER dimer formation (see, e.g., Agus et al, FIGS. 1A-B and WO01/00245 to Cancer Cell 2: 127-137 (2002)); reduction of HER ligand activation in cells expressing HER dimers (e.g., WO01/00245 and Agus et al, FIGS. 2A-B of Cancer Cell 2: 127-137 (2002)); blockade of HER ligand binding to HER dimer expressing cells (e.g., WO01/00245 and Agus et al, FIG. 2E of cancer cell 2: 127-137 (2002)); inhibition of Cell growth in the presence (or absence) of a HER ligand on Cancer cells expressing HER dimers (e.g., MCF7, MDA-MD-134, ZR-75-1, MD-MB-175, T-47D cells) (e.g., WO01/00245 and Agus et al, Cancer Cell 2: 127-137(2002) FIGS. 3A-D); inhibition of downstream signaling (e.g., inhibition of HRG-dependent AKT phosphorylation or inhibition of HRG-or TGF-dependent MAPK phosphorylation) (e.g., FIGS. 2C-D of WO01/00245 and Agus et al, Cancer Cell 2: 127-137 (2002)). Whether an antibody inhibits HER dimerization can also be assessed by studying the antibody-HER 2 binding site, for example by assessing the structure or model, such as crystal structure, of an antibody that binds to HER2 (see, e.g., Franklin et al, Cancer Cell 5: 317-.
A "heterodimer binding site" on HER2 refers to a region in the HER2 extracellular domain that contacts or interfaces with a region in the EGFR, HER3, or HER4 extracellular domain when HER2 forms a dimer with EGFR, HER3, or HER 4. Said region has been found in domain II of HER 2. Franklin et al, Cancer Cell 5: 317-328(2004).
The HER2 antibody may be "inhibit HRG-dependent AKT phosphorylation" and/or "inhibit HRG-or TGF-alpha-dependent MAPK phosphorylation" more effectively (e.g., at least 2-fold increase in efficacy) than trastuzumab (see, e.g., Agus et al, Cancer Cell 2: 127-137(2002) and WO 01/00245).
The HER2 antibody may be an antibody that "does not inhibit cleavage of the extracellular domain of HER 2" like Pertuzumab (Molina et al, Cancer Res.61: 4744-4749 (2001)). Trastuzumab, on the other hand, inhibits HER2 ectodomain cleavage.
A HER2 antibody that "binds to the heterodimer binding site of HER2 binds to residues in domain II (optionally also to other domains of the extracellular domain of HER2, such as residues in domains I and III) and may, at least to some extent, sterically hinder the formation of HER2-EGFR, HER2-HER3, or HER2-HER4 heterodimers. Franklin et al, Cancer Cell 5: 317-.
An antibody that "binds domain II of HER 2" binds to residues in domain II and optionally other domains of HER2, such as residues in domains I and III. Preferably, the antibody that binds domain II binds to the junction between domains I, II and III of HER 2.
Protein "expression" refers to the conversion of information encoded in a gene into messenger rna (mrna) and then into a protein.
As used herein, a sample or cell that "expresses" a protein of interest (such as HER3 and/or HER2) refers to a sample or cell in which the presence of mRNA encoding the protein or protein, including fragments thereof, is determined.
A sample, cell, tumor or cancer that "expresses HER3 at a level less than the median level of HER3 expression in a cancer type" refers to a sample, cell, tumor or cancer in which the level of HER3 expression is considered by the skilled artisan to be a "low HER3 level" for that cancer type. Generally, such levels will range from about 0 to less than about 50% relative to the level of HER3 in a population of samples, cells, tumors, or cancers of the same cancer type. For example, the population used to obtain the median expression level may be ovarian cancer samples in general, or a subset thereof, such as chemotherapy-resistant ovarian cancer, platinum-resistant ovarian cancer, and advanced, refractory, or recurrent ovarian cancer. The examples herein demonstrate how median expression levels can be determined. This may constitute expressing an absolute value. As such, referring to fig. 17 herein, the cutoff (cut off) for platinum-resistant ovarian cancer patients who express HER3 at low levels may be about 2.8 or less (less than the 60 th percentile); about 2.41 or less (less than the 55 th percentile); about 2.28 or less (less than the 50 th percentile); about 1.88 or less (less than the 45 th percentile); about 1.71 or less (less than the 40 th percentile); about 1.57 or less (less than the 35 th percentile); about 1.4 or less (less than the 30 th percentile); about 1.19 or less (less than the 25 th percentile); about 0.99 or less (less than the 20 th percentile); and the like. Such absolute values would be quantified in assays under defined assay conditions, such as qRT-PCR as disclosed herein, most preferably the qRT-PCR assay in example 1. Preferably, HER3 expression levels are less than the 50 th percentile, most preferably less than the 30 th or 25 th percentile.
The expressions "HER 2: HER 3" or "HER 2 versus HER 3" as used herein refer to the expression level of HER2 relative to the expression level of HER3 in a sample, cell, tumor or cancer. Such expression levels can be quantified using a variety of different techniques, such as those disclosed herein. While this may be calculated as the ratio of HER2 expression to HER3 expression, the present invention contemplates evaluating HER2 and HER3 levels in various other ways to select patients for therapy herein, including but not limited to using a decision tree (decision tree) in which patients are selected if their HER2 and/or HER3 expression is above or below a certain cutoff, etc. The phrases "HER 2: HER 3" or "HER 2 versus HER 3" herein encompass such various other means for comparing HER2 to HER 3.
A sample, cell, tumor or cancer that "expresses HER 2: HER3 at a level greater than the 25 th percentile for HER 2: HER3 expression in a cancer type refers to a sample, cell, tumor or cancer in which the ratio of HER2 expression to HER3 expression is not a" low HER 2: HER3 level "for that cancer type. Preferably, such levels will range from greater than about 25% to about 100% HER 2: HER3 levels relative to a population of samples, cells, tumors, or cancers of the same cancer type. For example, the population used to obtain the expression levels may be ovarian cancer samples in general, or a subset thereof, such as chemotherapy-resistant ovarian cancer, platinum-resistant ovarian cancer, and advanced, refractory, or recurrent ovarian cancer. The examples herein demonstrate how percentile expression levels can be determined. In one embodiment, HER 2: HER3 levels constitute absolute values of expression. Thus, referring to figure 29 herein, the cutoff for platinum-resistant ovarian cancer patients expressing HER 2: HER3 at this level can be about 0.82 or greater (greater than the 25 th percentile); about 0.90 or greater (greater than the 30 th percentile); about 1.06 or greater (greater than the 35 th percentile); about 1.13 or greater (greater than the 40 th percentile); about 1.26 or greater (greater than the 45 th percentile); about 1.53 or greater (greater than the 50 th percentile); about 1.70 or greater (greater than the 55 th percentile); about 1.86 or greater (greater than the 60 th percentile); about 2.15 or greater (greater than the 65 th percentile); about 2.49 or greater (greater than the 70 th percentile); about 2.62 or greater (greater than the 75 th percentile); about 2.92 or greater (greater than the 80 th percentile); and the like. Such absolute values would be quantified in assays under defined assay conditions, such as qRT-PCR as disclosed herein, most preferably the qRT-PCR assay in example 1. In one embodiment, the HER 2: HER3 level is greater than the 50 th percentile, preferably greater than the 70 th percentile, most preferably greater than the 75 th percentile. Patients whose cancers express HER 2: HER3 at the levels described herein may over-or under-express HER 2.
"polymerase chain reaction" or "PCR" techniques, as used herein, generally refer to procedures in which minute amounts of specific fragments of nucleic acid, RNA and/or DNA are amplified as described in U.S. Pat. No. 4,683,195, issued 7/28/1987. Generally, it is necessary to know sequence information at or beyond the end of the region of interest so that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to the opposite strand of the template to be amplified. The 5' terminal nucleotides of both primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences, etc. from total genomic DNA and cDNA, phage or plasmid sequences transcribed from total cellular RNA. See generally Mullis et al, Cold Spring harbor symp. quant. biol.51: 263 (1987); erlich ed., PCR Technology, Stockton Press, NY, 1989. As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, including the use of a known nucleic acid (DNA or RNA) as a primer and the use of a nucleic acid polymerase to amplify or generate a specific nucleic acid fragment, or to amplify or generate a specific nucleic acid fragment complementary to a specific nucleic acid.
"quantitative real-time polymerase chain reaction" or "qRT-PCR" refers to a form of PCR in which the amount of PCR product is measured at each step of the PCR reaction. This technique has been described in a number of publications including Cronin et al, am.j.pathol.164 (1): 35-42 (2004); ma et al, Cancer cell.5: 607-616(2004).
The term "microarray" refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.
The term "polynucleotide" when used in the singular or plural generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for example, a polynucleotide as defined herein includes, but is not limited to, single-and double-stranded DNA, DNA comprising single-and double-stranded regions, single-and double-stranded RNA, and RNA comprising single-and double-stranded regions, hybrid molecules comprising DNA and RNA, which may be single-stranded or, more typically, double-stranded, or comprise single-and double-stranded regions. In addition, the term "polynucleotide" as used herein refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The chains in such regions may be from the same molecule or from different molecules. The region may comprise the entire population of one or more molecules, but more typically is a region comprising only some molecules. One of the molecules of the triple-helical region is often an oligonucleotide. The term "polynucleotide" specifically includes cDNA. The term includes DNA (including cDNA) and RNA that contain one or more modified bases. Thus, a DNA or RNA whose backbone is modified for stability or other reasons is also a "polynucleotide" for which the term is intended herein. In addition, DNA or RNA comprising rare bases such as inosine or modified bases such as tritiated bases are also included within the term "polynucleotide" as defined herein. In general, the term "polynucleotide" encompasses all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.
The term "oligonucleotide" refers to relatively short polynucleotides, including but not limited to single-stranded deoxyribonucleotides, single-or double-stranded ribonucleotides, RNA, DNA hybrids, and double-stranded DNA. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using a commercially available automated oligonucleotide synthesizer. However, oligonucleotides can be prepared by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNA in cells and organisms.
The phrase "gene amplification" refers to the process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line. The replicated region (a stretch of amplified DNA) is often referred to as an "amplicon". Typically, the amount of messenger RNA (mRNA) produced is also increased in proportion to the number of copies of the particular gene expressed.
The "stringency" of the hybridization reaction can be readily determined by one of ordinary skill in the art, and is generally calculated empirically based on probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment less than their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it can be concluded that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For additional details and explanations of the stringency of hybridization reactions, see Ausubel et al, Current protocols in Molecular Biology, Wiley Interscience Publishers, 1995.
"stringent conditions" or "high stringency conditions", as defined herein, typically: (1) washing with low ionic strength and high temperature, e.g., 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate, at 50 ℃; (2) during hybridization, denaturing agents such as formamide, e.g., 50% (v/v) formamide and 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer pH 6.5 and 750mM sodium chloride, 75mM sodium citrate, at 42 ℃; or (3) washing in 0.2 XSSC (sodium chloride/citrate) and 50% formamide at 42 ℃ with 50% formamide, 5 XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS, and 10% dextran sulfate, followed by high stringency washing in 0.1 XSSC with EDTA at 55 ℃.
"moderately stringent conditions" can be as defined in Sambrook et al, Molecular Cloning: a laboratory Manual, New York, Cold Spring Harbor Press, 1989, including the use of less stringent wash solutions and hybridization conditions (e.g., temperature, ionic strength and% SDS) than those described above. An example of moderately stringent conditions is incubation overnight at 37 ℃ in a solution containing 20% formamide, 5 XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5 XDenhardt's solution, 10% dextran sulfate, and 20mg/ml denatured sheared salmon sperm DNA, followed by washing the filter in 1 XSSC at about 37-50 ℃. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc., as necessary to accommodate factors such as probe length.
A "native sequence" polypeptide refers to a polypeptide having the same amino acid sequence as a polypeptide derived from nature (e.g., a HER receptor or HER ligand), including naturally occurring or allelic variants. Such native sequence polypeptides may be isolated from nature, or may be produced by recombinant or synthetic means. Thus, a native sequence polypeptide can have the amino acid sequence of a naturally occurring human polypeptide, a murine polypeptide, or a polypeptide from any other mammalian species.
The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to substances that would interfere with the research, diagnostic, or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified to (1) greater than 95% by weight, and in some embodiments greater than 99% by weight, of the antibody as determined by, for example, the Lowry method, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using, for example, a rotor sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions and using, for example, coomassie blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, an isolated antibody will typically be prepared by at least one purification step.
"native antibody" refers to a heterotetrameric glycoprotein of about 150,000 daltons, typically composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (V) at one endH) Followed by a plurality of constant domains. Each light chain has a variable domain (V) at one endL) And the other end is a constant domain. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form the interface between the light and heavy chain variable domains.
An antibody "variable region" or "variable domain" refers to the amino-terminal domain of an antibody heavy or light chain. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are generally the most variable parts of an antibody and contain antigen binding sites.
The term "variable" refers to the fact that certain portions of the variable domains differ widely between antibody sequences and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of the antibodies. It is concentrated in three segments called hypervariable regions (HVRs) in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called the Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FRs, which mostly adopt a β -sheet conformation, connected by three HVRs that form loops connecting, and in some cases forming part of, the β -sheet structure. The HVRs in each chain are held together in close proximity by the FRs and, together with the HVRs of the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, National Institutes of Health, Bethesda, Md., 1991). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two distinct types, called kappa (κ) and lambda (λ), based on their constant domain amino acid sequences.
Antibodies (immunoglobulins) can be assigned to different classes depending on the amino acid sequence of their heavy chain constant domains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al, Cellular and mol. The antibody may be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
The terms "full-length antibody" and "intact antibody" are used interchangeably herein to refer to the antibody in substantially intact form, rather than to antibody fragments as defined below. The term specifically refers to antibodies in which the heavy chain comprises an Fc region.
"naked antibody" (naked antibody) "for the purposes of the present invention refers to an antibody which is not conjugated to a cytotoxic moiety or a radiolabel.
An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')2And Fv fragments; diabodies (diabodies); a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having an antigen-binding site, and a remaining "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produced an F (ab')2A fragment which has two antigen binding sites and is still capable of cross-linking antigens.
"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy and one light variable domain in tight, non-covalent association. In the single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain may be covalently linked by a flexible peptide linker, such that the light and heavy chains are joined in a "dimeric" structure analogous to that of a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact at V H-VLAn antigen binding site is defined on the surface of the dimer. Together, the six HVRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, with only less affinity than the entire binding site.
Fab fragments comprise the heavy and light chain variable domains, and further comprise the constant domain of the light chain and the heavy chainOf the first constant domain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant domain carry a free thiol group. F (ab')2Antibody fragments were originally generated as pairs of Fab 'fragments with a hinge cysteine between the pairs of Fab' fragments. Other chemically conjugated forms of antibody fragments are also known.
"Single chain Fv" or "scFv" antibody fragments comprise the V of an antibodyHAnd VLDomains, wherein the domains are present on a single polypeptide chain. In general, scFv polypeptides are at VHAnd VLFurther included between the domains is a polypeptide linker that enables the scFv to form the desired structure for binding to the antigen. For reviews of scFv see, for example, Pl ü ckthun, in The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, 1994, p.269-315. scFv fragments herein expressly include "small molecule immunopharmaceuticals" (SMIPs), such as those disclosed in US2005/0180970A1 and US2005/0186216A1 assigned to Trubion.
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments are in the same polypeptide chain (V)H-VL) Comprising a linked heavy chain variable domain (V)H) And a light chain variable domain (V)L). By using linkers that are too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain, thereby creating two antigen binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; hudson et al, nat. med.9: 129-134 (2003); and Hollinger et al, proc.natl.acad.sci.usa 90: 6444-6448(1993). Triabodies (triabodies) and tetrabodies (tetrabodies) are also described in Hudson et al, nat. med.9: 129-134(2003).
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates that the antibody is not characteristic of a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies typically comprise an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select unique clones from a collection of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the target binding sequence selected may be further altered, for example, to improve affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins.
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 to be used in accordance with the present invention may be generated by a variety of techniques, including, for example, the Hybridoma method (e.g., Kohler and Milstein, Nature, 256: 495-97 (1975); Hongo et al, Hybridoma, 14 (3): 253- -260(1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd edition 1988); Hammerling et al, Monoclonal Antibodies and T-cell Hybridoma 563-) (Elsevier, N.Y, 1981); the recombinant DNA method (see, for example, U.S. Pat. No.4,816,567), the phage display technique (see, for example, Nature 2004 352: 624-) (1991); Marks et al, J.mol.222: Sil 1247; phage display technique (see, 134; Legend et al., USA 134: 340; Legend, 2000-34. J.31; Legend et al; Legend; 132. J.31; Legend; U.31; 2000; USA; Legend; 132; U.32. Pat. No. 31; 35; Legend et al; Legend; No. 31; 35; Legend et al; No. 31; No. 3; 35; Legend; 35; No. 11; Legend; 35; No. 11; Legend; U.32; Legend; No. 11; Legend; No. 31; Leg, And techniques for generating human or human-like antibodies in animals having part or all of a human immunoglobulin locus or a gene encoding a human immunoglobulin sequence (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al, Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al, Nature 362: 255-258 (1993); Bruggemann et al, Yeast in Immuno.7: 33 (1993); U.S. Pat. No.5,545,807; 5,545, 806; 5,569, 825; 5,625,126; 5,633,425; and 5,661,016; Marks et al, Bio/Technology 10: 779-783 (1992); Lonberg et al, Nature 368: 1994-; Morrison, Nature: 812: 1995 (Fisherd et al; Nature wield et al, Biotech. 14: 1996; Neuberg 1996: 14: 1996; Lonberg 1996; Lorberg 11: 92: 1996; Regerg 11: 78, 1996).
Monoclonal antibodies specifically include "chimeric" antibodies wherein a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No.4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). Chimeric antibodies include "primatized" antibodies in which the antigen binding region of the antibody is derived from an antibody produced, for example, by immunizing macaques with an antigen of interest.
"humanized" forms of non-human (e.g., murine) antibodies refer to chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. In one embodiment, a humanized antibody is one in which residues from HVRs in a human immunoglobulin (recipient antibody) are replaced with residues from HVRs of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see, e.g., Jones et al, Nature 321: 522-525 (1986); riechmann et al, Nature 332: 323-329 (1988); and Presta, curr. op. struct.biol.2: 593-596(1992). See also, e.g., Vaswani and Hamilton, ann. 105-115 (1998); harris, biochem. soc. transactions 23: 1035-; hurle and Gross, curr.op.biotech.5: 428-; and U.S. patent nos. 6,982,321 and 7,087,409.
Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 or trastuzumab (HERCEPTIN)) As set forth in table 3 of U.S. patent No. 5,821,337, expressly incorporated herein by reference; humanized 520C9(WO 93/21319); and humanized 2C4 antibodies, such as pertuzamab, as described herein.
For the purposes of the present invention, "trastuzumab", "HERCEPTIN"and" huMAb4D5-8 "refer to antibodies comprising the light and heavy chain amino acid sequences shown in SEQ ID Nos. 15 and 16, respectively.
Herein, the textWherein "Pertuzumab" and "OMNITARGTM"refers to an antibody comprising the light and heavy chain amino acid sequences set forth in SEQ ID Nos. 13 and 14, respectively.
the functional differences between trastuzumab and Pertuzumab are shown in figure 6.
"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.mol.biol.227: 381 (1991); Marks et al, J.mol.biol.222: 581 (1991)). Also useful for the preparation of human monoclonal antibodies are the methods described in the following references: cole et al, Monoclonal Antibodies and cancer Therapy, Alan R.Liss, p.77 (1985); boerner et al, J.Immunol.147 (1): 86-95(1991). See also van Dijk and van de Winkel, curr, opin, pharmacol, 5: 368-74(2001). Human antibodies can be made by administering an antigen to a transgenic animal, such as an immunized XENOMOUSE (xenomice), that has been modified to produce human antibodies in response to antigenic stimuli but has had its endogenous genome disabled (see, e.g., 6,075,181 and 6,150,584 for xenomose TMA technique). See also, for example, Li et al, proc.natl.acad.sci.usa, 103: 3557-3562(2006) on human antibodies generated by the human B-cell hybridoma technique.
"framework" or "FR" residues refer to those residues in the variable domain other than the HVR residues as defined herein.
The term "variable domain residue numbering according to Kabat" or "amino acid position numbering according to Kabat" and variations thereof refers to Kabat et al, supra, for the numbering system used for antibody heavy chain variable domain or light chain variable domain editing. Using this numbering system, the actual linear amino acid sequence may comprise fewer or additional amino acids, corresponding to a shortening or insertion of the variable domain FR or HVR. For example, the heavy chain variable domain may comprise a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c according to Kabat, etc.) after heavy chain FR residue 82. The Kabat residue numbering for a given antibody can be determined by aligning the antibody sequence to the region of homology with a "standard" Kabat numbered sequence.
Throughout the present specification and claims, the Kabat numbering system is generally used when referring to residues in the variable domain (approximately light chain residues 1-107 and heavy chain residues 1-113) (e.g., Kabat et al, sequence of Immunological interest.5th ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
The "EU numbering system" or "EU index" is generally used when referring to residues in the constant region of an immunoglobulin heavy chain (e.g., the EU index reported in Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed. public Health Service, National Institutes of Health, Bethesda, MD (1991), expressly incorporated herein by reference). Unless otherwise indicated herein, reference to residue numbering in the variable domains of antibodies refers to residue numbering according to the Kabat numbering system. Unless otherwise indicated herein, reference to residue numbering in the constant domain of an antibody refers to residue numbering according to the EU numbering system (see, e.g., U.S. provisional application No.60/640,323, a figure for EU numbering).
An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more HVRs of the antibody that result in an improvement in the affinity of the antibody for an antigen as compared to a parent antibody without the alterations. In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies can be generated using certain procedures known in the art. For example Marks et al, Bio/Technology 10: 779-783(1992) describes affinity maturation by VH and VL domain shuffling. The following documents describe random mutagenesis of HVRs and/or framework residues: for example, Barbas et al, proc.nat.acad.sci.usa 91: 3809-3813 (1994); schier et al, Gene 169: 147-; yelton et al, J.Immunol.155: 1994-2004 (1995); jackson et al, j.immunol.154 (7): 3310-9 (1995); hawkins et al, J.mol.biol.226: 889-896(1992).
Antibody "effector functions" refer to those biological activities that can be attributed to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region) and vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.
The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as the segment from the amino acid residue at position Cys226 or Pro230 to the C-terminus thereof. The C-terminal lysine of the Fc region (residue 447, according to the EU numbering system) may be eliminated, for example, during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Thus, a complete antibody composition may include a population of antibodies that have eliminated all K447 residues, a population of antibodies that have not eliminated K447 residues, or a population of antibodies that have been mixed with antibodies with and without K447 residues.
Unless otherwise indicated, the numbering of residues in immunoglobulin heavy chains herein is that of the EU index as in Kabat et al, supra. "EU index as in Kabat" refers to the residue numbering of the human IgG1EU antibody.
A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions typically require that the Fc region be associated with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of published (e.g., in the definitions herein) assays.
A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc region (non-a and a allotypes), native sequence human IgG2 Fc region, native sequence human IgG3 Fc region, and native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.
A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution as compared to the native sequence Fc region or as compared to the Fc region of the parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, preferably from about 1 to about 5 amino acid substitutions, in the native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology, more preferably at least about 90% homology, and most preferably at least about 95% homology to the native sequence Fc region and/or the Fc region of the parent polypeptide.
"Fc receptor" and "FcR" describe receptors that bind to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds an IgG antibody (gamma receptor), including receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of those receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises in its cytoplasmic domain an immunoreceptor tyrosine-based activation motif (ITAM). The inhibitory receptor Fc γ RIIB comprises in its cytoplasmic domain an immunoreceptor tyrosine-based inhibitory motif (ITIM) (see, e.g., forAnnu, rev, immunol.15: 203-234(1997)). For reviews of FcR see, e.g., ravatch and Kinet, annu. 457-492 (1991); capel et al, immunolmethods 4: 25-34 (1994); de Haas et al, j.lab.clin.med.126: 330-41(1995). The term "FcR" encompasses other fcrs herein, including those that will be identified in the future.
The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117: 587(1976) and Kim et al, J.Immunol.24: 249(1994)) and for the regulation of the dynamic balance of immunoglobulins. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol Today 18 (12): 592-598 (1997); Ghetie et al, Nature Biotechnology, 15 (7): 637-640 (1997); Hinton et al, J.biol.Chem279 (8): 6213-6216 (2004); and WO2004/92219(Hinton et al)).
The in vivo binding and serum half-life of human FcRn high affinity binding polypeptides to human FcRn can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having variant Fc regions. WO2000/42072(Presta) describes antibody variants with improved or abolished binding to FcR. See also, e.g., Shields et al j.biol.chem.9 (2): 6591-6604(2001).
"human effector cells" refer to leukocytes which express one or more fcrs and which exert effector function. In certain embodiments, the cell expresses at least Fc γ RIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. The effector cells may be isolated from their natural source, e.g., blood.
"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells, followed by killing of the target cells with cytotoxins. The main cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravatch and Kinet, annu.rev.immunol.9: 457-92(1991) 464 Page table 3 summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. Pat. No.5,500,362 or 5,821,337 or U.S. Pat. No.6,737,056 (Presta). Effector cells useful in such assays include PBMC and NK cells. Alternatively/additionally, ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models, such as Clynes et al, pnas (usa) 95: 652-.
"complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that has bound to its cognate antigen. To assess complement activation, CDC assays can be performed, e.g., as described in Gazzano-Santoro et al, j.immunol.methods 202: 163 (1996). Polypeptide variants having altered Fc region amino acid sequences (polypeptides having variant Fc regions) and increased or decreased C1q binding ability are described, for example, in U.S. Pat. nos. 6,194,551B 1 and WO 1999/51642. See also, e.g., Idusogie et al, j. immunol.164: 4178-4184(2000).
"Fc region-containing antibody" refers to an antibody comprising an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be eliminated, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Thus, a composition comprising an antibody having an Fc region according to the invention may comprise a polypeptide having K447, a polypeptide that eliminates all K447, or a mixture of antibodies with and without the K447 residue.
The term "main species antibody" refers herein to the antibody structure in a composition as the quantitatively primary antibody molecule. In one embodiment, the main species antibody is a HER2 antibody, such as an antibody that binds domain II of HER2, an antibody that inhibits HER dimerization more effectively than Trastuzumab, and/or an antibody that binds to the heterodimeric binding site of HER 2. A preferred embodiment of the main species antibody herein is an antibody comprising the light and heavy chain variable region amino acid sequences of SEQ ID Nos. 3 and 4, most preferably the light and heavy chain amino acid sequences of SEQ ID Nos. 13 and 14 (Pertuzumab).
An "amino acid sequence variant" antibody is herein an antibody having an amino acid sequence that differs from that of the main species antibody. Typically, amino acid sequence variants will have at least about 70% homology with the main species antibody, and preferably, they will be at least about 80%, more preferably at least about 90% homologous with the main species antibody. Amino acid sequence variants have substitutions, deletions and/or additions at certain positions within or adjacent to the amino acid sequence of the main species antibody. Examples of amino acid sequence variants herein include acidic variants (e.g., deamidated antibody variants), basic variants, antibodies having an amino-terminal leader extension (e.g., VHS-) on one or both light chains thereof, antibodies having a C-terminal lysine residue on one or both heavy chains thereof, and the like, and include combinations of heavy and/or light chain amino acid sequence variations. Antibody variants of particular interest herein are antibodies comprising an amino-terminal leader extension on one or both of their light chains, optionally also comprising other amino acid sequences and/or glycosylation differences relative to the main species antibody.
"glycosylation variant" antibodies herein refer to antibodies having one or more carbohydrate moieties attached thereto and which carbohydrate is different from the one or more carbohydrate moieties attached to the main species of antibody. Examples of glycosylation variants herein include antibodies having an Fc region with a G1 or G2 oligosaccharide structure attached in place of the G0 oligosaccharide structure, antibodies having one or two carbohydrate moieties attached to one or two light chains, antibodies having one or two heavy chains without a carbohydrate moiety attached, and the like, as well as combinations of glycosylation alterations.
If the antibody has an Fc region, one or both heavy chains of the antibody, for example at residue 299 (298, Eu residue numbering), may have an oligosaccharide structure attached. For Pertuzumab, G0 is the predominant oligosaccharide structure, while other oligosaccharide structures such as G0-F, G-1, Man5, Man6, G1-1, G1(1-6), G1(1-3), and G2 are found in lesser amounts in Pertuzumab compositions.
Unless otherwise indicated, the "G1 oligosaccharide structures" include herein G1, G1-1, G1(1-6) and G1(1-3) structures.
An "amino-terminal leader extension" refers herein to one or more amino acid residues of an amino-terminal leader sequence present at the amino terminus of any one or more heavy or light chains of an antibody. Exemplary amino-terminal leader extensions comprise or consist of three amino acid residues, VHS, which are present on one or both light chains of an antibody variant.
"deamidated" antibody refers to an antibody in which one or more asparagine residues are derived, for example, as aspartic acid, succinimide, or isoaspartic acid.
The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. As used herein, "cancer type" refers to a particular class or indication of cancer. Examples of such cancer types include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumor (including carcinoid tumor, gastrinoma and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric (gastric or stomach cancer) including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (kidney or renal cancer), prostate cancer, vulval cancer, thyroid cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, cholangiocarcinoma, and head and neck cancer, and any such cancer of a subclass including, but not limited to, platinum-resistant, late-stage chemotherapy-resistant, and non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, Refractory, and/or recurrent types.
By "a type of cancer that is capable of responding to a HER inhibitor" is meant a type of cancer that exhibits therapeutically effective benefit in a patient when treated with a HER inhibitor, such as a HER2 antibody or small molecule inhibitor, in accordance with any criteria of effectiveness of the treatment known to the skilled oncologist, including those detailed herein, particularly in terms of survival, including Progression Free Survival (PFS) and/or Overall Survival (OS). Preferably, such cancer is selected from ovarian cancer, peritoneal cancer, fallopian tube cancer, Metastatic Breast Cancer (MBC), non-small cell lung cancer (NSCLC), prostate cancer, and colorectal cancer. Most preferably, the cancer is ovarian, peritoneal, or fallopian tube cancer, including platinum-resistant forms of such cancers, as well as advanced, refractory, or recurrent ovarian cancer.
By "type of cancer that is capable of responding to a HER dimerization inhibitor" is meant a type of cancer that exhibits therapeutically effective benefit in a patient when treated with a HER dimerization inhibitor, such as Pertuzumab, in accordance with any criteria of effectiveness of treatment known to the skilled oncologist, including those detailed herein, particularly in terms of survival, including Progression Free Survival (PFS) and/or Overall Survival (OS). Preferably, such cancer is selected from ovarian cancer, peritoneal cancer, fallopian tube cancer, Metastatic Breast Cancer (MBC), non-small cell lung cancer (NSCLC), prostate cancer, and colorectal cancer. Most preferably, the cancer is ovarian, peritoneal, or fallopian tube cancer, including platinum-resistant forms of such cancers, as well as advanced, refractory, or recurrent ovarian cancer.
"effective response" and similar terms refer to a response significantly higher than a response to a HER dimerization inhibitor, HER inhibitor or chemotherapeutic agent than a response from a patient that does not express HER3 at the desired level.
"advanced" cancer refers to cancer that spreads beyond the original site or organ by local invasion or metastasis.
"refractory" or "refractory" (refractory) cancer refers to a cancer that progresses even if an anti-neoplastic agent, such as a chemotherapeutic agent, is administered to a patient. An example of a refractory cancer is a platinum resistant cancer.
"recurrent" cancer refers to a cancer that regrows at the initial or distant site after responding to the initial treatment.
Herein, "patient" refers to a human patient. The patient may be a "cancer patient", i.e. a patient suffering from or at risk of suffering from one or more symptoms of cancer.
"tumor sample" as used herein refers to a sample derived from or comprising tumor cells from a patient's tumor. Examples of tumor samples herein include, but are not limited to, tumor biopsies, circulating tumor cells, circulating plasma proteins, ascites fluid, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.
A "fixed" tumor sample refers to a tumor sample that has been histologically preserved using a fixative.
A "formalin-fixed" tumor sample refers to a tumor sample that has been preserved using formaldehyde as a fixative.
An "embedded" tumor sample refers to a tumor sample surrounded by a strong and usually hard medium such as paraffin, wax, pyroxylin, or resin. Embedding makes it possible to cut out slices for microscopic examination or to generate Tissue Microarrays (TMAs).
"Paraffin-embedded" tumor samples refer to tumor samples surrounded by a purified mixture of petroleum-derived solid hydrocarbons.
Herein, "frozen" tumor sample refers to a frozen or frozen tumor sample.
A cancer or biological sample that "displays HER expression, amplification or activation" refers to a cancer or biological sample that expresses (including overexpresses) a HER receptor, has an amplified HER gene, and/or otherwise exhibits HER receptor activation or phosphorylation in a diagnostic test.
A cancer cell that "overexpresses or amplifies a HER receptor" refers to a cancer cell that has significantly higher levels of a HER receptor protein or gene as compared to a non-cancer cell of the same tissue type. Such overexpression may be caused by gene amplification or increased transcription or translation. Overexpression or amplification of a HER receptor can be determined in a diagnostic or prognostic assay by assessing an increase in the level of HER protein present on the surface of a cell (e.g., by an immunohistochemical assay; IHC). Alternatively, or in addition, the level of HER-encoding nucleic acid in the cell may be measured, for example, by fluorescence in situ hybridization (FISH; see WO98/45479 published 10.1998), Southern blotting or Polymerase Chain Reaction (PCR) techniques, such as quantitative real-time PCR (qRT-PCR). HER receptor overexpression or amplification can also be studied by measuring shed antigens (e.g., HER extracellular domains) in biological fluids such as serum (see, e.g., U.S. Pat. No. 4,933,294, published 6/12 in 1990; WO 91/05264, published 4/18 in 1991; U.S. Pat. No. 5,401,638, published 3/28 in 1995; Sias et al, J.Immunol. methods 132: 73-80 (1990)). In addition to the above assays, a variety of in vivo assays may be utilized by the skilled practitioner. For example, cells in a patient may be exposed to an antibody that is optionally labeled with a detectable label, such as a radioisotope, and binding of the antibody to cells in the patient may be assessed, such as by external scanning for radioactivity or by analysis of a biopsy sample taken from a patient that has been previously exposed to the antibody.
Conversely, a cancer in which the HER receptor is not overexpressed or amplified refers to a cancer that does not have higher than normal levels of HER receptor protein or gene as compared to noncancerous cells of the same tissue type. Antibodies that inhibit HER dimerization, such as Pertuzumab, can be used to treat cancers that do not overexpress or amplify HER2 receptor.
As used herein, "antineoplastic agent" refers to a drug used to treat cancer. Non-limiting examples of antineoplastic agents herein include chemotherapeutic agents, HER inhibitors, HER dimerization inhibitors, HER antibodies, antibodies against tumor-associated antigens, anti-hormonal compounds, cytokines, EGFR-targeted drugs, anti-angiogenic agents, tyrosine kinase inhibitors, growth inhibitors and growth inhibitory antibodies, cytotoxic agents, antibodies that induce apoptosis, COX inhibitors, farnesyl transferase inhibitors, antibodies that bind oncofetal protein CA125, HER2 vaccine, Raf or ras inhibitors, doxorubicin liposomes, topotecan, taxanes (taxanes), dual tyrosine kinase inhibitors, TLK286, EMD-7200, Pertuzumab, trastuzumab, erlotinib, and bevacizumab.
An "approved antineoplastic agent" refers to a Drug that has been marketed by regulatory agencies, such as the Food and Drug Administration (FDA) or its equivalent foreign agency, for the treatment of cancer.
If the HER inhibitor or HER dimerization inhibitor is administered as a "single antineoplastic agent," it is the only antineoplastic agent administered to treat cancer, i.e., it is not administered in combination with another antineoplastic agent, such as chemotherapy.
By "standard of care" is meant herein one or more antineoplastic agents conventionally used to treat a particular form of cancer. For example, for platinum-resistant ovarian cancer, the standard treatment is topotecan or liposomal doxorubicin.
"growth inhibitory agent" as used herein refers to a compound or composition that inhibits the growth of a cell, particularly a HER-expressing cancer cell, in vitro or in vivo. As such, the growth inhibitory agent may be one that significantly reduces the percentage of HER-expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a position outside the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes (taxanes), and topoisomerase II inhibitors such as doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), etoposide (etoposide), and bleomycin (bleomycin). Those agents that block G1 also spill over into S phase arrest, for example, DNA alkylating agents such as tamoxifen (tamoxifen), prednisone (prednisone), dacarbazine (dacarbazine), mechlorethamine (mechlorethamine), cisplatin (cissplatin), methotrexate (methotrexate), 5-fluorouracil (5-fluorouracil), and ara-C. For more information see the molecular Basis of Cancer, edited by Mendelsohn and Israel, chapter 1, entitled "Cell cycle expansion, oncogenes, and anti-cosmetic drugs", Murakami et al, WB Saunders, Philadelphia, 1995, especially page 13.
Examples of "growth inhibitory" antibodies are those that bind to HER2 and inhibit the growth of cancer cells that overexpress HER 2. The preferred growth inhibitory HER2 antibody inhibits the growth of SK-BR-3 breast tumor cells in cell culture by more than 20%, preferably more than 50% (e.g., about 50% to about 100%) at an antibody concentration of about 0.5-30 μ g/ml, wherein growth inhibition is determined 6 days after exposure of the SK-BR-3 cells to the antibody (see U.S. Pat. No. 5,677,171 issued 10/14/1997). The SK-BR-3 cell growth inhibition assay is described in more detail in this patent and below. A preferred growth inhibitory antibody is a humanized variant of murine monoclonal antibody 4D5, such as trastuzumab.
An antibody that "induces apoptosis" refers to an antibody that induces programmed cell death as measured by annexin V binding, DNA fragmentation, cell contraction, endoplasmic reticulum expansion, cell rupture, and/or membrane vesicle (referred to as apoptotic bodies) formation. The cell is typically a cell that overexpresses the HER2 receptor. Preferably, the cell is a tumor cell, such as a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder tumor cell. In vitro, the cell may be an SK-BR-3, BT474, Calu 3 cell, MDA-MB-453, MDA-MB-361 or SKOV3 cell. There are a variety of methods available for assessing cellular events associated with apoptosis. For example, Phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed by DNA laddering (laddering); and nuclear/chromatin condensation accompanying DNA fragmentation can be assessed by any increase in hypodiploid cells. Preferably, the antibody that induces apoptosis is one that results in about 2 to 50 fold, preferably about 5 to 50 fold, most preferably about 10 to 50 fold induction of binding to annexin relative to untreated cells in an annexin binding assay using BT474 cells (see below). Examples of HER2 antibodies that induce apoptosis are 7C2 and 7F 3.
"epitope 2C 4" refers to the region in the extracellular domain of HER2 to which antibody 2C4 binds. To screen for Antibodies that bind the epitope of 2C4, a conventional cross-blocking assay can be performed, such as that described in Antibodies, antigen Manual, Cold Spring Harbor Laboratory, Ed Harbor and David Lane, 1988. Preferably, the antibody blocks binding of 2C4 to HER2 by about 50% or more. Alternatively, epitope mapping can be performed to assess whether an antibody binds the 2C4 epitope of HER 2. Epitope 2C4 comprises residues from domain II in the extracellular domain of HER 2. 2C4 and Pertuzumab bind the extracellular domain of HER2 at the junction of domains I, II and III. Franklin et al, Cancer Cell 5: 317-328(2004).
"epitope 4D 5" refers to the region in the extracellular domain of HER2 to which antibodies 4D5(ATCC CRL 10463) and trastuzumab bind. This epitope is close to the transmembrane domain of HER2 and within domain IV of HER 2. To screen for Antibodies that bind the epitope of 4D5, a conventional cross-blocking assay can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, EdHarlow and David Lane, 1988. Alternatively, epitope mapping can be performed to assess whether an antibody binds to the 4D5 epitope of HER2 (e.g., any one or more residues within the region of about residue 529 to about residue 625 of HER2ECD, both endpoints, with the residue numbering including the signal peptide).
"epitope 7C2/7F 3" refers to the region within domain I of the extracellular domain of HER2 to which the 7C2 and/or 7F3 antibodies (each deposited with the ATCC, see below) bind at the N-terminus. To screen for Antibodies that bind the epitope 7C2/7F3, a conventional cross-blocking assay can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harbor and David Lane, 1988. Alternatively, epitope mapping can be performed to determine whether an antibody binds to the 7C2/7F3 epitope on HER2 (e.g., any one or more residues within the region of about residue 22 to about residue 53 of HER2 ECD, residue numbering including signal peptide).
"treatment" and "treatment" (treatment) refer to both therapeutic treatment and prophylactic or preventative measures. Subjects in need of treatment include those already with cancer as well as those in which cancer is to be prevented. Thus, a patient to be treated herein may have been diagnosed as having cancer or may have a predisposition to or susceptibility to developing cancer.
The term "therapeutically effective amount" or "effective amount" refers to an amount of a drug effective to treat cancer in a patient. The effective amount of the drug can reduce the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow to some extent and preferably prevent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and/or to alleviate one or more symptoms associated with cancer to some extent. Depending on the extent to which the drug can prevent the growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic (cytotoxic). An effective amount may prolong progression-free survival (e.g., as measured by Response assessment criteria for Solid Tumors (RECIST) or CA-125 changes), result in an objective Response (including partial Response, PR or complete Response, CR), improve survival (including overall survival and progression-free survival), and/or improve one or more symptoms of cancer (e.g., as assessed by FOSI). Most preferably, the therapeutically effective amount of the drug is effective to improve Progression Free Survival (PFS) and/or Overall Survival (OS).
"survival" (survival) refers to the patient remaining alive, including overall survival (overall survival) and progression free survival (progress free survival).
"overall survival" refers to patients who remain alive for a period of time, such as 1 year, 5 years, etc., calculated from the time of diagnosis or treatment.
"progression-free survival" refers to a patient that remains alive without progression or worsening of cancer.
By "extended survival" is meant an extension of the overall survival or progression-free survival of a patient receiving treatment relative to a patient not receiving treatment (i.e. relative to a patient not treated with a HER inhibitor, a HER dimerization inhibitor such as Pertuzumab) or a patient not expressing HER3 or HER 2: HER3 at a desired level, and/or relative to a patient treated with an approved antineoplastic agent such as topotecan or liposomal doxorubicin where the cancer is ovarian cancer.
"objective response" refers to a measurable response, including a Complete Response (CR) or a Partial Response (PR).
"complete response" (complete response) or "CR" means that all signs of cancer disappear in response to treatment. This does not always mean that the cancer has cured.
"partial response" or "PR" means that the size of one or more tumors or lesions or the extent of cancer in vivo decreases in response to treatment.
The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of a cell and/or causes destruction of a cell. The term is intended to include radioisotopes (e.g., At)211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32And radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and/or variants thereof.
"chemotherapeutic agent" refers to a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and cyclophosphamide (cycloxan)) (ii) a Alkyl sulfonates such as busulfan(buffalfan), improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodepa), carboquone (carboquone), metoclopramide (meteredepa), and uretepa (uredepa); ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimetalmamine; annonaceous acetogenins (especially bullatacin and bullatacin); delta-9-tetrahydrocannabinol (dronabinol), MARINOL ) (ii) a Beta-lapachone (lapachone); lapachol (lapachol); colchicines (colchicines); betulinic acid (betulinic acid); camptothecin (camptothecin) (including synthetic analogue topotecan (HYCAMTIN)) CPT-11 (irinotecan), CAMPTOSAR) Acetyl camptothecin, scopoletin (scopoletin), and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); podophyllotoxin (podophylotoxin); podophyllinic acid (podophyllic acid); teniposide (teniposide); cryptophycins (especially cryptophycins 1 and 8); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); pancratistatin; sarcodictyin; spongistatin (spongistatin); nitrogen mustards (nitrogen mustards), such as chlorambucil (chlorembucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamineMethylamine (mechlorothiamine), mechlorothiamine oxide hydrochloride (hydroxychloride), melphalan (melphalan), neomustard (novembichin), benzene mustard cholesterol (phenesterine), prednimustine (prednimustine), trofosfamide (trofosfamide), uracil mustard (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranimustine); antibiotics such as enediynes antibiotics (enediynes) (e.g., calicheamicins, particularly calicheamicin γ 1I and calicheamicin ω I1 (see, e.g., Nicolaou et al, Angew. chem. int. Ed. Engl. 33: 183-186 (1994)); CDP323, an oral α -4 integrin inhibitor, anthracyclines (dynemicin), including dynemicin A; esperamicin (esperamicin), and neocarzines (neocarzinostatin) and related chromogenes of chromenes, clarithromycin (acrinomycin), actinomycins (actinomycin), anthranomycin (anthramycin), azamycins (anthramycin), azaserines (bleomycin), actinomycins (actinomycins), carminomycin (carmycin), carminomycin (monochromycin), monochromycin (monochromycin), daunomycin (monochromycin), monochromycin (monochromycin), monochromycin (daunomycin), daunomycin (daunomycin), doxorubicin (doxorubicin) (including ADRIAMYCIN) Morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrol doxorubicin, doxorubicin hydrochloride liposome injection (DOXIL)) Liposomal doxorubicin TLC D-99 (MYOCET)) PEGylated liposomal doxorubicin (CAELYX)) And doxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcellomycin), mitomycins (mitomycins) such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (peplomycin), pofiomycin (potfiromycin), puromycin (puromycin), triiron doxorubicin (quelamycin), rodobicin (rodorubicin), streptonigrin (streptonigrin), streptozocin (streptozotocin), tubercidin (tubicidin), ubenimex (enimebendamusex), purified staudin (zinostatin), zorubicin (zorubicin); antimetabolites, such as methotrexate (methotrexate), gemcitabine (gemcitabine) (GEMZAR)) Tegafur (UFTORAL)) Capecitabine (XELODA)) Epothilone (epothilone) and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (azauridine), carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (folinic acid); acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); enluracil (eniluracil) ) (ii) a Amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defosfamide); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etitanium acetate; etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidamine); maytansinoids (maytansinoids) such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine); PSKPolysaccharide complex (JHS natural products, Eugene, OR); razoxane (rizoxane); rhizomycin (rhizoxin); sizofuran (sizofiran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2, 2', 2 "-trichlorotriethylamine; trichothecenes (trichothecenes), especially the T-2 toxin, verrucin A, rorodin A and snake-fish (anguidin); urethane (urethan); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); thiotepa (thiotepa); taxols (taxoid), e.g. paclitaxel (paclitaxel) (TAXOL) ) Albumin-engineered nanoparticle dosage form (ABRAXANE) of paclitaxelTM) And docetaxel (docetaxel) (TAXOTERE)) ); chlorambucil (chlorambucil); 6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); (ii) a platinum-containing agent,such as cisplatin (cissplatin), oxaliplatin (oxa iplatin) and carboplatin (carboplatin); vinblastines (vincas), which prevent tubulin polymerization to form microtubules, include Vinblastine (VELBAN)) Vincristine (vincristine) (ONCOVIN)) Vindesine (elderine)，FILDESIN) And vinorelbine (vinorelbine) (NAVELBINE)) (ii) a Etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); leucovorin (leucovorin); oncostatin (novantrone); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); ibandronate (ibandronate); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids (retinoids), such as retinoic acid (retinoic acid), including bexarotene (TARGRETIN)) (ii) a Diphosphonates (bisphosphates), such as clodronate (e.g. BONEFOS) Or OSTAC) Etidronate (DIDROCAL)) NE-58095, zoledrineAcid/zoledronic acid salt (ZOMETA)) Alendronate (FOSAMAX)) Pamidronate (AREDIA)) Tiludronate (SKELID)) Or risedronate (actone)) (ii) a Troxacitabine (1, 3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC- α, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines, e.g. THERATOPEVaccines and gene therapy vaccines, e.g. ALLOVECTINVaccine, LEUVECTINVaccine and VAXIDA vaccine; topoisomerase 1 inhibitors (e.g. luttotecan)) (ii) a rmRH (e.g. ABARELIX)) (ii) a BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine (perifosine), COX-2 inhibitors (e.g., celecoxib (celecoxib) or etoricoxib (etoricoxib)), proteosome inhibitors (e.g., PS 341); bortezomib (VELCADE)) (ii) a CCI-779; tipifarnib (R11577); orafenaib; ABT 510; bcl-2 inhibitors, such as hamaman (oblimersen sodium) (GENASENSE) ) (ii) a pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combination therapy) and FOLFOX (oxaliplatin)TM) Abbreviation for treatment regimen combining 5-FU and folinic acid).
As used herein, a chemotherapeutic agent includes the class of "anti-hormonal agents" or "endocrine therapeutic agents" that act to modulate, reduce, block or inhibit the effects of hormones that promote cancer growth. They may themselves be hormones, including but not limited to: antiestrogens with mixed agonist/antagonist properties including tamoxifen (tallifen) (NOLVADEX), 4-hydroxyttamoxifen, toremifene (toremifene) (FARESTON)) Idoxifene (idoxifene), droloxifene (droloxifene), raloxifene (raloxifene) (EVISTA)) Trovaxifene (trioxifene), naloxifene (keoxifene), and Selective Estrogen Receptor Modulators (SERMs), such as SERM 3; pure antiestrogens without agonist properties, such as Fulvestrant (FASLODEX) ) And EM800 (such agents may block Estrogen Receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors, such as formestane and exemestane (AROMASIN)) And non-steroidal aromatase inhibitors such as Anastrozole (ARIMIDEX)) Letrozole (FEMARA)) And aminoglutethimide (aminoglutethimide), and other aromatase inhibitors, including vorozole (rivsor)) Megestrol acetate (MEGASE)) Fadrozole and 4(5) -imidazole; luteinizing hormone releasing hormone agonists, including Leuprolide (LUPRON)And ELIGARD) Goserelin (goserelin), buserelin (buserelin) and triptorelin (triptorelin); sex steroids (sex steroids) including pregnanins (progestines) such as megestrol acetate and medroxyprogesterone acetate (medroxyprogesterone acetate), estrogens such as diethylstilbestrol (diethylstilbestrol) and pramelin (premarin), and androgens/retinoids such as fluoxymesterone (fluoxymesterone), all trans-retinoids Acid (transrotinic acid) and fenretinide (fenretinide); onapristone (onapristone); anti-pregnenones; estrogen receptor down-regulators (ERD); anti-androgens such as flutamide (flutamide), nilutamide (nilutamide), and bicalutamide (bicalutamide); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the foregoing.
An "antimetabolite chemotherapeutic agent" refers to an agent that is structurally similar to a metabolite but cannot be utilized by the body in a productive manner. Many antimetabolite chemotherapeutic agents interfere with the production of nucleic acids, RNA and DNA. Examples of antimetabolite chemotherapeutic agents include gemcitabine (gemcitabine) (GEMZAR ar)) 5-fluorouracil (5-FU), capecitabine (Xeloda)TM) 6-mercaptopurine, methotrexate (methotrexate), 6-thioguanine, pemetrexed (pemetrexed), raltitrexed (raltitrexed), cytarabine (arabinosylcytine ARA-C cytarabine) (CYTOSAR-U)) Dacarbazine (dacarbazine) (DTIC-DOME)) Azo cytosine (azocytosine), deoxy cytosine (deoxycytosine), pyrididylene, fludarabine (fludarabine) (FLUDARA)) Cladribine (cladribine), 2-deoxy-D-glucose and the like. The preferred antimetabolite chemotherapeutic agent is gemcitabine.
"Gemcitabine" or "2 ' -deoxy-2 ', 2 ' -difluorocytidine monohydrochloride (b-isomer)" are nucleoside analogs that exhibit antitumor activity. The empirical formula for gemcitabine hydrochloride is C9H11F2N3O4 & HCl. Gemcitabine hydrochloride manufactured by Eli Lilly under the trademark GEMZARAnd (5) selling.
"platinum-based chemotherapeutic agents" include organic compounds that contain platinum as the major part of the molecule. Examples of platinum-based chemotherapeutic agents include carboplatin (carboplatin), cisplatin (cissplatin), and oxaliplatin (oxaliplatinum).
"platinum-based chemotherapy" refers to therapy with one or more platinum-based chemotherapeutic agents, optionally in combination with one or more other chemotherapeutic agents.
"chemotherapy-resistant" cancer refers to a cancer patient that has progressed while receiving a chemotherapy regimen (i.e., the patient is "chemotherapy refractory"), or the patient has progressed within 12 months (e.g., within 6 months) after completion of a chemotherapy regimen (cancer).
"platinum-resistant" cancer refers to a cancer patient that has progressed while receiving platinum-based chemotherapy (i.e., the patient is "platinum refractory"), or the patient has progressed within 12 months (e.g., within 6 months) after completion of a platinum-based chemotherapy regimen (cancer).
An "anti-angiogenic agent" refers to a compound that blocks or interferes to some extent with vascular development. The anti-angiogenic factor can be, for example, a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. Preferred anti-angiogenic factors in this context are antibodies that bind to Vascular Endothelial Growth Factor (VEGF), such as bevacizumab (AVASTIN) )。
The term "cytokine" is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; (ii) a relaxin; a prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); a liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian (Mullerian) inhibitory substances; mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-derived growth factor; transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO); osteoinductive factor (osteoinductive factor); interferons such as interferon- α, - β, and- γ; colony Stimulating Factors (CSFs), such as macrophage CSFs (M-CSF), granulocyte-macrophage CSFs (GM-CSF), and granulocyte CSFs (G-CSF); interleukins (IL), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; tumor necrosis factors such as TNF- α or TNF- β; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
As used herein, the term "EGFR-targeting agent" refers to a therapeutic agent that binds to EGFR and optionally inhibits EGFR activation. Examples of such agents include antibodies and small molecules that bind EGFR. Examples of EGFR-binding antibodies include MAb 579(ATCC CRL HB 8506), MAb 455(ATCC CRLHB 8507), MAb 225(ATCC CRL 8508), MAb 528(ATCC CRL 8509) (see U.S. Pat. No. 4,943,533, Mendelsohn et al) and variants thereof, such as chimera 225(C225 or Cetuximab; ERBUTIX)) And reconstituted human 225(H225) (see WO 96/40210, imclonessystems Inc.); IMC-11F8, a fully human EGFR-targeting antibody (Imclone); antibodies that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind EGFR, as described in U.S. patent No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF (see WO 98/50433, Abgenix); EMD 55900 (Straglioto et al, Eur. J. cancer 32A: 636-640 (1996)); EMD7200(matuzumab), a humanized EGFR antibody directed against EGFR, which competes for EGFR binding with both EGF and TGF- α; and mAb 806 or humanized mAb 806(Johns et al, J.biol.chem.279 (29): 30375-30384 (2004)). anti-EGFR antibodies can be conjugated to cytotoxic agents, thus generating immunoconjugates (see e.g. EP 659,439a2, Merck patent gmbh). Examples of small molecules that bind EGFR include ZD1839 or Gefitinib (IRESSA) TM(ii) a AstraZeneca); CP-358774 or Erlotinib (TARCEVA)TM(ii) a Genentech/OSI); and AG1478, AG1571(SU 5271; Sugen); EMD-7200.
"tyrosine kinase inhibitor" refers to a molecule that inhibits the tyrosine kinase activity of a tyrosine kinase, such as a HER receptor. Examples of such inhibitors include the EGFR-targeting drugs mentioned in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; CP-724,714, an oral ErbB2 receptor tyrosine kinase selective inhibitor (Pfizer and OSI); dual HER inhibitors that preferentially bind EGFR but inhibit both HER2 and EGFR overexpressing cells, such as EKB-569 (available from Wyeth); GW572016 (available from Glaxo), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan (pan) HER inhibitors such as canertinib (CI-1033; Pharmacia); raf-1 inhibitors, such as the antisense agent ISIS-5132, available from ISIS Pharmaceuticals, which inhibits Raf-1 signaling; non-HER targeted TK inhibitors, such as Imatinib mesylate (GLEEVAC), available from GlaxoTM) (ii) a MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035, 4- (3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261, and CGP 62706; pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolo [2, 3-d ]A pyrimidine; curcumin (diferuloylmethane, 4, 5-bis (4-fluoroanilino) -phthalimide); tyrphostins containing nitrothiophene moieties; PD-0183805 (Warner-Lambert); antisense molecules (such as those that bind to HER-encoding nucleic acids); quinoxalines (U.S. patent No. 5,804,396); trypostins (America)National patent No. 5,804,396); ZD6474(Astra Zeneca); PTK-787(Novartis/Schering AG); pan HER inhibitors such as CI-1033 (Pfizer); affinitac (ISIS 3521; ISIS/Lilly); imatinib mesylate (Gleevac; Novartis); PKI 166 (Novartis); GW2016(Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (sugen); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); INC-1C11 (Imclone); or any of the following patent publications: U.S. patent No. 5,804,396; WO 99/09016(American Cyanimid); WO98/43960(American Cyanamid); WO 97/38983(Warner Lambert); WO99/06378(Warner Lambert); WO 99/06396(Warner Lambert); WO 96/30347(Pfizer, Inc.); WO 96/33978 (Zeneca); WO 96/3397 (Zeneca); WO 96/33980 (Zeneca).
Herein, a "fixed" (fixed) or "constant" (flat) dose of a chemotherapeutic agent refers to a dose that is administered to a human patient without regard to the patient's body Weight (WT) and Body Surface Area (BSA). Thus, a fixed or constant dose is not intended as a mg/kg dose or mg/m 2The dosage is provided, but rather is provided as an absolute amount of the therapeutic agent.
A "loading" dose herein generally includes an initial dose of a therapeutic agent administered to a patient, followed by one or more maintenance doses thereof. Generally, a single loading dose is administered, but multiple loading doses are also contemplated herein. Typically, the amount of loading dose administered exceeds the amount of maintenance dose administered, and/or the loading dose is administered more frequently than the maintenance dose, thereby achieving the desired steady-state concentration of the chemotherapeutic agent earlier than the maintenance dose is used.
A "maintenance" (maintenance) dose refers herein to one or more doses of a therapeutic agent administered to a patient during treatment. Typically, the maintenance dose is administered at a therapeutic interval, for example, about once every week, about once every two weeks, about once every three weeks, or about once every four weeks.
"drug" refers to an active agent for the treatment of cancer, such as a HER inhibitor, a HER dimerization inhibitor (such as Pertuzumab), or a chemotherapeutic agent (such as gemcitabine).
"target audience" refers to a group or institution of people who receive or are about to receive a particular drug promotion, such as by promotion or advertising (particularly for a particular use, treatment, or indication), such as individual patients, groups of patients, newspapers, medical literature, and magazine readers, television or internet viewers, radio or internet listeners, physicians, pharmaceutical companies, and the like.
"package insert" is used to refer to instructions typically included in commercial packaging for therapeutic products that contain information regarding indications, usage, dosage, administration, contraindications, other therapeutic products associated with the packaged product, and/or warnings relating to the use of such therapeutic products.
Generation of antibodies
Since in preferred embodiments the HER inhibitor is an antibody, exemplary techniques for generating a HER antibody for use in accordance with the present invention are described below. The HER antigen used to generate the antibody may be, for example, a soluble form of the HER receptor extracellular domain or a portion thereof comprising the desired epitope. Alternatively, cells expressing HER on their cell surface (e.g., NIH-3T3 cells transformed to overexpress HER 2; or cancer cell lines such as SK-BR-3 cells, see Stancovski et al, PNAS (USA) 88: 8691-8695(1991)) can be used to generate antibodies. Other forms of HER receptor useful for generating antibodies will be apparent to those skilled in the art.
(i) Polyclonal antibodies
Polyclonal antibodies are preferably generated by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant in the animal. Using bifunctional or derivatizing reagents, e.g. maleimidobenzoyl sulphosuccinimide ester (coupled via cysteine residues), N-hydroxysuccinimide (coupled via lysine residues), glutaraldehyde, succinic anhydride, SOCl 2Or R1N ═ C ═ NR (where R and R are1Are different hydrocarbon groups) it may be useful to couple the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g. keyhole limpetHemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.
Animals are immunized against an antigen, immunogenic conjugate or derivative by mixing, for example, 100 or 5 μ g of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, animals were boosted with an initial amount of 1/5-1/10 of the peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, blood was collected from the animals, and the antibody titer of the serum was determined. Animals were boosted until the titer reached a plateau (pateau). Preferably, the animal is boosted with a conjugate of the same antigen but conjugated to a different protein and/or via a different cross-linking agent. Conjugates can also be prepared as protein fusions in recombinant cell culture. Also, a coagulant such as alum is suitably used to enhance the immune response.
(ii) Monoclonal antibodies
There are a variety of methods available in the art for preparing monoclonal antibodies herein. For example, monoclonal antibodies may be used as originally produced by Kohler et al, Nature 256: 495(1975) by a recombinant DNA method (U.S. Pat. No. 4,816,567).
In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1986).
The hybridoma cells so prepared are seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these cells, preferred myeloma Cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center (San Diego, California, USA) and SP-2 or X63-Ag8-653 cells available from the American Type culture Collection (American Type culture Collection, Rockville, Maryland, USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human Monoclonal antibodies (Kozbor, J.Immunol.133: 3001 (1984); Brodeur et al, Monoclonal antibody production Techniques and Applications, pp.51-63, Marcel Dekker, Inc., NewYork, 1987).
The medium in which the hybridoma cells are growing can be assayed for production of monoclonal antibodies to the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
The binding affinity of monoclonal antibodies can be determined, for example, by Munson et al, anal. biochem.107: 220(1980) by Scatchard analysis.
After identification of hybridoma cells producing Antibodies with the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, academic Press, 1986). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo in animals as ascites tumors.
The monoclonal antibodies secreted by the subclones can be suitably separated from the culture medium, ascites fluid or serum by conventional antibody purification procedures, such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells are preferred sources of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell that does not otherwise produce antibody protein, such as an escherichia coli cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell, to obtain synthesis of monoclonal antibodies in the recombinant host cell. A review of recombinant expression of DNA encoding antibodies in bacteria includes Skerra et al, curr. opinion in immunol.5: 256-262(1993) and Pl ü ckthun, Immunol. Revs.130: 151-188(1992).
In another embodiment, the compounds may be prepared from compounds using McCafferty et al, Nature 348: 552 (1990) and isolating monoclonal antibodies or antibody fragments from phage antibody libraries. Clackson et al, Nature 352: 624-628(1991) and Marks et al, J.mol.biol.222: 581-597(1991) describes the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the generation of high affinity (nM range) human antibodies by chain shuffling (Marks et al, Bio/Technology 10: 779-. As such, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.
The DNA may also be modified, for example, by replacing homologous murine sequences with the coding sequences for the constant domains of the human heavy and light chains (U.S. Pat. No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA 81: 6851(1984)), or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.
Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-binding site of an antibody, to produce a chimeric bivalent antibody comprising one antigen-binding site with specificity for one antigen and another antigen-binding site with specificity for a different antigen.
(iii) Humanized antibodies
Methods for humanizing non-human antibodies have been described in the art. Preferably, the humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be performed essentially following the method of Winter and co-workers (Jones et al, Nature 321: 522-525 (1986); Riechmann et al, Nature 332: 323-327 (1988); Verhoeyen et al, Science 239: 1534-1536(1988)), by replacing the corresponding human antibody sequences with hypervariable region sequences. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which essentially less than the entire human variable domain is replaced with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.
The choice of human variable domains, both light and heavy, used to construct humanized antibodies is important for reducing antigenicity. The entire library of known human variable domain sequences is screened with the variable domain sequences of rodent antibodies according to the so-called "best-fit" method. The closest human sequence to rodents is then selected as the human Framework Region (FR) of the humanized antibody (Sims et al, J.Immunol.151: 2296 (1993); Chothia et al, J.mol.biol.196: 901 (1987)). Another approach uses specific framework regions derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA 89: 4285 (1992); Presta et al, J. Immunol.151: 2623 (1993)).
More importantly, the antibodies retain high affinity for the antigen and other favorable biological properties after humanization. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a method of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available and familiar to those skilled in the art. Computer programs are available that illustrate and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these display images allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the recipient and import sequences and combined to obtain desired antibody characteristics, such as increased affinity for the target antigen. In general, hypervariable region residues are directly and most substantially involved in the effect on antigen binding.
WO01/00245 describes the generation of exemplary humanized HER2 antibodies that bind to HER2 and block ligand activation of the HER receptor. Humanized antibodies of particular interest herein block EGF, TGF- α and/or HRG mediated MAPK activation substantially as effectively as murine monoclonal antibody 2C4 (or a Fab fragment thereof) and/or bind HER2 substantially as effectively as murine monoclonal antibody 2C4 (or a Fab fragment thereof). The humanized antibodies herein may, for example, comprise non-human hypervariable region residues which incorporate the human heavy chain variable domain, and may further comprise Framework Region (FR) substitutions at positions selected from 69H, 71H and 73H, using the variable domain numbering system set forth in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed., public Health Service, National Institutes of Health, Bethesda, MD, 1991. In one embodiment, the humanized antibody comprises FR substitutions at two or all positions 69H, 71H and 73H.
An exemplary humanized antibody of interest herein comprises the heavy chain variable domain complementarity determining residues GFTFTDYTMX, wherein X is preferably D or S (SEQ ID NO: 7); DVNPNSGGSIYNQRFKG (SEQ ID NO: 8); and/or NLGPSFYFDY (SEQ ID NO: 9), optionally comprising amino acid modifications of those CDR residues, e.g., wherein the modifications substantially maintain or improve the affinity of the antibody. For example, an antibody variant of interest may have from about 1 to about 7 or about 5 amino acid substitutions in the heavy chain variable domain CDR sequences described above. Such antibody variants can be prepared by affinity maturation, e.g., as described below. Most preferred humanized antibodies comprise the amino acid sequence of SEQ ID NO: 4, and (b) a heavy chain variable domain amino acid sequence in (4).
For example, in addition to those heavy chain variable domain CDR residues in the preceding paragraph, the humanized antibody may further comprise light chain variable domain complementarity determining residue KASQDVSIGVA (SEQ ID NO: 10); SASYX1X2X3Wherein X is1Preferably R or L, X2Preferably Y or E, and X3Preferably T or S (SEQ ID NO: 11); and/or QQYYIYPYT (SEQ ID NO: 12). Such humanized antibodies optionally comprise amino acid modifications of the CDR residues described above, e.g., wherein the modifications substantially maintain or improve the affinity of the antibody. For example, an antibody variant of interest may have from about 1 to about 7 or about 5 amino acid substitutions in the light chain variable domain CDR sequences described above. Such antibody variants can be prepared by affinity maturation, e.g., as described below. Most preferred humanized antibodies comprise the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain amino acid sequence.
Affinity matured antibodies that bind to HER2 and block ligand activation of HER receptors are also contemplated. The parent antibody may be a human or humanized antibody, e.g. comprising light chain variable domain and/or heavy chain variable domain sequences of SEQ ID NOs: 3 and 4 (i.e., VL and/or VH comprising Pertuzumab). The affinity matured antibody preferably binds HER2 receptor with an affinity superior to that of murine 2C4 or Pertuzumab (e.g., an increase in affinity of about 2-fold or about 4-fold to about 100-fold or about 1000-fold as assessed by using HER2 extracellular domain (ECD) ELISA). Exemplary heavy chain variable domain CDR residues for substitution include H28, H30, H34, H35, H64, H96, H99, or a combination of two or more of the foregoing residues (e.g., a combination of 2, 3, 4, 5, 6, or 7 of these residues). Examples of light chain variable domain CDR residues for alteration include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97, or a combination of two or more of the foregoing residues (e.g., a combination of 2 to 3, 4, 5, or up to about 10 of these residues).
Various forms of humanized antibodies or affinity matured antibodies are contemplated. For example, the humanized antibody or affinity matured antibody may be an antibody fragment, such as a Fab, which is optionally conjugated to one or more cytotoxic agents to produce an immunoconjugate. Alternatively, the humanized antibody or affinity matured antibody may be an intact antibody, such as an intact IgG1 antibody. A preferred intact IgG1 antibody comprises SEQ ID NO: 13 and the light chain sequence in SEQ ID NO: 14, or a light chain sequence of seq id No. 14.
(iv) Human antibodies
As an alternative to humanization, human antibodies can be generated. For example, it is now possible to generate transgenic animals (e.g., mice) that are capable of generating a complete repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, the region of the antibody heavy chain junction (J) has been described in chimeric and germline mutant miceH) Homozygous deletion of the gene results in complete suppression of endogenous antibody production. Transfer of large numbers of human germline immunoglobulin genes in such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, proc.natl.acad.sci.usa 90: 2551 (1993); jakobovits et al, Nature 362: 255-258 (1993); bruggermann et alYear in immune.7: 33 (1993); and U.S. patent nos. 5,591,669, 5,589,369, and 5,545,807.
Alternatively, phage display technology (McCafferty et al, Nature 348: 552-553(1990)) can be used to generate human antibodies and antibody fragments in vitro from a repertoire of immunoglobulin variable (V) domain genes from an unimmunized donor. According to this technique, antibody V domain genes are cloned in-frame into the major or minor coat protein genes of filamentous phage such as M13 or fd and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous phage particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody displaying those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for reviews see, e.g., Johnson, Kevin s. and Chiswell, David j., Current Opinion in Structural Biology 3: 564-571(1993). Several sources of V gene segments are available for phage display. Clackson et al, Nature 352: 624-628(1991) a large number of different anti-oxazolone antibodies were isolated from a random combinatorial library of small V genes derived from the spleen of immunized mice. Marks et al, j.mol.biol.222: 581-597(1991) or Griffith et al, EMBO J.12: 725-734(1993) by constructing a V gene repertoire from non-immunized human donors and isolating antibodies against a number of different antigens, including self-antigens. See also U.S. Pat. nos. 5,565,332 and 5,573,905.
Human antibodies can also be generated by activating B cells in vitro, as described above (see U.S. Pat. nos. 5,567,610 and 5,229,275).
Human HER2 antibody is described in U.S. patent No. 5,772,997 issued at 30.6.1998 and WO 97/00271 published at 3.1.1997.
(v) Antibody fragments
Have been developed for generating antibodies comprising one or more antigen binding regionsVarious techniques for body fragmentation. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); Brennan et al, Science 229: 81 (1985)). However, these fragments can now be produced directly from recombinant host cells. For example, antibody fragments can be isolated from phage antibody libraries as discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab')2Fragments (Carter et al, Bio/Technology 10: 163-. According to another method, F (ab') can be isolated directly from recombinant host cell cultures2And (3) fragment. Other techniques for generating antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. patent No. 5,571,894; U.S. Pat. No. 5,587,458. The antibody fragment may also be a "linear antibody," for example as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.
(vi) Bispecific antibodies
Bispecific antibodies refer to antibodies having binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the HER2 protein. Other such antibodies may combine the HER2 binding site with the binding site of EGFR, HER3, and/or HER 4. Alternatively, the HER2 arm may be combined with an arm that binds to a triggering molecule on leukocytes, such as a T cell receptor molecule (e.g., CD2 or CD3) or an Fc receptor of IgG (Fc γ R), such as Fc γ RI (CD64), Fc γ RII (CD32), and Fc γ RIII (CD16), such that the cellular defense mechanisms focus on cells expressing HER 2. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing HER 2. These antibodies possess a HER2 binding arm and an arm that binds a cytotoxic agent (e.g., saporin, anti-interferon-alpha, vinca alkaloids, ricin a chain, methotrexate or radioisotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab')2Bispecific antibodies).
WO 96/16673 describes a bispecific HER 2/FcyRIII antibody and U.S. Pat. No. 5,837,234 discloses a bispecific HER 2/FcyRI antibody IDM1 (Osidem). WO98/02463 shows a bispecific HER2/Fc α antibody. U.S. Pat. No. 5,821,337 teaches a bispecific HER2/CD3 antibody. MDX-210 is a bispecific HER2/Fc γ RIII antibody.
Methods for constructing bispecific antibodies are known in the art. The traditional generation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, Nature 305: 537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually performed by an affinity chromatography step, is rather cumbersome and the product yield is low. WO 93/08829 and Traunecker et al, EMBO J.10: 3655-3659(1991) a similar procedure is disclosed.
According to a different approach, antibody variable domains with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant domain sequences. The fusion preferably uses an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. Preferably, in at least one of the fusions, the first heavy chain constant region (CH1) comprises the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains used for construction provide optimal yields, this provides great flexibility in adjusting the mutual ratios of the three polypeptide fragments. However, where expression of at least two polypeptide chains in the same ratio results in high yields or where the ratio is of no particular significance, it is possible to insert the coding sequences for two or all three polypeptide chains into the same expression vector.
In a preferred embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. Since the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient way of isolation, it was found that this asymmetric structure facilitates the separation of the desired bispecific complex from the unwanted immunoglobulin chain combinations. This method is disclosed in WO 94/04690. For further details on the generation of bispecific antibodies see, e.g., Suresh et al, methods in Enzymology 121: 210(1986).
According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces comprise a C of at least part of the antibody constant domainH3 domain. In this method, one or more small amino acid side chains of the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size for large side chains are created at the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.
Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one antibody of the heterologous conjugate may be conjugated to avidin and the other antibody to biotin. For example, such antibodies have been proposed for targeting immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treating HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of crosslinking techniques.
The literature also describesTechniques for generating bispecific antibodies from antibody fragments are described. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science 229: 81(1985) describes the proteolytic cleavage of intact antibodies to F (ab')2And (5) flow of fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then reverted back to Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as selective immobilization reagents for enzymes.
Recent advances have facilitated the direct recovery of Fab' -SH fragments from E.coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al, j.exp.med.175: 217-225(1992) describes the generation of fully humanized bispecific antibodies F (ab')2A molecule. Each Fab' fragment was separately secreted by E.coli and subjected to directed chemical coupling in vitro to form bispecific antibodies. The bispecific antibody so formed was able to bind to cells overexpressing the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for the direct preparation and isolation of bispecific antibody fragments from recombinant cell cultures are also described. For example, leucine zippers have been used to generate bispecific antibodies. Kostelny et al, J.Immunol.148 (5): 1547-1553(1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. Prepared from Hollinger et al, proc.natl.acad.sci.usa 90: 6444-. The fragment comprises heavy chain variable domains (V) connected by a linker H) And a light chain variable domain (V)L) The linker is too short to allow pairing between the two domains on the same strand. Thus, V on a segment is forcedHAnd VLDomain and complementary V on another fragmentLAnd VHThe domains pair, thereby forming two antigen binding sites. Another strategy for constructing bispecific antibody fragments by using single chain fv (sfv) dimers has also been reported. See Gruber et al, j.immunol.152: 5368(1994).
Antibodies with more than two titers are contemplated. For example, trispecific antibodies can be prepared. Tutt et al, j.immunol.147: 60(1991).
(vii) Other amino acid sequence modifications
Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, so long as the final construct possesses the desired properties. Amino acid changes can also alter post-translational processing of the antibody, such as changing the number or position of glycosylation sites.
One method that can be used to identify certain residues or regions of an antibody that are preferred mutagenesis positions is referred to as "alanine scanning mutagenesis," e.g., Cunningham and Wells, Science 244: 1081-. Here, a residue or set of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid positions exhibiting functional sensitivity to substitution are then refined by introducing more or other variants at or for the substitution site. Thus, although the site of introduction of an amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the consequences of a mutation at a given site, alanine scanning mutagenesis or random mutagenesis is performed at the target codon or region and the expressed antibody variants are screened for the desired activity.
Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include fusion enzymes at the N-or C-terminus of the antibody (e.g., for ADEPT) or polypeptides that increase the serum half-life of the antibody.
Another class of variants are amino acid substitution variants. These variants have at least one amino acid residue in the antibody molecule replaced with a different residue. Sites of most interest for substitutional mutagenesis include hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in table 1 under the heading "preferred substitutions". If such substitutions result in a change in biological activity, more substantial changes, referred to as "exemplary substitutions" in Table 1, or as described further below with respect to amino acid species, can be introduced and the products screened.
TABLE 1
Original residues Example alternatives Preferred alternatives
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp；Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu; val; met; ala; phe; norleucine Leu
Leu(L) Norleucine; ile; val; met; ala; phe (Phe) Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile; leu; met; phe; ala; norleucine Leu
Substantial modification of antibody biological properties is accomplished by selecting substitutions that differ significantly in their effectiveness in maintaining: (a) the structure of the polypeptide backbone of the surrogate region, e.g., as a folded sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Amino acids can be grouped according to their similarity in side chain properties (A.L. Lehninger, in: Biochemistry, 2nd ed., pp.73-75, Worth Publishers, New York, 1975):
(1) Non-polar: ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)
(2) Uncharged, polar: gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)
(3) Acidic: asp (D), Glu (E)
(4) Basic: lys (K), Arg (R), His (H)
Alternatively, based on common side chain properties, naturally occurring residues may be grouped as follows:
(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;
(3) acidic: asp and Glu;
(4) basic: his, Lys, Arg;
(5) residues that influence chain orientation: gly, Pro;
(6) aromatic: trp, Tyr, Phe.
Non-conservative substitutions will entail replacing one member of one of these classes for another.
Any cysteine residue not involved in maintaining the correct conformation of the antibody may also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bonds may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).
A particularly preferred class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Typically, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were produced. One convenient method of generating such surrogate variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) were mutated to generate all possible amino acid substitutions at each site. The antibody variants so produced are displayed in monovalent form on filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues which contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the contact point between the antibody and human HER 2. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants is screened as described herein and antibodies with superior properties in one or more relevant assays can be selected for further development.
Another class of amino acid sequence variants of antibodies alters the original glycosylation pattern of the antibody. An alteration means the deletion of one or more carbohydrate moieties found in the antibody, and/or the addition of one or more glycosylation sites not present in the antibody.
Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate module to an asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.
The addition of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence to include one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding or replacing one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
If the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. For example, U.S. patent application No. US 2003/0157108a1(Presta, L.) describes antibodies with a mature carbohydrate structure lacking fucose attached to the Fc region of the antibody. See also US 2004/0093621a1(Kyowa hakko kogyo co., Ltd.). Antibodies having an aliquot of N-acetylglucosamine (GlcNAc) in a carbohydrate attached to the Fc region of the antibody are mentioned in WO 03/011878(Jean-Mairet et al) and U.S. Pat. No. 6,602,684 (Umana et al). Antibodies having at least one galactose residue in an oligosaccharide attached to the Fc region of an antibody are reported in WO 97/30087(Patel et al). For antibodies with altered carbohydrate attachment to their Fc region see also WO 98/58964(Raju, S.) and WO 99/22764(Raju, S.).
It may be desirable to modify an antibody of the invention with respect to effector function, e.g., to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively, or in addition, cysteine residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibody so produced may have improved internalization capacity and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, j.exp.med.176: 1191-1195(1992) and shop, B., J.Immunol.148: 2918-2922(1992). Homodimeric antibodies with enhanced anti-tumor activity can also be used, for example, in Wolff et al, Cancer Research 53: 2560, 2565 (1993). Alternatively, antibodies can be engineered to have dual Fc regions, and thus can have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer drug design 3: 219-230(1989).
WO 00/42072(Presta, L.) describes antibodies with improved ADCC function in the presence of human effector cells, wherein the antibodies comprise amino acid substitutions in their Fc region. Preferably, antibodies with improved ADCC comprise substitutions at positions 298, 333 and/or 334(Eu residue numbering) of the Fc region. Preferably, the altered Fc region is a human IgG1 Fc region comprising, replacing, or consisting of at one, two, or three of these positions. Such substitutions are optionally combined with one or more substitutions that increase the combination of C1q and/or CDC.
Antibodies with altered C1q binding and/or Complement Dependent Cytotoxicity (CDC) are described in WO 99/51642, U.S. patent No. 6,194,551B1, U.S. patent No. 6,242,195B1, U.S. patent No. 6,528,624B1, and U.S. patent No. 6,538,124 (Idusogie et al). The antibody comprises an amino acid substitution at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333 and/or 334(Eu residue numbering) of its Fc region.
To extend the serum half-life of the antibody, a salvage receptor binding epitope can be incorporated into the antibody (particularly an antibody fragment), as described, for example, in U.S. patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an IgG molecule (e.g., IgG) 1、IgG2、IgG3Or IgG4) Is responsible for extending the serum half-life of the IgG molecule in vivo.
Antibodies with improved binding to neonatal Fc receptor (FcRn) and prolonged half-life are described in WO 00/42072(Presta, L.) and US2005/0014934A1(Hinton et al). These antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. For example, the Fc region may have substitutions at one or more of the following positions: 238. 250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428 or 434(Eu residue number). Preferred Fc-region-containing antibody variants with improved FcRn binding comprise amino acid substitutions at one, two or three of positions 307, 380 and 434(Eu residue numbering) of their Fc region.
Engineered antibodies having three or more (preferably four) functional antigen binding sites are also contemplated (U.S. application No. US 2002/0004587a1, Miller et al).
Nucleic acid molecules encoding antibody amino acid sequence variants can be prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants), or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or non-variant version of the antibody.
(viii) Screening for antibodies with desired Properties
Techniques for generating antibodies have been described above. Antibodies having certain biological properties may further be selected, if desired.
To identify an antibody that blocks activation of a HER receptor by a ligand, the ability of the antibody to block binding of the HER ligand to a cell expressing a HER receptor (e.g., along with another HER receptor that forms a HER heteromer with the HER receptor of interest) can be determined. For example, cells that naturally express or are transfected to express HER receptors of HER hetero-oligomers may be incubated with antibodies and then exposed to labeled HER ligands. The ability of the antibody to block ligand binding to HER receptors in HER heteromers can then be assessed.
For example, inhibition of HRG binding to MCF7 breast tumor cell lines by HER2 antibody can be performed on ice using a monolayer of MCF7 culture in 24-well plates, essentially as described in WO 01/00245. The HER2 monoclonal antibody can be added to each well and incubated for 30 minutes. Then can be added125I-tagged rHRG beta 1177-224(25pm) and incubation can be continued for 4-16 hours. Dose-response curves can be plotted and the IC of the antibody of interest can be calculated50The value is obtained. In one embodiment, an antibody that blocks ligand activation of HER receptor inhibits the IC of HRG binding to MCF7 cells in this assay 50About 50nM or less, more preferably 10nM or less. IC for inhibiting HRG binding to MCF7 cells in this assay if the antibody is an antibody fragment such as a Fab fragment50May be, for example, about 100nM or less, more preferably 50nM or less.
Alternatively, or in addition, the ability of an antibody to block HER ligand-stimulated tyrosine phosphorylation of HER receptors present in HER heteromers can be assessed. For example, cells endogenously expressing or transfected to express HER receptor can be incubated with the antibody and then HER ligand-dependent tyrosine phosphorylation activity measured using an anti-phosphotyrosine monoclonal, optionally conjugated with a detectable label. The kinase receptor activation assay described in U.S. patent No. 5,766,863 can also be used to determine HER receptor activation and blocking of this activity by antibodies.
In one embodiment, antibodies can be screened for inhibition of HRG-stimulated p180 tyrosine phosphorylation in MCF7 cells, substantially as described in WO 01/00245. For example, MCF7 cells can be dispensed into 24-well plates and a monoclonal antibody to HER2 added to each well and incubated for 30 minutes at room temperature; then, rHRG beta 1 can be added to each well177-244To a final concentration of 0.2nM and incubation can be continued for 8 minutes. The medium was aspirated from each well and the reaction was stopped by adding 100. mu.l SDS sample buffer (5% SDS, 25mM DTT, 25mM Tris-HCl, pH 6.8). Each sample (25. mu.l) can be electrophoresed on a 4-12% gradient gel (Novex) and then transferred to a polyvinylidene fluoride membrane by electrophoresis. Immunoblots of anti-phosphotyrosine (at 1. mu.g/ml) were developed and the intensity of the major reactive band with a molecular weight of about 180,000 was quantified by reflection densitometry. In this assay, the selected antibody preferably significantly inhibits HRG-stimulated p180 tyrosine phosphorylation to approximately 0-35% of the control. The dose-response curve for inhibition of HRG-stimulated p180 tyrosine phosphorylation as determined by reflection densitometry can be plotted and the IC of the antibody of interest can be calculated 50. In one embodiment, an antibody that blocks ligand activation of HER receptor inhibits the IC of HRG stimulation of p180 tyrosine phosphorylation in this assay50About 50nM or less, more preferably 10nM or less. IC for inhibiting HRG stimulation of p180 tyrosine phosphorylation in this assay if the antibody is an antibody fragment such as a Fab fragment50May be, for example, about 100nM or less, more preferably 50nM or less.
The growth inhibitory effect of the antibodies on MDA-MB-175 cells may also be assessed, for example, essentially as described by Schaefer et al, Oncogene 15: 1385-1394 (1997). According to this assay, MDA-MB-175 cells may be treated with HER2 monoclonal antibody (10. mu.g/mL) for 4 days and stained with crystal violet. Incubation with the HER2 antibody may show similar growth inhibitory effects on this cell line as demonstrated using monoclonal antibody 2C 4. In yet another embodiment, exogenous HRG does not significantly reverse this inhibitory effect. Preferably, the antibody will be capable of inhibiting cell proliferation of MDA-MB-175 cells to a greater extent than monoclonal antibody 4D5 (and optionally to a greater extent than monoclonal antibody 7F 3) in both the presence and absence of exogenous HRG.
In one embodiment, the HER2 antibody of interest blocks heregulin-dependent HER2 binding to HER3 in MCF7 and SK-BR-3 cells substantially more effective than monoclonal antibody 4D5, preferably substantially more effective than monoclonal antibody 7F3, as determined by co-immunoprecipitation experiments, such as described in WO 01/00245.
To identify growth inhibitory HER2 antibodies, antibodies can be screened for inhibition of growth of HER2 overexpressing cancer cells. In one embodiment, the growth inhibitory antibody is selected to inhibit the growth of SK-BR-3 cells in cell culture by about 20-100%, preferably about 50-100%, at an antibody concentration of about 0.5-30 μ g/ml. To identify such antibodies, the SK-BR-3 assay described in U.S. Pat. No. 5,677,171 can be performed. According to this assay, SK-BR-3 cells were cultured in a 1: 1 mixture of DMEM and F12 medium supplemented with 10% fetal bovine serum, glutamine and penicillin streptomycin. SK-BR-3 cells were dispensed into 35mm cell culture dishes (2ml/35mm dish) at 20,000 cells. The HER2 antibody was added at 0.5-30. mu.g/ml per dish. After 6 days, the electronic COULTER was usedTMThe cell counter counts the number of cells compared to untreated cells. Those that inhibit the growth of SK-BR-3 cells by about 20-100% or about 50-100% can be selected as growth inhibitory antibodies. For assays used to screen for growth inhibitory antibodies such as 4D5 and 3E8, see U.S. patent No. 5,677,171.
To select antibodies that induce apoptosis, an annexin binding assay using BT474 cells can be used. BT474 cells were cultured and seeded in petri dishes as discussed in the previous paragraph. The medium was then removed and replaced with fresh medium alone or medium containing 10. mu.g/ml monoclonal antibody. At 3 days warm After the incubation period, the cell monolayer was washed with PBS and detached by trypsinization. The cells were then centrifuged and resuspended in Ca as discussed above for the cell death assay2+Buffer was combined and aliquoted into tubes. Labelled annexin (e.g.annexin V-FTIC) (1. mu.g/ml) is then added to the tube. The FACSCAN can be usedTMFlow cytometer and FACSCOPERTTMCellQuest software (Becton Dickinson) was used to analyze the samples. Those antibodies that induced a statistically significant level of annexin binding relative to the control were selected as apoptosis-inducing antibodies. In addition to the annexin binding assay, a DNA staining assay using BT474 cells can also be utilized. To perform this assay, BT474 cells, which had been treated with the antibody of interest as described in the preceding two paragraphs, were incubated with 9. mu.g/ml HOECHST 33342TMIncubate at 37 ℃ for 2 hours, then EPICS ELITETMMODFIT LT was used on a flow cytometer (Coulter Corporation)TMThe Software (Verity Software House) performs the analysis. Antibodies that induce a change in the percentage of apoptotic cells in this assay to 2-fold or greater (preferably 3-fold or greater) over untreated cells (up to 100% apoptotic cells) may be selected as pro-apoptotic antibodies. For assays for screening antibodies that induce apoptosis, such as 7C2 and 7F3, see WO 98/17797.
To screen for Antibodies that bind to the epitope bound by the antibody of interest on HER2, a conventional cross-blocking assay, such as that described in Antibodies, a Laboratory Manual, Cold Spring harbor Laboratory, Ed Harlow and David Lane, 1988, can be performed to assess whether the Antibodies cross-block the binding of an antibody, such as 2C4 or Pertuzumab, to HER 2. Alternatively/additionally, epitope mapping and/or the antibody-HER 2 structure (Franklin et al, Cancer Cell 5: 317-.
(ix) Pertuzumab composition
In one embodiment of the HER2 antibody composition, the composition comprises the main species Pertuzumab antibodyAnd mixtures of one or more variants thereof. Preferred embodiments of Pertuzumab primary species antibodies herein are antibodies comprising the light and heavy chain variable domain amino acid sequences of SEQ ID nos. 3 and 4, most preferably comprising the light chain amino acid sequence selected from SEQ ID nos. 13 and 17 and the heavy chain amino acid sequence selected from SEQ ID nos. 14 and 18 (including deamidated and/or oxidized variants of those sequences). In one embodiment, the composition comprises a mixture of the major species Pertuzumab antibody and an amino acid sequence variant thereof comprising an amino-terminal leader extension. Preferably, the amino-terminal leader extension is located on a light chain of the antibody variant (e.g., on one or both light chains of the antibody variant). The main species HER2 antibody or antibody variant may be a full-length antibody or antibody fragment (e.g. Fab or F (ab') 2Fragments), but preferably both are full length antibodies. The antibody variants herein may comprise an amino-terminal leader extension on any one or more of its heavy or light chains. Preferably, the amino-terminal leader extension is located on one or both light chains of the antibody. The amino-terminal leader extension preferably comprises or consists of VHS-. The presence of the amino-terminal leader extension in the composition can be detected by a variety of analytical techniques, including but not limited to N-terminal sequence analysis, assays for charge heterogeneity (e.g., cation exchange chromatography or capillary zone electrophoresis), mass spectrometry, and the like. The amount of antibody variant in the composition will generally be in the range from a detection limit constituting any assay for detecting the variant, preferably N-terminal sequence analysis, to an amount of less than the main species of antibody. Typically, about 20% or less (e.g., from about 1% to about 15%, e.g., from 5% to about 15%) of the antibody molecules in the composition comprise an amino-terminal leader extension. Such percentage amounts are preferably determined using quantitative N-terminal sequence analysis or cation exchange analysis (preferably using a high resolution, weak cation exchange column such as PROPAC WCX-10TMCation exchange column). In addition to amino-terminal leader extension variants, other amino acid sequence alterations of the main class of antibodies and/or variants are contemplated, including but not limited to antibodies comprising a C-terminal lysine residue on one or both of its heavy chains, deamidated antibody variants, and the like.
In addition, the main species antibody or variant may also comprise glycosylation variations, non-limiting examples of which include antibodies comprising a G1 or G2 oligosaccharide structure attached to its Fc region, antibodies comprising carbohydrate moieties attached to its light chain (e.g., one or two carbohydrate moieties, such as glucose or galactose, attached to one or both light chains of the antibody, e.g., attached to one or more lysine residues), antibodies comprising one or two non-glycosylated heavy chains, or antibodies comprising sialylated oligosaccharides attached to one or both heavy chains thereof, and the like.
The compositions may be recovered from genetically engineered cell lines expressing the HER2 antibody, such as Chinese Hamster Ovary (CHO) cell lines, or may be prepared by peptide synthesis.
(x) Immunoconjugates
The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a toxin (e.g., a small molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof), or a radioisotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Also contemplated herein are conjugates of the antibody with one or more small molecule toxins, such as calicheamicin (calicheamicin), maytansine (maytansine) (U.S. Pat. No. 5,208,020), trichothecene (trichothene), and CC 1065.
In a preferred embodiment of the invention, the antibody is conjugated to one or more maytansine molecules (e.g., about 1 to about 10 maytansine molecules per antibody molecule). For example, maytansine can be converted to May-SS-Me, which can be reduced to May-SH3 and reacted with modified antibodies (Chari et al, Cancer Research 52: 127-131(1992)) to generate maytansinoid-antibody immunoconjugates.
Another interesting exemptConjugates comprise an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. Structural analogs of calicheamicin that can be used include, but are not limited to, gamma1 I、α2 I、α3 IN-acetyl-gamma1 IPSAG and θI 1(Hinman et al, Cancer Research 53: 3336-. See also U.S. Pat. nos. 5,714,586; nos. 5,712,374; 5,264,586 and 5,773,001, expressly incorporated herein by reference.
Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii toxoprotein, Dianthus caryophyllus (dianhin) toxoprotein, Phytolacca americana (Phytolacca americana) toxoprotein (PAPI, PAPII, and PAP-S), Momordica charantia (momordia) inhibitor, Jatrophin (curcin), crotin (crotin), Saponaria officinalis (saparonia officinalis) inhibitor, gelonin (gelonin), mitomycin (restrictocin), crotin (restrictocin), trichothecene (enomycin), and enomycin (enomycin). See, for example, WO 93/21232 published on 10/28/1993.
The invention also contemplates immunoconjugates formed between an antibody and a compound having nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
There are a variety of radioisotopes available for use in the generation of radioconjugated HER2 antibody. Examples include At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32And radioactive isotopes of Lu.
Conjugates of the antibody with cytotoxic agents can be made using a variety of bifunctional protein-coupling agents, such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexylamine-1-carboxylate, Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (such as toluene-2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, ricin immunotoxins may be prepared, such as vietta et al, Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Research 52: 127-.
Alternatively, fusion proteins comprising the antibody and cytotoxic agent may be prepared, for example, by recombinant techniques or peptide synthesis.
Other immunoconjugates are also contemplated herein. For example, the antibody can be linked to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol. The antibody may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.ed., 1980.
The antibodies disclosed herein can also be formulated as immunoliposomes. Antibody-containing liposomes can be prepared by methods known in the art, such as Epstein et al, proc.natl.acad.sci.usa 82: 3688 (1985); hwang et al, proc.natl.acad.sci.usa 77: 4030 (1980); U.S. patent nos. 4,485,045 and 4,544,545; WO 97/38731 published on 23/10/1997. Liposomes with extended circulation time are disclosed in U.S. patent No. 5,013,556.
Particularly useful liposomes can be formed by reverse phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter having a defined pore size to produce liposomes having a desired diameter. Fab' fragments of the antibodies of the invention can be coupled to liposomes via disulfide exchange reactions, such as Martin et al, j.biol.chem.257: 286 and288 (1982). Optionally, a chemotherapeutic agent is included in the liposomes. See Gabizon et al, j. national cancer inst.81 (19): 1484(1989).
Diagnostic method
In a first aspect, the invention herein provides a method for selecting a therapy for a patient with a type of cancer (e.g. ovarian cancer) that is capable of responding to a HER inhibitor or a HER dimerization inhibitor (e.g. Pertuzumab), comprising determining HER3 expression in a cancer sample from the patient, and selecting a HER inhibitor or a HER dimerization inhibitor as the therapy if the cancer sample expresses HER3 at a level less than the median level for HER3 expression in the cancer type and/or the cancer sample expresses HER 2: HER3 at a level greater than the 25 th percentile (or greater than the median level) for HER 2: HER3 expression in the cancer type.
In a second aspect, the invention provides a method for selecting a therapy for a patient with a cancer type (e.g. ovarian cancer) that is capable of responding to a chemotherapeutic agent, comprising determining HER3 expression in a cancer sample from the patient, and selecting a chemotherapeutic agent (e.g. gemcitabine) as the therapy if the cancer sample expresses HER3 at a level greater than the median level for HER3 expression in the cancer type.
The median or percentile expression level may be determined substantially simultaneously with the measurement of HER3 expression (or HER2 and HER3 expression), or may be determined in advance.
Prior to the treatment methods described below, the level of HER3 expression and optionally HER2 expression in the patient's cancer is assessed. Generally, a biological sample is obtained from a patient in need of treatment, and the sample is subjected to one or more diagnostic assays, typically at least one In Vitro Diagnostic (IVD) assay. However, other forms of assessing HER3 and/or HER2 expression, such as in vivo diagnostics, are expressly contemplated herein. The biological sample is typically a tumor sample, preferably from an ovarian, peritoneal, fallopian tube, Metastatic Breast (MBC), non-small cell lung (NSCLC), prostate, or colorectal tumor sample.
The biological sample herein may be a fixed sample, such as a formalin fixed, paraffin embedded (FFPE) sample, or a frozen sample.
There are a variety of methods for determining mRNA or protein expression, including but not limited to gene expression profiling, Polymerase Chain Reaction (PCR) including quantitative real-time PCR (qRT-PCR), microarray analysis, Serial Analysis of Gene Expression (SAGE), MassARRAY, gene expression analysis by Massively Parallel Signature Sequencing (MPSS), proteomics, Immunohistochemistry (IHC), and the like. Preferably, mRNA is quantified. The mRNA analysis is preferably performed using Polymerase Chain Reaction (PCR) techniques or by microarray analysis. If PCR is used, the preferred form of PCR is quantitative real-time PCR (qRT-PCR). The preferred qRT-PCR assay is described in example 1 below.
The steps of a representative gene expression profiling scheme using fixed, paraffin-embedded tissues as a source of RNA are given in various published journal papers (e.g., Godfrey et al, J.Molec.diagnostics 2: 84-91 (2000); Specht et al, am.J.Pathol.158: 419-29 (2001)): including mRNA isolation, purification, primer extension and amplification. Briefly, one representative method begins by cutting a paraffin-embedded tumor tissue sample into sections approximately 10 microns thick. RNA is then extracted and proteins and DNA are removed. After analysis of the RNA concentration, RNA repair and/or amplification steps may be included, if desired, and the RNA is reverse transcribed using a gene-specific promoter, followed by PCR. Finally, the data is analyzed to determine the best treatment options available to the patient based on the characteristic gene expression patterns identified in the tumor samples examined.
Various exemplary methods for determining gene expression will now be described in more detail.
(i) Profiling of gene expression
Generally, gene expression profiling (gene expression profiling) methods can be divided into two major groups: methods based on polynucleotide hybridization analysis and methods based on polynucleotide sequencing. The most common Methods known in the art for quantifying mRNA expression in a sample include northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106: 247-283 (1999)); RNase protection assay (Hod, Biotechniques 13: 852-854 (1992)); and Polymerase Chain Reaction (PCR) (Weis et al, Trends in Genetics 8: 263-264 (1992)). Alternatively, antibodies that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes may be used. Representative methods of sequencing-based Gene Expression Analysis include Gene Expression Sequential Analysis (SAGE) and Gene Expression Analysis by Massively Parallel Signature Sequencing (MPSS).
(ii) Polymerase Chain Reaction (PCR)
In the techniques listed above, PCR is a sensitive and flexible quantitative method that can be used to compare mRNA levels in different sample populations, in normal and tumor tissues, with or without drug treatment, to characterize gene expression patterns, to distinguish closely related mrnas, and to analyze RNA structures.
The first step is to isolate mRNA from the target sample. The starting material is typically total RNA isolated from human tumors or tumor cell lines and corresponding normal tissues or cell lines, respectively. Thus, RNA can be isolated from a variety of primary tumors, including tumors or tumor cell lines of the breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, and the like, as well as pooled DNA from healthy donors. If the source of the mRNA is a primary tumor, the mRNA can be extracted from, for example, a frozen or archived paraffin-embedded and fixed (e.g., formalin-fixed) tissue sample. General methods for extracting mRNA are well known in the art and are disclosed in standard textbooks of Molecular Biology, including Ausubel et al, Current Protocols of Molecular Biology, John Wiley and Sons, 1997. Methods for extracting RNA from paraffin-embedded tissue are disclosed, for example, in Rupp and Locker, Lab invest.56: a67 (1987); de Andre et al, BioTechniques 18: 42044(1995). Specifically, RNA isolation can be performed using purification kits, buffer sets, and proteases from commercial manufacturers such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cultured cells can be isolated using Qiagen RNeasy mini columns. Other commercial RNA isolation kits include MASTERPURE Whole DNA and RNA purification kit (EPICENTRE)Madison, Wis.) and paraffin block RNA isolation kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumors can be isolated by, for example, cesium chloride density gradient centrifugation.
Since RNA cannot serve as a template for PCR, the first step in gene expression profiling by PCR is reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. Two of the most commonly used reversalsThe transcriptases are avian myeloblastosis Virus reverse transcriptase (AMV-RT) and Moloney murine leukemia Virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed with specific primers, random hexamers or oligo-dT primers, depending on the context and target of the expression profiling. For example, the extracted RNA can be GENEAMPTMThe RNA PCR kit (Perkin Elmer, calif., USA) was reverse transcribed following the manufacturer's instructions. The derived cDNA can then be used as a template for subsequent PCR reactions. Although the PCR step may employ a variety of thermostable DNA-dependent DNA polymerases, typically Taq DNA polymerase is used, which has 5 '-3' nuclease activity but lacks 3 '-5' proofreading endonuclease activity. Thus, TAQMAN PCR typically utilizes the 5 '-nuclease activity of Taq or Tth polymerase to hydrolyze hybridization probes bound to their target amplicons, but any enzyme with equivalent 5' nuclease activity can be used. Two oligonucleotide primers are used to generate the amplicon, typically of a PCR reaction. A third oligonucleotide or probe is designed to detect the nucleotide sequence located between the first two PCR primers. The probe is Taq DNA polymerase non-extendable and labeled with a reporter fluorescent dye and a quencher fluorescent dye. When the two dyes are positioned close together as they are on the probe, any laser-induced emission from the reporter dye is quenched by the quenching dye. During the amplification reaction, Taq DNA polymerase cleaves the probe in a template-dependent manner. The resulting probe fragments dissociate in solution and the signal from the released expressed dye is no longer subject to the quenching effect of the second fluorophore. The detection of the unquenched reporter dye provides the basis for quantitative elucidation of the data.
TAQMANPCR can be performed using commercially available equipment, such as, for example, ABI PRISM7700Sequence detection systems (Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA) or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In a preferred embodiment, the 5' nuclease procedure is run on a real-time quantitative PCR device, such as the ABI PRISM7700 A sequence detection system. The system consists of a thermal cycler, a laser, a Charge Coupled Device (CCD), a camera and a computer. The system amplifies samples in 96-well format on a thermal cycler. During amplification, laser-induced fluorescence signals were collected in real time from all 96 wells via a fiber optic cable and detected at the CCD. The system includes software for running the device and analyzing the data.
The 5' -nuclease assay data was originally expressed as Ct, or cycle threshold (threshold cycle). As discussed above, fluorescence values were recorded during each cycle and represent the amount of product amplified up to that point in the amplification reaction. The first point at which a statistically significant fluorescence signal is recorded is the cycle threshold (Ct).
In order to minimize the effects of errors and sample-to-sample variation, PCR is typically performed using internal standards. The ideal internal standard is expressed at a constant level between different tissues and is not affected by experimental treatments. The most frequently used RNAs for normalization of gene expression patterns are the mRNA for the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P-actin.
A recent variation of the PCR technique is quantitative real-time PCR (qRT-PCR), which generates probes by dual-labeled fluorescence (i.e., TAQMAN) Probe) to measure PCR product accumulation. Real-time PCR is compatible with both quantitative competitive PCR (where internal competitors for each target sequence are used for normalization) and quantitative comparative PCR (where normalization genes or housekeeping genes contained within a sample are used for PCR). More detailsSee, e.g., Held et al, Genome Research 6: 986-994(1996).
Several published journal papers show steps of representative protocols for gene expression profiling using fixed, paraffin-embedded tissues as a source of RNA, including mRNA isolation, purification, primer extension and amplification (e.g., Godfrey et al, j.molec. diagnostics 2: 84-91 (2000); Specht et al, am.j.pathol.158: 419-29 (2001)). Briefly, one representative method begins by cutting out approximately 10 micron thick sections of paraffin-embedded tumor tissue samples. Then, RNA is extracted, and proteins and DNA are removed. After analysis of the RNA concentration, RNA repair and/or amplification steps may be included, if necessary, and the RNA is reverse transcribed using gene specific promoters, followed by PCR.
According to one aspect of the invention, PCR primers and probes are designed based on intron sequences present in the gene to be amplified. In this embodiment, the first step in primer/probe design is to delineate intron sequences within the gene. This can be done by publicly available software, such as that available by Kent, w., genomes res.12 (4): 656-64(2002) DNA BLAT software or BLAST software, including software different therefrom. The subsequent steps follow a fully established PCR primer and probe design approach.
To avoid non-specific signals, it is important to mask the repeated sequences within the (mask) intron when designing the primers and probes. This can be readily accomplished by using the Repeat Masker program, which is available on-line by the Baylor College of Medicine (Baylor College of Medicine), which screens DNA sequences against libraries of Repeat elements and returns query sequences with Repeat elements masked therein. The masked intron sequences can then be used to design Primer and probe sequences using any commercially available Primer/probe design package or by other means, such as Primer Express (Applied Biosystems); MGBassay-by-design (applied biosystems); primer3(Rozen and Squaletsky (2000) Primer3 is available on the world Wide Web for general users and biologist programmers in "Bioinformatics Methods and Protocols: Methods in Molecular Biology", ed. by Krawetz S, Miseners, Humana Press, Totowa, N.J., pp. 365-.
Factors considered in PCR primer design include primer length, melting temperature (Tm), G/C content, specificity, complementary primer sequence, and 3' -end sequence. In general, optimal PCR primers are typically 17-30 bases long, comprising about 20-80% such as, for example, about 50-60% G + C bases. Typical preferred Tm is between 50 and 80 ℃, e.g., about 50-70 ℃.
For further guidance on PCR Primer and probe Design see, e.g., Dieffenbach et al, "General Concepts for PCR Primer Design", in PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, p. 133-155; innis and Gelfand, "Optimization of PCRs" (Optimization of PCR), in PCR Protocols, A Guide to Methods and Applications, CRCPress, London, 1994, pages 5-11; and platter, t.n., Primerselect: primer and probe design. methods mol. biol.70: 520-527(1997), the entire disclosure of which is expressly incorporated herein by reference.
Preferred conditions, primers, probes, and internal reference (G6PDH) are described in example 1 below.
(iii) Microarray
Differential gene expression can also be identified or verified using microarray technology. Thus, the expression profile of breast cancer-associated genes can be measured in fresh or paraffin-embedded tumor tissue using microarray technology. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are coated (plate) or arrayed (array) on a microchip substrate. The aligned sequences are then hybridized to specific DNA probes from the cell or tissue of interest. As in the PCR method, the source of mRNA is typically total RNA isolated from human tumors or tumor cell lines and corresponding normal tissues or cell lines. Thus, RNA can be isolated from a variety of primary tumors or tumor cell lines. If the source of the mRNA is a primary tumor, the mRNA can be extracted from, for example, frozen or archived paraffin-embedded and fixed (e.g., formalin-fixed) tissue samples that are routinely prepared and stored in routine clinical practice.
In one specific embodiment of microarray technology, inserts of PCR amplified cDNA clones are applied to a substrate in a dense array. Preferably, at least 10,000 nucleotide sequences are applied to the substrate. Microarray genes immobilized on microchips with 10,000 components per substrate are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes can be generated by incorporating fluorescent nucleotides into reverse transcription of RNA extracted from the tissue of interest. Labeled cDNA probes applied to the chip hybridize specifically to each DNA spot on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by other detection methods such as a CCD camera. Quantification of hybridization of each array component allows assessment of the abundance of the corresponding mRNA. Separately labeled cDNA probes generated from both RNA sources were hybridized in pairs to the array by virtue of dual colored fluorescence. The relative abundance of transcripts from both sources corresponding to each designated gene was thus determined simultaneously. Miniaturized scale hybridization provides convenient and rapid assessment of large numbers of gene expression patterns. Such methods have shown at least about 2-fold differences in the sensitivity and reproducible detection expression levels required to detect rare transcripts expressed in a small number of copies per cell (Schena et al, Proc. Natl. Acad. Sci. USA 93 (2): 106-. Microarray analysis can be performed using commercial equipment following the manufacturer's protocol, such as using Affymetrix GENCHIP TMThe technique or Incyte's microarray technique.
Microarray methods developed for large-scale analysis of gene expression make it possible to systematically search for molecular markers for cancer classification and outcome prediction in a variety of tumor types.
(iv) Continuous analysis of Gene expression (SAGE)
Sequential Analysis of Gene Expression (SAGE) is a method that allows simultaneous and quantitative analysis of a large number of gene transcripts without the need to provide individual hybridization probes for each transcript. First, a short sequence tag (about 10-14bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is derived from a unique position within each transcript. Many transcripts are then joined to form long, contiguous molecules that can be sequenced to reveal the identity (identity) of multiple tags simultaneously. The expression pattern of any transcript population can be quantitatively assessed by determining the abundance of individual tags and identifying the genes corresponding to each tag. For more details see, e.g., Velculescu et al, Science 270: 484- & 487 (1995); velculescu et al, Cell 88: 243-51(1997).
(v) MassARRAY technology
The MassARRAY (Sequenom, San Diego, Calif.) technique is an automated, high throughput method of gene expression analysis that uses Mass Spectrometry (MS) for detection. According to this method, after RNA isolation, reverse transcription and PCR amplification, cDNA is subjected to primer extension. The cDNA-derived primer extension products were purified and distributed onto chip arrays pre-loaded with the components required for MALTI-TOF MS sample preparation. The various cDNAs present in the reaction were quantified by analyzing the peak areas in the resulting mass spectra.
(vi) Gene expression analysis by Massively Parallel Signature Sequencing (MPSS)
By Brenner et al, Nature Biotechnology 18: the method described in 630-634(2000) is a sequencing method that combines non-gel based signature sequencing with in vitro cloning of millions of templates on separate 5 micron diameter microbeads. First, a bead library of DNA templates was constructed by in vitro cloning. Then, in a flow cell at high density (typically greater than 3X 10)6Micro bead/cm2) A planar array of template-containing microbeads was assembled. The free ends of the cloned templates on each microbead were simultaneously analyzed using a fluorescence-based signature sequencing method that does not require separation of the DNA fragments. This approach has been shown to provide numbers from a yeast cDNA library simultaneously and accurately in a single operation toOne hundred thousand gene signature sequences.
(vii) Immunohistochemistry
Immunohistochemical methods are also suitable for detecting the expression level of the prognostic markers of the present invention. Thus, expression is detected using antibodies or antisera, preferably polyclonal antisera, most preferably monoclonal antibodies, specific for each marker. The antibody may be detected by directly labeling the antibody itself, for example with a radiolabel, a fluorescent label, a hapten label such as biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibodies are used in combination with labeled secondary antibodies, including antisera, polyclonal antisera, or monoclonal antibodies specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.
(viii) Proteomics
The term "proteome" is defined as the totality of proteins present in a sample (e.g., a tissue, organism or cell culture) at a point in time. Proteomics includes, among other things, the global variation of protein expression in a study sample (also referred to as "expression proteomics"). Proteomics typically includes the following steps: (1) separating the various proteins in the sample by two-dimensional gel electrophoresis (2-D PAGE); (2) identifying the various proteins recovered from the gel, for example by mass spectrometry or N-terminal sequencing; and (3) analyzing the data using bioinformatics. Proteomics methods are useful complements of other gene expression profiling methods, either alone or in combination with other methods, for detecting the products of the prognostic markers of the present invention.
(ix) General description of mRNA isolation, purification and amplification
Several published journal papers show steps of representative protocols for gene expression profiling using fixed, paraffin-embedded tissues as a source of RNA, including mRNA isolation, purification, primer extension and amplification (e.g., Godfrey et al, j.molec. diagnostics 2: 84-91 (2000); Specht et al, am.j.pathol.158: 419-29 (2001)). Briefly, one representative method begins by cutting out approximately 10 micron thick sections of paraffin-embedded tumor tissue samples. Then, RNA is extracted, and proteins and DNA are removed. After analysis of the RNA concentration, RNA repair and/or amplification steps may be included, if necessary, and the RNA is reverse transcribed using gene specific promoters, followed by PCR. Finally, based on the characteristic gene expression pattern identified in the tumor sample examined, the data is analyzed to identify the best treatment options available to the patient.
In one embodiment, in addition to expressing HER3 at a certain level and/or HER 2: HER3 at a certain level, the patient treated herein does not further overexpress HER 2. HER overexpression can be analyzed by IHC, e.g. using HERCEPTEST(Dako). Paraffin-embedded tissue sections from tumor biopsies can be subjected to IHC assays and against the following HER2 protein staining intensity criteria:
score 0: no staining was observed or membrane staining was observed in less than 10% of the tumor cells.
Score 1 +: faint/barely detectable membrane staining was detected in more than 10% of tumor cells. The cells have staining in only a portion of their membranes.
And the score is 2 +: weak to moderate complete membrane staining was observed in more than 10% of tumor cells.
Score 3 +: moderate to intense complete membrane staining was observed in more than 10% of tumor cells.
Those tumors with HER2 overexpression assessed to score 0 or 1+ may be characterized as not overexpressing HER2, while those with a score of 2+ or 3+ may be characterized as overexpressing HER 2.
HER2 overexpressing tumors can be ranked according to an immunohistochemical score corresponding to the number of copies of HER2 molecule expressed per cell, and can be biochemically determined:
0-10,000 copies/cell,
1+ ═ at least about 200,000 copies per cell,
2+ at least about 500,000 copies/cell,
at least about 2,000,000 copies per cell.
Overexpression of HER2 at 3+ levels leading to ligand-dependent activation of tyrosine kinases (Hudziak et al, Proc. Natl. Acad. Sci. USA 84: 7159-7163(1987)) occurs in approximately 30% of breast cancers and in these patients, relapse-free survival and overall decreased survival (Slamon et al, Science 244: 707-712 (1989); Slamon et al, Science 235: 177-182 (1987)). Alternatively, or in addition, FISH assays such as INFORM may be performed on formalin fixed, paraffin embedded tumor tissueTM(Ventana, Arizona) or PATHVISIONTM(Vysis, Illinois) to determine the extent, if any, of HER2 amplification in tumors.
HER3 and/or HER2 expression may also be assessed using in vivo diagnostic assays, for example by administering a molecule (such as an antibody) that binds to the molecule to be detected and is labeled with a detectable label (e.g. a radioisotope) and then externally scanning the patient to locate the label.
Pharmaceutical formulations
Therapeutic formulations of HER inhibitors, HER dimerization inhibitors, or chemotherapeutic agents for use in accordance with the invention are prepared for storage by mixing the antibody of the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16 th edition, Osol, a. eds. (1980)), typically in the form of a lyophilized formulation or an aqueous solution. Antibody crystals are also contemplated (see, e.g., U.S. patent application 2002/0136719). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl chloride) Benzyl ammonium; (ii) hexanediamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, such as TWEENTM、PLURONICSTMOr polyethylene glycol (PEG). Lyophilized antibody formulations are described in WO 98/56418, expressly incorporated herein by reference.
A preferred Pertuzumab formulation for therapeutic use comprises 30mg/mL Pertuzumab in 20mM histidine acetate, 120mM sucrose, 0.02% polysorbate 20, pH 6.0. Another Pertuzumab formulation comprised 25mg/mL Pertuzumab, 10mM histidine-HCl buffer, 240mM sucrose, 0.02% polysorbate 20, pH 6.0.
The formulations herein may also contain more than one active compound necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Various drugs that can be combined with HER inhibitors or HER dimerization inhibitors are described in the therapeutic section below. Suitably, such molecules are present in combination in amounts effective for the intended purpose.
The active ingredient may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's pharmaceutical sciences, 16 th edition, Osol, A. eds (1980).
Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid with gamma ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRONDEPOT TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid.
Formulations for in vivo administration must be sterile. This can be easily achieved by filtration using sterile filtration membranes.
Thus, there is provided a method for the manufacture of a HER inhibitor, or a HER dimerization inhibitor (such as Pertuzumab), or a pharmaceutical composition thereof, the method comprising combining in a package the inhibitor or pharmaceutical composition and a label stating that the inhibitor or pharmaceutical composition is indicated for use in treating a patient with a type of cancer that is capable of responding to the inhibitor (e.g., ovarian cancer), wherein the patient's cancer expresses HER3 at a level less than the median level for HER3 expression in the type of cancer and/or a cancer sample of the patient expresses HER 2: HER3 at a level greater than the 25 th percentile for HER 2: HER3 expression in the type of cancer.
Additionally, a method for the manufacture of a chemotherapeutic agent (such as gemcitabine) or a pharmaceutical composition thereof is provided, wherein the method comprises combining in a package said chemotherapeutic agent or pharmaceutical composition and a label stating that said chemotherapeutic agent or pharmaceutical composition is indicated for treating a patient having a type of cancer, wherein said patient's cancer expresses HER3 at a level greater than the median level for HER3 expression in the type of cancer.
Treatment with HER inhibitors
The invention herein provides a method for treating a patient having a type of cancer which is capable of responding to a HER inhibitor or a HER dimerization inhibitor comprising administering to the patient a therapeutically effective amount of the inhibitor, wherein the patient's cancer expresses HER3 at a level less than the median level for HER3 expression in the type of cancer and/or wherein a cancer sample of the patient expresses HER 2: HER3 at a level greater than the 25 th percentile for HER 2: HER3 expression in the type of cancer. Preferably, the patient's cancer expresses HER3 at a level less than the 25 th percentile for HER3 expression in the cancer type and/or HER 2: HER3 at a level greater than the median level for HER 2: HER3 expression in the cancer type (most preferably greater than the 75 th percentile for HER 2: HER3 expression in the cancer type).
In a particularly preferred embodiment, the invention provides a method for treating a patient with ovarian, peritoneal, or fallopian tube cancer comprising administering a therapeutically effective amount of Pertuzumab to the patient, wherein the patient's cancer expresses HER3 at a level less than the median level for HER3 expression in ovarian, peritoneal, or fallopian tube cancer, and/or wherein a cancer sample of the patient expresses HER 2: HER3 at a level greater than the 25 th percentile for HER 2: HER3 expression in ovarian, peritoneal, or fallopian tube cancer. In this embodiment, preferably the patient's cancer expresses HER3 at a level less than the 25 th percentile for HER3 expression in ovarian, peritoneal, or fallopian tube cancer and/or expresses HER 2: HER3 at a level greater than the median level for HER 2: HER3 expression in ovarian, peritoneal, or fallopian tube cancer (most preferably greater than the 75 th percentile for HER 2: HER3 expression in ovarian, peritoneal, or fallopian tube cancer).
In another aspect, the invention provides a method for selecting a therapy for a patient with a type of cancer that is capable of responding to a chemotherapeutic agent, comprising determining HER3 expression in a cancer sample from the patient, and selecting a chemotherapeutic agent as therapy if the cancer sample expresses HER3 at a level greater than the median level for HER3 expression in the cancer type. In this embodiment, preferably the cancer type is ovarian, peritoneal, or fallopian tube cancer, including platinum-resistant ovarian, peritoneal, or fallopian tube cancer, as well as advanced, refractory, and/or recurrent ovarian cancer. The chemotherapeutic agent is preferably an antimetabolite, such as gemcitabine. As such, in this embodiment, high HER3 is associated with improved response to chemotherapy agent (such as gemcitabine) therapy.
Examples of various cancers that can be treated with a HER inhibitor or a HER dimerization inhibitor are listed in the definitions section above. Preferred cancer types include ovarian cancer, peritoneal cancer, fallopian tube cancer, breast cancer including Metastatic Breast Cancer (MBC), lung cancer including non-small cell lung cancer (NSCLC), prostate cancer and colorectal cancer. In one embodiment, the cancer treated is an advanced, refractory, relapsed, chemotherapy-resistant and/or platinum-resistant cancer.
HER inhibitors, HER dimerization inhibitors and/or chemotherapeutic agent therapies preferably prolong survival, including progression free survival (PGS) and/or Overall Survival (OS). In one embodiment, the HER inhibitor or HER dimerization inhibitor therapy extends survival by at least about 20% over that achieved by administration of an approved antineoplastic agent or standard treatment for the cancer being treated.
In preferred embodiments, the methods relate to treating a patient having ovarian, peritoneal, or fallopian tube cancer. The patient may have advanced, refractory, recurrent, chemotherapy-resistant and/or platinum-resistant ovarian, peritoneal or fallopian tube cancer. Administration of Pertuzumab to a patient can, for example, extend survival by at least about 20% over that achieved by administration of topotecan or liposomal doxorubicin to such a patient.
The HER inhibitor, HER dimerization inhibitor and/or chemotherapeutic agent is administered to the human patient according to known methods, such as intravenous administration, e.g. bolus injection or continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical or inhalation routes. Intravenous administration of the antibody is preferred.
For the prevention or treatment of cancer, the dosage of the HER inhibitor, HER dimerization inhibitor and/or chemotherapeutic agent will depend on the type of cancer to be treated, as defined above, the severity and course of the cancer, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the drug, and the discretion of the attending physician.
In one embodiment, a fixed dose of inhibitor is administered. Suitably, the fixed dose may be administered to the patient at one time or over a series of treatments. If a fixed dose is administered, the dose is preferably in the range of about 20mg to about 2000mg of the inhibitor. For example, the fixed dose may be about 420mg, about 525mg, about 840mg, or about 1050mg of an inhibitor, such as Pertuzumab.
If multiple doses are administered, they may be administered, for example, about weekly, about every two weeks, about every three weeks, or about every four weeks, but preferably about every three weeks. The fixed dose may, for example, be administered continuously until the disease progression, adverse event, or other time as determined by the physician. For example, a fixed dose of about 2, 3, or 4 doses, up to about 17 or more doses, may be administered.
In one embodiment, one or more loading doses (loading doses) of antibody are administered followed by one or more maintenance doses (maintenance doses) of antibody. In another embodiment, multiple identical doses are administered to the patient.
According to a preferred embodiment of the invention, a fixed dose of about 840mg (loading dose) of a HER dimerization inhibitor (e.g. Pertuzumab) is administered, followed by one or more doses of about 420mg (maintenance dose) of the antibody. The maintenance dose is preferably administered about every three weeks for a total of at least 2 doses up to 17 or more doses.
According to another preferred embodiment of the invention, one or more HER dimerization inhibitors (e.g. Pertuzumab) is administered at a fixed dose of about 1050mg, e.g. every three weeks. According to this embodiment, one, two or more fixed doses are administered, which may be, for example, up to 1 year (17 cycles) and longer as desired.
In another embodiment, a fixed dose of about 1050mg of a HER dimerization inhibitor (e.g., Pertuzumab) is administered as a loading dose, followed by one or more maintenance doses of about 525 mg. According to this embodiment, about 1, 2 or more maintenance doses are administered to the patient every three weeks.
Although the HER inhibitor, HER dimerization inhibitor or chemotherapeutic agent may be administered as a single anti-tumor agent, the patient is optionally treated with a combination of the inhibitor (or chemotherapeutic agent) and one or more (additional) chemotherapeutic agents. Exemplary chemotherapeutic agents herein include: gemcitabine, carboplatin, paclitaxel, docetaxel, topotecan, and/or liposomal doxorubicin. Preferably, the at least one chemotherapeutic agent is an antimetabolite chemotherapeutic agent, such as gemcitabine. Co-administration includes co-administration or simultaneous administration using separate formulations or a single pharmaceutical formulation, as well as sequential administration in either order, wherein preferably all two (or more) active agents exert their biological activities simultaneously over a period of time. As such, the antimetabolite chemotherapeutic agent may be administered before or after the inhibitor. In this embodiment, the timing between the administration of the at least one antimetabolite chemotherapeutic agent and the administration of the at least one inhibitor is preferably about 1 month or less, most preferably about 2 weeks or less. Alternatively, the antimetabolite chemotherapeutic agent and the inhibitor are administered to the patient simultaneously in a single formulation or in separate formulations. Combination therapy of a chemotherapeutic agent (e.g., an antimetabolite chemotherapeutic agent such as gemcitabine) with an inhibitor (e.g., Pertuzumab) can produce a synergistic, or greater than additive, therapeutic benefit to a patient.
Chemotherapeutic agents particularly desirable for use in combination with inhibitors (e.g., for the therapy of ovarian cancer) include: antimetabolite chemotherapeutic agents such as gemcitabine; platinum compounds such as carboplatin; taxoids (taxoid), such as paclitaxel or docetaxel; topotecan; or a liposome of doxorubicin.
Antimetabolite chemotherapyThe agent, if administered, is typically administered at its known dose, or optionally reduced by the combined effect of the drugs or by adverse side effects resulting from administration of the antimetabolite chemotherapeutic agent. The formulation and dosing schedule for such chemotherapeutic agents may be used according to the manufacturer's instructions, or may be determined empirically by the skilled practitioner. If the antimetabolite chemotherapeutic agent is gemcitabine, it is preferred, for example, to be present at about 600mg/m on days 1 and 8 of the three week cycle2To 1250mg/m2Between (e.g., about 1000 mg/m)2) The dosage of (a).
In addition to the inhibitor and antimetabolite chemotherapeutic agents, other treatment regimens may also be combined. For example, a second (third, fourth, etc.) chemotherapeutic agent may be administered, wherein the second chemotherapeutic agent is either another different antimetabolite chemotherapeutic agent or a non-antimetabolite chemotherapeutic agent. For example, the second chemotherapeutic agent may be a taxane (such as paclitaxel or docetaxel), capecitabine or platinum-based chemotherapeutic (such as carboplatin, cisplatin or oxaliplatin), an anthracycline (such as doxorubicin, including liposomal doxorubicin), topotecan, pemetrexed, vinca alkaloids (such as vinorelbine), and TLK 286. A "cocktail" of different chemotherapeutic agents may be administered.
Other therapeutic agents that may be combined with the inhibitor and/or chemotherapeutic agent include any one or more of the following: a second, different HER inhibitor, a HER dimerization inhibitor (e.g., a growth inhibitory HER2 antibody such as trastuzumab or a HER2 antibody such as 7C2, 7F3 or humanized variants thereof that induces apoptosis of HER2 overexpressing cells); antibodies against different tumor associated antigens such as EGFR, HER3, HER 4; anti-hormonal compounds, for example anti-estrogenic compounds such as tamoxifen, or aromatase inhibitors; cardioprotective agents (to prevent or alleviate any myocardial dysfunction associated with treatment); a cytokine; EGFR-targeting drugs (such as TARCEVA)IRESSAOr cetuximab); anti-angiogenic agents (in particular Genentech under the trade mark AVASTIN)TMBevacizumab sold); tyrosine kinase inhibitors; COX inhibitors (e.g., COX-1 or COX-2 inhibitors); nonsteroidal anti-inflammatory drugs, Celecoxib (CELEBREX)) (ii) a Farnesyl transferase inhibitors (e.g., Tipifarnib/ZARNESTRA available from Johnson and JohnsonR115777 or LonafarnibSCH66336 available from Schering-Plough); antibodies that bind oncofetal protein CA 125, such as oregovmab (MoAb B43.13); HER2 vaccine (such as the HER2 Auto Vac vaccine from Pharmexia, or the APC8024 protein vaccine from Dendreon, or the HER2 peptide vaccine from GSK/Corixa); other HER-targeted therapies (e.g., trastuzumab, cetuximab, ABX-EGF, EMD7200, gefitinib, erlotinib, CP724714, CI1033, GW572016, IMC-11F8, TAK165, etc.); raf and/or ras inhibitors (see, e.g., WO 2003/86467); doxil (DOXIL) liposome injection ) (ii) a Topoisomerase I inhibitors such as topotecan; a taxane; HER2 and EGFR dual tyrosine kinase inhibitors such as lapatinib/GW 572016; TLK286 (TELCYTA)) (ii) a EMD-7200; drugs that treat nausea, such as serotonin antagonists, steroids, or benzodiazepines; drugs to prevent or treat skin rash or standard acne therapy including topical or oral antibiotics; a medicament for treating or preventing diarrhea; hypothermic drugs such as acetaminophen (acetaminophen), diphenhydramine (diphenhydramine), or meperidine (meperidine); hematopoietic growth factors, and the like.
Suitable dosages for any of the above co-administered agents are those presently used and may be reduced by the combined action (synergy) of the agent and the inhibitor.
In addition to the above treatment regimens, the patient may also be subjected to surgical removal of cancer cells and/or radiation therapy.
If the inhibitor is an antibody, preferably, the antibody administered is a naked antibody. However, the administered inhibitor may be conjugated to a cytotoxic agent. Preferably, the conjugated inhibitor and/or antigen to which it is conjugated is subject to cellular internalization resulting in increased therapeutic efficacy of the conjugate to kill the cancer cells to which it is bound. In a preferred embodiment, the cytotoxic agent targets or interferes with nucleic acid in cancer cells. Examples of such cytotoxic agents include maytansinoids (maytansinoids), calicheamicin (calicheamicin), ribonucleases and DNA endonucleases.
The application also encompasses administration of the inhibitor by gene therapy. See, e.g., WO 96/07321 published 3/14 in 1996, which focuses on the use of gene therapy to generate intrabodies.
There are two main approaches to the entry of nucleic acids (optionally contained in a vector) into the cells of a patient, i.e., in vivo (in vivo) and ex vivo (ex vivo). For in vivo delivery, the nucleic acid is typically injected directly into the patient at the site where the antibody is desired. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are either administered directly to the patient, or, for example, are packed into a porous membrane and implanted into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acids into viable cells. These techniques vary depending on whether the nucleic acid is transferred to cultured cells in vitro or to cells in vivo of the host of interest. Suitable techniques for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. A commonly used vector for ex vivo delivery of genes is a retrovirus.
Presently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, herpes simplex I virus or adeno-associated virus) and lipid-based systems (lipids that can be used for lipid-mediated gene transfer are e.g. DOTMA, DOPE and DC-Chol). In some instances, it is desirable to provide a source of nucleic acid with an agent that targets a target cell, such as an antibody specific for a cell surface membrane protein or target cell, a ligand for a receptor on a target cell, and the like. Where liposomes are employed, proteins that bind to cell surface membrane proteins associated with endocytosis can be used to target and/or facilitate uptake, such as capsid proteins or fragments thereof that are tropic for a particular cell type, antibodies to proteins that internalize in the circulation, and proteins that target intracellular localization and prolong intracellular half-life. Receptor-mediated endocytosis techniques are described, for example, in Wu et al, j.biol.chem.262: 4429-4432 (1987); wagner et al, proc.natl.acad.sci.usa 87: 3410-3414(1990). For a review of currently known gene markers and gene therapy protocols see Anderson et al, Science 256: 808-813(1992). See also WO 93/25673 and references cited therein.
VI. product
In another embodiment of the invention, an article of manufacture comprising a substance useful for treating the diseases or conditions described above is provided. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, vials, cartridges, syringes, and the like. The container may be made of a variety of materials, such as glass or plastic. The container contains a composition effective for treating the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a HER dimerization inhibitor (such as Pertuzumab) or a chemotherapeutic agent (such as gemcitabine).
The article of manufacture may further comprise a second container having a pharmaceutically acceptable dilution buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further comprise other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
The kits and articles of manufacture of the invention also include information, e.g., in the form of a package insert or label, indicating that the composition is for use in treating cancer, wherein the patient's cancer expresses HER3 and/or HER 2: HER3 at levels defined according to the drug. The insert or label may take any form, such as paper or on an electronic medium, such as a magnetic recording medium (e.g., floppy disk) or a CD-ROM. The label or insert may also include other information regarding the pharmaceutical composition and dosage form in the kit or article of manufacture.
Generally, such information aids patients and physicians in the efficient and safe use of packaged pharmaceutical compositions and dosage forms. For example, the following information about HER dimerization inhibitors or chemotherapeutic agents may be provided in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, prophylaxis, adverse reactions, overdose, correct dosage and administration, how to replenish, correct storage conditions, literature references, and patent information.
In a specific embodiment of the invention, there is provided an article of manufacture comprising, packaged together, a pharmaceutical composition comprising a HER inhibitor or a HER dimerization inhibitor in a pharmaceutically acceptable carrier and a label stating that the inhibitor or pharmaceutical composition is indicated for use in treating a patient with a type of cancer which is capable of responding to the HER inhibitor or the HER dimerization inhibitor, wherein the patient's cancer expresses HER3 at a level less than the median level for HER3 expression in the type of cancer and/or the patient's cancer expresses HER 2: HER3 at a level greater than the 25 th percentile for HER 2: HER3 expression in the type of cancer.
In an optional embodiment of this aspect of the invention, the article of manufacture herein further comprises a container containing a second drug, wherein the HER inhibitor or HER dimerization inhibitor is the first drug, and the article of manufacture further comprises instructions on the package insert for treating the patient with an effective amount of the second drug. The second drug may be any of those listed above, with an exemplary second drug being another HER2 antibody or chemotherapeutic agent.
In another aspect, there is provided an article of manufacture comprising a pharmaceutical composition comprising a chemotherapeutic agent (such as gemcitabine) in a pharmaceutically acceptable carrier and a label packaged together stating that the chemotherapeutic agent or pharmaceutical composition is indicated for use in treating a patient with a type of cancer, wherein the patient's cancer expresses HER3 at a level greater than the median level for HER3 expression in the type of cancer.
The package insert is on or attached to the container. Suitable containers include, for example, vials, cartridges, syringes, and the like. The container may be made of a variety of materials, such as glass or plastic. The container contains a composition effective for the treatment of the type of cancer and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a HER inhibitor, a HER dimerization inhibitor, or a chemotherapeutic agent. The label or package insert indicates that the composition is used to treat cancer in a subject eligible for treatment and specific instructions regarding the amount and interval of administration of the provided inhibitor and any other drugs. The article of manufacture may further comprise another container in which is contained a pharmaceutically acceptable dilution buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and/or dextrose solution. The article of manufacture may further comprise other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
In practicing the present invention, many alternative experimental Methods known in the art may be successfully substituted for those specifically described herein, such as those described in many of the excellent handbooks and texts available in the art to which the present invention pertains (e.g., Using Antibodies, A Laboratory Manual, Harlow, E. and Lane, eds., 1999, Cold Spring Harbor Laboratory Press, (e.g., ISBN 0-87969-544-7); Roe B.A. et al, 1996, DNA Isolation and Sequencing (Essential technologies Series), John Wiley & Sons. (e.g., ISBN 0-471 Molecular 97324-0); Methods in Enzymology: Chimergenes and Proteins, 2000, J.Abelson, M.Simon, S.Man, J.organ. Acerand; sample 35573, J.Abelson; sample 35573, calcium 0; such as journal technologies 573-7; calcium 577; such as Molecular dynamics, edit M.Ausubel et al, John Wiley & Sons (e.g., ISBN 0-471-; current Protocols in protein science, ed.g., John E.Coligan, John Wiley & Sons (e.g., ISBN 0-471-; and Methods in Enzymology: guide to protein Purification, 1990, Vol.182, eds Deutscher, M.P., academic Press, Inc. (e.g., ISBN 0-12-213585-7)), or many university and commercial websites devoted to the description of molecular biology experimental methods.
VII advertising method
The invention herein also encompasses a method for advertising a HER inhibitor, a HER dimerization inhibitor (e.g. Pertuzumab), or a pharmaceutically acceptable composition thereof, comprising promoting to a target audience the use of the inhibitor or a pharmaceutical composition thereof for treating a population of patients with a cancer type, such as ovarian cancer, wherein the patient's cancer expresses HER3 at a level less than the median level for HER3 expression in the cancer type and/or the patient's cancer sample expresses HER 2: HER3 at a level greater than the 25 th percentile for HER 2: HER3 expression in the cancer type.
In yet another embodiment, the invention provides a method of advertising a chemotherapeutic agent (such as gemcitabine) or a pharmaceutically acceptable composition thereof, comprising promoting the use of the chemotherapeutic agent or pharmaceutical composition thereof to a target audience for treating a population of patients having a cancer type (such as ovarian cancer), wherein the patients' cancer expresses HER3 at a level greater than the median level for HER3 expression in the cancer type.
Advertising is a communication, typically paid for, via non-personal media, where the originator is authenticated and the information is controlled. Advertising for purposes herein includes promotions (publicity), public relations (public relations), product placement (product placement), sponsorships (sponsorship), insurance (underpurwritting), and promotions (sales promotion). The term also includes commercial information announcements appearing in any printed distribution medium designed to appeal to the general public to persuade, inform, promote, encourage, or otherwise alter behavior, evolve toward purchasing, supporting, or recognizing the benefits of the invention herein.
The advertising and promotion of the diagnostic methods herein may be accomplished by any means. Examples of advertising media used to deliver such information include television, radio, movies, magazines, newspapers, the internet, and billboards, including commercial, i.e., information that appears in broadcast media. Advertisements also include those on food truck seats, on airport walkway walls, and on the sides of buses, or on telephone waiting messages or heard in the in-store PA system, or anywhere where visual or auditory communications may be placed. More specific examples of promotional or advertising means include television, radio, movies, the internet such as web posters and web seminars, interactive computer networks intended for synchronous users, fixed or electronic billboards and other public signs, posters, traditional or electronic documents such as magazines and newspapers, other media channels, lectures, or personal contact, for example by email, telephone, instant messaging, mail, courier, mass, or carrier mail, in person access, and the like.
The type of advertising used will depend on many factors, such as the nature of the targeted audience to be communicated, e.g., hospitals, insurance companies, clinics, doctors, nurses, and patients, and the relevant jurisdictional laws and regulations governing cost considerations and advertising for pharmaceuticals and diagnostics. The advertising may be personalized or customized based on user characteristics defined by service interactions and/or other data, such as user demographics and geographic positioning.
Material Collection
The following hybridoma cell lines have been deposited with the American type culture Collection (American type culture Collection, 10801 University Boulevard, Manassas, VA 20110-:
antibody name ATCC deposit date
7C2 ATCC HB-12215 1996.10.17
7F3 ATCC HB-12216 1996.10.17
4D5 ATCC CRL 10463 1990.05.24
2C4 ATCC HB-12697 1999.04.08
The following non-limiting examples illustrate further details of the invention. The disclosures of all citations in the specification are expressly incorporated herein by reference.
Examples
Example 1: pertuzumab and gemcitabine for the treatment of platinum-resistant ovarian, primary peritoneal, or fallopian tube cancer
This example provides the results of a phase III clinical trial evaluating the safety, tolerability, and efficacy of Pertuzumab in combination with gemcitabine in patients with platinum-resistant ovarian, primary peritoneal, or fallopian tube cancer. Pertuzumab represents a new class of targeting agents, called HER Dimerization Inhibitors (HDIs), which inhibit HER2 dimerization with EGFR, HER3 and HER4, and inhibit signaling via MAP and P13 kinases. Pertuzumab binds at the dimer-dimer interaction site, has a profound effect on HER2 function as a co-receptor (co-receptor), prevents EGFR/HER2 and HER3/HER2 dimerization, and inhibits multiple HER-mediated signaling pathways.
The effect of Pertuzumab and gemcitabine on Progression Free Survival (PFS) and Overall Survival (OS) was evaluated in all patients, in a subset of patients whose tumors contained markers indicative of HER2 activation. The study design/protocol is shown in figure 9.
Patients who progressed on receiving a platinum-based chemotherapeutic regimen or within 6 months of receiving a platinum-based chemotherapeutic regimen met the criteria of this study. Patients were randomized to receive gemcitabine in combination with Pertuzumab or gemcitabine in combination with a placebo. Patients treated herein include those that have never received a prior remedial treatment regimen for platinum-resistant disease prior to study entry, and those that have received 1 prior treatment regimen for platinum-resistant disease.
At 1000mg/m on days 1 and 8 of each 21-day cycle2Gemcitabine is administered. Gemcitabine was first infused for 30 minutes. Allowing for reduced dosages due to toxicity issues. Placebo or Pertuzumab was administered on day 1 of the 21 day cycle. Subjects randomized to Pertuzumab administered an initial loading dose of 840mg (cycle 1), followed by 420mg in cycle 2 and subsequent cycles. Subjects randomized to placebo were administered placebo in the same volume as the Pertuzumab group in cycle 1, cycle 2 and subsequent cycles. Subjects without progressive disease receive treatment for up to 17 cycles or 1 year. Due to the decrease in blood cells, patients decreased the standard gemcitabine dose and the maintenance dose (hold doses). Pertuzumab was also maintained for any day 1 gemcitabine maintenance dose. Subsequent doses are reduced doses and no longer elevated. If the dose reduction or maintenance dose is required for more than 4 times, or if the maintenance dose exceeds 3 weeks, gemcitabine is discontinued and blinded medication continues until disease progression with the consent of the attending physician and medical monitor. If the gemcitabine dose is maintained on day 8, the dose on day 8 may be omitted and subsequent treatments started on the next cycle (day 22 of the previous cycle).
The gemcitabine dose was maintained and reduced as recommended in the following table:
absolute granulocyte count (x 10)6/L) Platelet count (x 10)6/L) % of the total dose
＞1000 And ＞100,000 100
500-999 or 50,000-99,000 75
＜500 Or ＜50,000 Maintenance of
The subsequent dose to any patient requiring a reduced dose is a reduced dose. If the maintenance dose exceeds 3 weeks due to cytopenia, the patient is presumed to have unacceptable toxicity and gemcitabine is discontinued. If there is no additional grade III or IV toxicity, administration of the blinded medication is continued at the discretion of the physician and medical monitor. The hematological toxicity of gemcitabine is related to the rate of administration. Gemcitabine administration continued for 30 minutes regardless of the total dose. Colony stimulating agents were used for NCI-CTC grade 2 cytopenia at the discretion of the attending physician.
An option is provided for exchange with the single agent Pertuzumab (crossover). A loading dose of 840mg was administered at the next cycle, with subsequent cycles every 21 days continuing at 420 mg.
Responses were evaluated at the end of cycles 2, 4, 6, 8, 12 and 17. Measurable disease is assessed by solid tumor Response assessment Criteria (Response assessment Criteria for solid Tumors, RECIST) by clinical assessment and CT scanning or equivalent means. The response of subjects with evaluable disease is assessed based on changes in CA-125 and clinical and radiological evidence of disease. Responses were confirmed 4-8 weeks after initial recording of responses.
The following result measurements were evaluated.
Primary Efficacy Endpoint (Primary Efficacy Endpoint)
Progression-free survival was performed after initiation of the indicated study treatment in all subjects in each group, as determined by investigator evaluation using RECIST or CA-125 changes.
Progression-free survival, as determined by investigator's assessment using RECIST or CA-125 changes, was performed after initiation of the indicated study treatment in each arm of the following subgroups:
subject capable of detecting a marker for HER2 activation
Subjects who have no detectable marker of HER2 activation
Secondary Efficacy Endpoints (Secondary Efficacy Endpoints)
Objective response (PR or CR)
Duration of response
Time to live
No progress for 4 months
These endpoints were evaluated in all subjects in the following subgroups and in each branch:
subject capable of detecting a marker for HER2 activation
Subjects who have no detectable marker of HER2 activation
To prevent or treat possible nausea and vomiting, a serotonin antagonist, a steroid and/or a benzodiazepine is administered to the patient in a prodromal. To prevent or treat possible skin rashes, standard acne therapies are used, including topical and/or oral antibiotics. Other possible concomitant medications are any prescribed drugs or over-the-counter drug formulations, used by subjects during the interval starting 7 days before study day 1 and continuing to the last day of the follow-up period. Subjects with infusion-related temperature increases to > 38.5 ℃ or other infusion-related symptoms are symptomatic treatment with acetaminophen (acetaminophen), diphenhydramine (diphenhydramine), or meperidine (meperidine). Non-experimental hematopoietic growth factors were administered to NCI-CTC grade 2 cytopenia.
Formalin-fixed paraffin-embedded tissue (FFPET) samples obtained from this clinical trial were analyzed by qRT-PCR for EGFR, HER2, HER3, two HER ligands (amphiregulin and betacytokinin), and G6PDH (a housekeeping gene). The qRT-PCR assay was performed by TARGOS molecular Pathology GmbH (Kassel, Germany) using a laboratory type kit from Roche diagnostics. The workflow and analysis of performing qRT-PCR assays on clinical samples is depicted in fig. 27 and 28 herein.
mRNA analyses of EGFR, HER2, HER3, amphiregulin, and betacytokinin were performed in duplicate. To allow quantitative data analysis, G6PDH was also analyzed as an internal reference. The primers and probes are designed to amplify only mRNA and not DNA. qRT-PCR was performed separately for each marker and G6PDH in a two-step protocol.
In the first step, cDNA was reverse transcribed from 5 μ l total RNA using AMV reverse transcriptase and primers specific for each marker and G6 PDH. The temperature spectrum is annealing 10min./25 deg.C, reverse transcription 60min./42 deg.C and enzyme inactivation 5min./94 deg.C.
In the second step, LIGHTCYCLER is usedThe apparatus (Roche Applied Science, Mannheim, Germany) amplifies 100-and 120-bp fragments of the marker and G6PDH mRNA from 5. mu.l cDNA. The amplicons are detected by fluorescence using specific pairs of labeled hybridization probes (principle of fluorescence resonance energy transfer). All reagents used for qRT-PCR were from Roche Applied Science, Mannheim, Germany. The temperature spectra were 10min./95 ℃ for initial denaturation and 10sec./62 ℃ for 45 cycles of annealing, 9sec./72 ℃ for extension, and 10sec./95 ℃ for denaturation. The primer/probe sequences used are seen in the table below.
Calibrator RNA (RNA purified from HT29 cell line) was included in each run to allow relative quantitation, using positive and negative controls to check workflow and reagents.
Use LIGHTCYCLERData analysis was performed by relative quantification software (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. The result is a "calibrator normalized ratio (calibrated ratio)" for each marker per patient sample.
qRT-PCR values were obtained for 119/130 patients (92%). The dynamic range is: EGFR-about 10 fold, HER 2-about 10 fold, HER 3-about 20 fold. The principle of "relative quantification" is used. Gene expression (mRNA levels) in a sample was relatively quantified with reference to housekeeping gene expression in the same sample (reference G6 PDH). This relative gene expression is then normalized to the relative gene expression in the calibrator. For each marker, the "calibrator normalization ratio" was calculated as follows:
target-gene of interest
Reference substance (G6PDH) as housekeeping gene
Calibrator HT29 colorectal cancer cell line RNA
Efficacy results were evaluated when median values (range 1.3-20.3) were followed at 7.1 months. There were 101 Progression Free Survivors (PFS) at that time. Figures 10A and B present PFS in all patients treated with gemcitabine and placebo or gemcitabine and Pertuzumab. P-values were estimated by randomization of stratification factors (ECOG PS, number of prior treatment regimens for platinum-resistant disease, and disease testability) using a stratified Cox model (normalized Cox model) and a stratified log-rank test (normalized log-rank test).
PFS by predicting pHER2 status is shown in fig. 11A and B, comparing PFS in patients predicted to pHER2 negative and patients predicted to pHER2 positive. A prediction algorithm was developed using 80 commercial ovarian cancer samples. HER2, HER3 and amphiregulin expression combinations predicted 30% of the highest pHER samples with an accuracy of 80%. If amphiregulin, HER2, and HER3 are greater than or equal to the 70 th percentile, then the patient is predicted to be pHER2 positive, and other patients are considered to be pHER2 negative.
Figures 12A and B present PFS based on qRT-PCR EGFR retention; figures 13A and B present PFS based on qRT-PCR HER2 retention; while figures 14A and B present PFS based on qRT-PCR HER3 retention. Patients with low HER3 had better results in terms of PFS. These data are shown in more detail in fig. 15A and B. As shown in those figures, Pertuzumab activity is greatest in patients with HER3 low expressing tumors and tends to increase with decreasing levels of HER3 gene expression. These figures include the absolute values of HER3 expression as quantified in the qRT-PCR assay.
Figures 16A and B illustrate PFS by HER3 subgroup. These data show that there may be a negative interaction between Pertuzumab and gemcitabine in patients with tumors with high expression of HER 3.
Figures 17A and B are further tables summarizing data for PFS obtained by HER3 subgroups for both high HER3 expression and low HER3 expression. Figures 18A and B present PFS based on four different percentile subgroups of HER 3. Patients with HER3 expressed in the range of 0 to less than 50 th percentile, in particular 0 to 25 th percentile, have an improved Hazard Ratio (HR) for PFS. (lower HR correlates with improved outcome as measured by PFS.)
Figures 19A and B provide data showing PFS, 50/50 resolution by HER3 qRT-PCR. Patients with low HER3 expression (less than the 50 th percentile) had an extended PFS duration (in months) compared to patients with high HER3 expression (greater than and equal to the 50 th percentile). This correlation is more evident in figures 20A and B, where patients with low HER3 expression are characterized as those in less than the 25 th percentile, while patients with high HER3 expression are those in greater than or equal to the 25 th percentile. The P-value for HR difference between these two diagnostic subgroups was 0.0007. The 25 th percentile is equal to 1.19 CNR.
Preliminary data for Overall Survival (OS) may be obtained. Such data for all patients is provided in fig. 21A and B. Figures 22A and B compare OS obtained by HER3 qRT-PCR with lower HER3 expression (less than 50 th percentile) and high HER3 expression (greater than or equal to 50 th percentile).
Figures 23A and B illustrate PFS, 50/50 resolution, high-to-low Hazard Ratio (HR) by HER3 qRT-PCR. Full PFS data (including percentiles from 5% to 95%) are shown in figures 24A and B.
HER3 calibration normalization ratio expression ranges are shown in figure 26. This range is about 20-80 times.
PFS results were further evaluated in terms of HER 2: HER3 ratio. The results of these further analyses are depicted in FIGS. 29-31. As these figures show, Pertuzumab activity is greatest in patients with a high HER 2: HER3 ratio.
Conclusion
Pertuzumab activity is greatest in patients with HER3 low-expressing cancers and tends to increase with decreased levels of HER3 gene expression. Pertuzumab activity is also greatest in patients with high HER 2: HER3 expressing cancers and tends to increase with increased levels of HER 2: HER3 gene expression. Most patients with low HER3 expression levels who respond to Pertuzumab therapy also have a high HER 2: HER3 ratio.
In patients with tumors with high expression of HER3, there may be a negative interaction between Pertuzumab and gemcitabine.
HER3 expression may be prognostic in a chemotherapy setting, with highly expressed tumors being preferred.
The above results are surprising and unexpected.
Example 2: pertuzumab for the treatment of advanced, refractory, or recurrent ovarian cancer
This example relates to a single-arm open-multi-center (multi-center) phase II clinical trial of ovarian cancer patients. Patients with advanced, refractory, or recurrent ovarian cancer were treated with Pertuzumab, a humanized HER2 antibody.
Patients with recurrent ovarian cancer were enrolled to receive treatment with "low dose" single agent Pertuzumab; pertuzumab is administered Intravenously (IV) at a loading dose of 840mg followed by 420mg every 3 weeks.
A second group of patients received "high dose" Pertuzumab therapy; 1050mg every 3 weeks, administered as a single dose.
Tumor assessments were obtained after 2, 4, 6, 8, 12 and 16 cycles. The Response Rate (RR) according to RECIST is the primary endpoint. Safety and tolerability were also evaluated. Secondary endpoints (secondary endpoints) are TTP, duration of response, duration of survival, Pharmacokinetics (PK), and FOSI (group 2).
The qRT-PCR assay was performed on archived formalin-fixed, paraffin-embedded tissues. Assay data was available for 46/117 patients. PFS and OS by HER3 qRT-PCR are shown in figure 25, where 25/75 was selected as the best resolution. Here, high HER3 is expressed in greater than and equal to the 75 th percentile, while low HER3 is expressed in less than the 75 th percentile.
Also, patients treated with Pertuzumab with low HER3 expression showed better results in PFS and OS.
Example 3: pertuzumab for treatment of platinum-resistant recurrent ovarian cancer
In this randomized, open phase II clinical study, the efficacy and safety of Pertuzumab treatment in combination with standard carboplatin-based chemotherapy was investigated in patients with platinum-sensitive, recurrent ovarian cancer. The target sample volume is 100-500 individuals. The target sample volume is 148.
Selection criteria:
histologically confirmed ovarian, primary peritoneal, or fallopian tube cancer;
only 1 previous treatment regimen, which must be platinum-based;
platinum-sensitive disease, defined as a no progression interval of greater than 6 months after completion of platinum-based chemotherapy.
Exclusion criteria:
prior radiotherapy;
previous treatment with an anti-cancer vaccine or any targeted therapy;
major surgery or traumatic injury within 4 weeks of study;
history or evidence of central nervous system metastases.
The results are shown in FIGS. 32-35. The results of this trial further demonstrate that Pertuzumab activity is greatest in patients with cancers with low expression of HER3 and tends to increase with decreased expression levels of the HER3 gene. Pertuzumab activity is also greatest in patients with cancers with high HER 2: HER3 expression, and tends to increase with increased expression levels of HER 2: HER3 genes. Most patients with low HER3 expression levels who respond to Pertuzumab therapy also have a high HER 2: HER3 ratio.
In patients with tumors with high expression of HER3, there may be a negative interaction between Pertuzumab and gemcitabine.
HER3 expression may be prognostic in a chemotherapy setting, with highly expressed tumors being preferred.
Example 4: analysis of HER pathway gene expression in phase II studies of Pertuzumab + gemcitabine versus gemcitabine + placebo in platinum-resistant epithelial ovarian cancer patients
Background: a randomized phase II trial (N-130) with Pertuzumab + gemcitabine versus gemcitabine + placebo in patients with platinum resistant (CDDP-R) Epithelial Ovarian Cancer (EOC) suggested that Pertuzumab prolonged PFS (HR 0.66, 95% CI 0.43, 1.03) and that PFS duration may be correlated with HER3 gene expression (see examples 2 and 3).
The method comprises the following steps: CDDP-R EOC patients were randomized to G + P or G + placebo. Treatment is given until progression occurs or until unacceptable toxicity occurs. The primary endpoint was PFS. A secondary objective was to assess efficacy outcomes in patients with an expression profile associated with HER2 activation. As described above, qRT-PCR assays allowing mRNA expression analysis of HER pathway genes, including HER1, HER2, HER3, amphiregulin, and betacytokinin, were performed using archived formalin-fixed, paraffin-embedded tissue (FFPET). Results are described as low gene expression (< median) and high gene expression (> median).
As a result: of the 5 biomarkers tested, only HER3 gene expression suggests a subgroup of patients with different PFS and OS outcomes based on low versus high outcome. The final PFS and OS results for all patients obtained by qRT-PCR HER3 results were as follows:
G+P g + placebo Hazard ratio (95% CI)
PFS (middle, moon)
All patients (130 ═ n) 2.9 2.6 0.66*(0.43，1.03)
Low HER3 (N61) 5.3 1.4 0.34(0.18，0.63)
High HER3 (N61) 2.8 5.5 1.48(0.83，2.63)
OS (middle, moon)
All patients (130 ═ n) 13.0 13.1 0.91*(0.58，1.41)
Low HER3 (N61) 11.8 8,4 0.62(0.35，1.11)
High HER3 (N61) 16.1 18.2 1.59(0.8，3.2)
All patient analyses were stratified for CDDP-R disease based on ECOG status, disease testability and prior treatment regimen #.
And (4) conclusion: this exploratory analysis suggests that low tumor HER3 gene expression levels may be useful as a prognostic indicator in CDDP-REOC patients. Pertuzumab treatment can increase gemcitabine clinical activity in patients with tumors with low HER3 gene expression. These data suggest that HER3 mRNA expression levels can be used as prognostic and predictive diagnostic biomarkers.

Claims (12)

  1. Use of a HER2 dimerization inhibitor for the manufacture of a medicament for the treatment of a cancer that is capable of responding to a HER2 dimerization inhibitor, wherein the cancer expresses HER3 at a level less than the median level for HER3 expression in the cancer type, the treatment comprising administering to a patient with the cancer a therapeutically effective amount of the HER2 dimerization inhibitor, wherein the HER2 dimerization inhibitor is a HER2 antibody that binds to the HER2 heterodimer binding site.
  2. 2. The use of claim 1 wherein the cancer expresses HER3 at a level that is less than the 25 th percentile for HER3 expression levels in the cancer type.
  3. 3. The use of claim 1 or 2, wherein the HER2 antibody comprises Complementarity Determining Regions (CDRs) in the variable light and heavy chain amino acid sequences of SEQ ID nos. 3 and 4.
  4. 4. The use of claim 1 or 2, wherein the HER2 antibody comprises the variable light and heavy chain amino acid sequences in SEQ ID No.3 and 4.
  5. 5. The use of claim 1 or 2 wherein the HER2 antibody is Pertuzumab.
  6. 6. The use of any one of claims 1-5, wherein the treatment comprises administering to the patient a second therapeutic agent, wherein the second therapeutic agent is an antimetabolite.
  7. 7. The use of claim 6 wherein the antimetabolite is gemcitabine.
  8. 8. The use of any one of claims 1-7, wherein the cancer is selected from the group consisting of: breast, ovarian, peritoneal, fallopian tube, and lung cancers.
  9. 9. The use of claim 8, wherein the cancer is metastatic breast cancer, non-small cell lung cancer (NSCLC), or advanced, refractory, or recurrent ovarian cancer.
  10. 10. The use of claim 8, wherein the cancer is a platinum-resistant form of ovarian, peritoneal, or fallopian tube cancer.
  11. Use of a HER3 primer, probe or antibody in the manufacture of an agent for an in vitro method of selecting a therapy for a patient with a type of cancer that is capable of responding to a HER2 dimerization inhibitor, the method comprising determining in vitro HER3 expression in a cancer sample from the patient and, if the cancer sample expresses HER3 at a level less than the median level for HER3 expression in the type of cancer, selecting as therapy a HER2 dimerization inhibitor, wherein the HER2 dimerization inhibitor is a HER2 antibody that binds to the HER2 heterodimer binding site.
  12. 12. The use of claim 11, wherein the cancer sample expresses HER3 at a level that is less than the 25 th percentile for HER3 expression levels in the cancer type.
HK10107891.3A 2007-03-02 2008-02-29 Predicting response to a her dimerisation inhibitor based on low her3 expression HK1141586B (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US89264007P 2007-03-02 2007-03-02
US60/892,640 2007-03-02
US91205307P 2007-04-16 2007-04-16
US60/912,053 2007-04-16
US2974808P 2008-02-19 2008-02-19
US61/029,748 2008-02-19
PCT/US2008/055502 WO2008109440A2 (en) 2007-03-02 2008-02-29 Predicting response to a her dimerisation inhibitor based on low her3 expression

Publications (2)

Publication Number Publication Date
HK1141586A1 HK1141586A1 (en) 2010-11-12
HK1141586B true HK1141586B (en) 2014-07-25

Family

ID=

Similar Documents

Publication Publication Date Title
AU2008223069B2 (en) Predicting response to a HER dimerisation inhibitor based on low HER3 expression
AU2006216732C1 (en) Extending time to disease progression or survival in cancer patients using a HER dimerization inhibitor
TW200815472A (en) Extending survival of cancer patients with elevated levels of EGF or TGF-alpha
WO2011146568A1 (en) Predicting response to a her inhibitor
HK1141586B (en) Predicting response to a her dimerisation inhibitor based on low her3 expression
AU2013200248A1 (en) Predicting response to a HER dimerisation inhibitor based on low HER3 expression
HK1112588B (en) Extending time to disease progression or survival in cancer patients using a her dimerization inhibitor
HK1113499A (en) Selecting patients for therapy with a her inhibitor
HK1187814A (en) Extending time to disease progression or survival in cancer patients using a her dimerization inhibitor