Uses of her2 dimerization inhibitors pertuzumab and articles of manufacture comprising pertuzumab

Abstract

To provide uses of pertuzumab, which is a first in-class HER2 dimerization inhibitors, and articles of manufacture comprising the same.SOLUTION: A method for extending progression free survival in a population of HER2 + breast carcinoma patients; a method for combining two HER2 antibodies to treat HER2 + breast carcinoma without increasing cardiotoxicity; a method for treating early HER2 + breast carcinoma; a method for treating HER2 + breast carcinoma by co-administering a mixture of pertuzumab and trastuzumab from the same I. v. bag; a method for treating HER2 + metastatic gastric carcinoma; a method for treating HER2 + breast carcinoma with pertuzumab, trastuzumab and vinorelbine; Methods for treating HER2 positive breast cancers with trastuzumab and aromatase inhibitors; and methods for treating low HER3 ovarian, primary peritoneal, or fallopian tube cancers are provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This nonprovisional application, filed under 37 CFR § 1.53(b), claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61 / 547,535, filed October 14, 2011, U.S. Provisional Application No. 61 / 567,015, filed December 5, 2011, U.S. Provisional Application No. 61 / 657,669, filed June 8, 2012, U.S. Provisional Application No. 61 / 682,037, filed August 10, 2012, and U.S. Provisional Application No. 61 / 694,584, filed August 29, 2012. These applications are incorporated herein by reference in their entireties.

[0002] The present invention relates to the use of pertuzumab, a first-in-class HER2 dimerization inhibitor, and to articles of manufacture comprising pertuzumab.

[0003] In particular, the invention relates to extending progression-free survival in a population of HER2-positive breast cancer patients; combining two HER2 antibodies to treat HER2-positive cancer without increasing cardiotoxicity; treating early stage HER2-positive breast cancer; treating HER2-positive cancer by co-administering a mixture of pertuzumab and trastuzumab from the same intravenous infusion bag; treating HER2-positive metastatic gastric cancer; treating HER2-positive breast cancer with pertuzumab, trastuzumab and vinorelbine; treating HER2-positive breast cancer with pertuzumab, trastuzumab and an aromatase inhibitor; and treating HER3-low ovarian, primary peritoneal or fallopian tube cancer.

[0004] The present invention also relates to an article of manufacture comprising a vial containing pertuzumab and a package insert providing safety and / or efficacy data; a method for producing said article of manufacture; and methods for ensuring the safe and effective use of pertuzumab related thereto.

[0005] Additionally, the present invention relates to an intravenous (IV) bag containing a stable mixture of pertuzumab and trastuzumab suitable for administration to cancer patients. [Background technology]

[0006] Members of the HER family of receptor tyrosine kinases are important mediators of cell proliferation, differentiation, and survival. The receptor family includes the epidermal growth factor receptor (EGFR, ErbB1 or HER1), HER2 (ErbB2 or p185), and neu HER2 (ErbB3), HER3 (ErbB3), and HER4 (ErbB4 or tyro2). Members of the receptor family have been implicated in various types of human malignancies.

[0007] The recombinant humanized murine anti-HER2 antibody 4D5 (huMAb4D5-8, rhuMAbHER2, trastuzumab, or HERCEPTIN®; U.S. Pat. No. 5,821,337) is clinically active in patients with HER2-overexpressing metastatic breast cancer who have been extensively pretreated with anticancer therapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)).

[0008] Trastuzumab received marketing approval from the Food and Drug Administration on September 25, 1998, for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein. Currently, trastuzumab is approved for use as a single agent or in combination with chemotherapy or hormonal therapy in the metastatic setting, and for use as a single agent or in combination with chemotherapy as adjuvant treatment for patients with early-stage HER2-positive breast cancer. Trastuzumab-based therapy is currently the recommended treatment for patients with HER2-positive early-stage breast cancer who have no contraindications to its use (Herceptin® prescribing information; NCCN Guidelines, 2011, 2nd edition). Trastuzumab plus docetaxel (or paclitaxel) is the registered standard of care in the setting of first-line metastatic breast cancer (MBC) treatment (Slamon et al. N Engl J Med. 2001;344(11):783-792; Marty et al. J Clin Oncol. 2005;23(19):4265-4274).

[0009] Although trastuzumab has been associated with excellent results in the treatment of breast cancer, recent data from clinical trials of lapatinib seem to suggest that HER2 plays an active role in tumor biology even in the setting of trastuzumab (Geyer et al., N Engl J Med 2006;355:2733-2743).

[0010] Patients treated with the HER2 antibody trastuzumab are selected for therapy based on HER2 expression. 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.). See also U.S. Patent Nos. 6,573,043, 6,905,830, and US2003 / 0152987 (Cohen et al.), which discuss immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for detecting HER2 overexpression and amplification. Thus, optimal management of metastatic breast cancer now takes into account not only the patient's general condition, medical history, and receptor status, but also HER2 status.

[0011] Pertuzumab (also known as recombinant humanized monoclonal antibody 2C4 (rhuMAb 2C4); Genentech, Inc., South San Francisco) is the first in a new class of agents known as HER dimerization inhibitors (HDIs), which act to inhibit the ability of HER2 to form active heterodimers or homodimers with other HER receptors (e.g., EGFR / HER1, HER2, HER3, and HER4). 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 Struct Biol 10:158-9 (2003); Cho et al. Nature 421:756-60 (2003); and Malik et al. Pro Am Soc Cancer Res 44:176-7 (2003).

[0012] Blockade of HER2-HER3 heterodimer formation in tumor cells by pertuzumab has been demonstrated to inhibit critical cell signaling that reduces tumor growth and survival (Agus et al., Cancer Cell 2:127-37 (2002)).

[0013] Pertuzumab has been tested as a single agent in the clinic in Phase Ia trials in patients with advanced cancer and in 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 had progressed during or after standard therapy were treated with pertuzumab administered intravenously every 3 weeks. Pertuzumab was generally well tolerated. Tumor shrinkage was achieved in 3 of 20 patients evaluable for response. Two patients had confirmed partial responses. Stable disease lasting more than 2.5 months was observed in 6 of 21 patients (Agus et al., Pro Am Soc Clin Oncol 22:192 (2003)). At doses of 2.0 to 15 mg / kg, the pharmacokinetics of pertuzumab was linear, with mean clearance ranging from 2.69 to 3.74 mL / day / kg and a mean terminal elimination half-life ranging from 15.3 to 27.6 days. No antibodies to pertuzumab were detected (Allison et al., Pro Am Soc Clin Oncol 22:197 (2003)).

[0014] US2006 / 0034842 describes a method for treating ErbB-expressing cancers with a combination of anti-ErbB2 antibodies. US2008 / 0102069 describes the use of trastuzumab and pertuzumab in the treatment of HER2-positive metastatic cancers, such as breast cancer. Baselga et al. (J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I, Col. 25, No. 18S (June 20 Supplement), 2007: 1004) report the combination treatment of trastuzumab and pertuzumab in patients with previously treated HER2-positive breast cancer who had progressed during trastuzumab treatment. Portera et al. (J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I. Vol. 25, No. 18S (June 20 Supplement), 2007: 1028) evaluated the efficacy and safety of the combination of trastuzumab and pertuzumab in patients with HER2-positive breast cancer who had progressive disease on trastuzumab-based therapy. The authors concluded that further evaluation of the efficacy of the combination is needed to clarify the overall risks and benefits of this treatment regimen.

[0015] Pertuzumab has been evaluated in combination with trastuzumab in phase II studies in patients with HER2-positive metastatic breast cancer who had previously received trastuzumab for metastatic disease. One study conducted by the National Cancer Institute (NCI) enrolled 11 patients with previously treated HER2-positive metastatic breast cancer. Two of the 11 patients demonstrated a partial response (PR) (Baselga et al., J Clin Oncol 2007 ASCO Annual Meeting Proceedings;25:18S (June 20 Supplement):1004). Results from a phase II neoadjuvant study evaluating the efficacy of a novel combination regimen of pertuzumab and trastuzumab plus the chemotherapy drug docetaxel in women with early-stage HER2-positive breast cancer, presented at the CTRC-AACR San Antonio Breast Cancer Symposium (SABCS) held December 8-12, 2010, showed that the two HER2 antibodies plus docetaxel administered in the neoadjuvant setting before surgery significantly improved the rate of complete tumor disappearance in the breast (pathological complete response rate (pCR) 45.8%) by more than half compared with trastuzumab plus docetaxel (pCR 29.0%) (p=0.014).

[0016] Patent publications related to HER2 antibodies include the following: U.S. Patent Nos. 5,677,171; 5,720,937; 5,720,954; 5,725,856; 5,770,195; 5,772,997; 6,165,464; 6,387,371; 6,399,063; 6,015,567; 6,333,169; 4,968,603; 5,821,337; 6,054,297; 6,407,213; 6,639,055; 6,719,971; 6,800,738; 6,000,800; No. 75,890; No. 5,648,237; No. 7,018,809; No. 6,267,958; No. 6,685,940; No. 6,821 ,515;No.7,060,268;No.7,682,609;No.7,371,376;No.6,127,526;No.6,333,39 No. 8; No. 6,797,814; No. 6,339,142; No. 6,417,335; No. 6,489,447; No. 7,074,404 ;No. 7,531,645;No. 7,846,441;No. 7,892,549;No. 8,075,892;No. 6,573,043;No. 6 ,905,830;No.7,129,051;No.7,344,840;No.7,468,252;No.7,674,589;No.7,9 No. 19,254; No. 6,949,245; No. 7,485,302; No. 7,498,030; No. 7,501,122; No. 7,537, No. 931; No. 7,618,631; No. 7,862,817; No. 7,041,292; No. 6,627,196; No. 7,371,37 No. 9; No. 6,632,979; No. 7,097,840; No. 7,575,748; No. 6,984,494; No. 7,279,287; No. 7,811,773; No. 7,993,834; No. 8,076,066; No. 8,044,017; No. 7,435,797; No. 7 ,850,966;No.7,485,704;No.7,807,799;No.8,142,784;No.7,560,111;No.7,87 Nos. 9,325; 8,241,630; 7,449,184; 8,163,287; 7,700,299; 7,981,418; 8,247,397; and US2010 / 0016556; US2005 / 0244929; US2001 / 0014326;US2003 / 0202972;US2006 / 0099201;US2010 / 0158899;US2011 / 0236383;US2011 / 0033460;US2008 / 0286280;US2005 / 0063972;US2006 / 0182739;US2009 / 0220492;US2003 / 0147884;US2004 / 0037823;US2005 / 0002928;US2007 / 0292419;US2008 / 0187533;US2011 / 0250194;US2012 / 0034213;US2003 / 0152987;US2005 / 0100944;US2006 / 0183150;US2008 / 0050748;US2009 / 0155803;US2010 / 0120053;US2005 / 0244417;US2007 / 0026001;US2008 / 0160026;US2008 / 0241146;US2005 / 0208043;US2005 / 0238640;US2006 / 0034842;US2006 / 0073143;US2006 / 0193854;US2006 / 0198843;US2011 / 0129464;US2007 / 0184055;US2007 / 0269429;US2008 / 0050373;US2006 / 0083739;US2009 / 0087432;US2006 / 0210561;US2002 / 0035736;US2002 / 0001587;US2008 / 0226659;US2002 / 0090662;US2006 / 0046270;US2008 / 0108096;US2007 / 0166753;US2008 / 0112958;US2009 / 0239236;US2012 / 0034609;US2012 / 0093838;US2004 / 0082047;US2012 / 0065381;US2009 / 0187007;US2011 / 0159014;US2004 / 0106161;US2011 / 0117096;US2004 / 0258685;US2009 / 0148402;US2009 / 0099344;US2006 / 0034840;US2011 / 0064737;US2005 / 0276812;US2008 / 0171040;US2009 / 0202536;US2006 / 0013819;US2012 / 0107391;US2006 / 0018899;US2009 / 0285837;US2011 / 0117097;US2006 / 0088523;US2010 / 0015157;US200 6 / 0121044;US2008 / 0317753;US2006 / 0165702;US2009 / 0081223;US2006 / 0188509;US2009 / 0155 259;US2011 / 0165157;US2006 / 0204505;US2006 / 0212956;US2006 / 0275305;US2012 / 0003217;U S2007 / 0009976;US2007 / 0020261;US2007 / 0037228;US2010 / 0112603;US2006 / 0067930;US2007 / 0224203;US2011 / 0064736;US2008 / 0038271;US2008 / 0050385;US2010 / 0285010;US2011 / 02231 59;US2008 / 0102069;US2010 / 0008975;US2011 / 0245103;US2011 / 0246399;US2011 / 0027190;US2 010 / 0298156;US2011 / 0151454;US2011 / 0223619;US2012 / 0107302;US2009 / 0098135;US2009 / 01 48435;US2009 / 0202546;US2009 / 0226455;US2009 / 0317387;US2011 / 0044977;US2012 / 0121586. ; Summary of the Invention

[0017] In a first aspect, the present invention relates to a method for extending progression-free survival by 6 months or more in a population of patients with HER2-positive breast cancer, comprising administering to the patients in the population pertuzumab, trastuzumab, and a chemotherapeutic agent (e.g., a taxane, e.g., docetaxel). Optionally, the method results in a response rate of 80% or more in the patients in the population. Optionally, the breast cancer is metastatic or locally recurrent unresectable breast cancer or de novo Stage IV disease. In one embodiment, the patients in the population have not been previously treated or have relapsed after adjuvant therapy; have a left ventricular ejection fraction (LVEF) of 50% or greater at baseline; and / or have an Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 or 1. Optionally, HER2-positive breast cancer is defined as immunohistochemistry (IHC) 3+ and / or a fluorescence in situ hybridization (FISH) amplification ratio of 2.0 or greater. In some cases, this method reduces the risk of death by about 34% or more compared to patients treated with trastuzumab and a chemotherapy agent.

[0018] In another aspect, the invention relates to a method of combining two HER2 antibodies to treat HER2-positive cancer without increasing cardiotoxicity in a population of HER2-positive cancer patients, comprising administering pertuzumab, trastuzumab, and a chemotherapeutic agent to patients in said population. Optionally, cardiotoxicity in said patient population is monitored for the incidence of symptomatic left ventricular systolic dysfunction (LVSD) or congestive heart failure (CHF), or a decrease in left ventricular ejection fraction (LVEF). The HER2-positive cancer is optionally breast cancer, for example, metastatic or locally recurrent unresectable breast cancer, or de novo Stage IV disease.

[0019] In another aspect, the invention relates to an article of manufacture comprising a vial containing pertuzumab and a package insert, wherein the package insert provides the safety data in Table 3 or Table 4 and / or the efficacy data in Table 2, Table 5, Figure 8 or Figure 10.

[0020] The invention further relates to a method of manufacturing an article of manufacture comprising packaging a vial containing pertuzumab together with a package insert, wherein the package insert provides the safety data in Table 3 or Table 4 and / or the efficacy data in Table 2, Table 5, Figure 8 or Figure 10.

[0021] In a related aspect, the invention relates to a method for ensuring the safe and effective use of pertuzumab, comprising packaging a vial containing pertuzumab together with a package insert, wherein the package insert provides the safety data in Table 3 or Table 4 and / or the efficacy data in Table 2, Table 5, Figure 8 or Figure 10.

[0022] In some cases, the article of manufacture includes a single-dose vial containing about 420 mg of pertuzumab.

[0023] Optionally, the package insert further includes the boxed warning of Example 4.

[0024] In some cases, the package insert further provides overall survival (OS) efficacy data in Example 9 or Table 14.

[0025] In another aspect, the present invention relates to a method for treating early-stage HER2-positive breast cancer, comprising administering pertuzumab, trastuzumab, and a chemotherapy agent to a breast cancer patient, wherein the chemotherapy agent comprises an anthracycline chemotherapy agent (e.g., 5-FU, epirubicin, and cyclophosphamide (FEC)) or a carboplatin chemotherapy agent (e.g., docetaxel and carboplatin). Optionally, pertuzumab is administered simultaneously with the anthracycline chemotherapy agent or the carboplatin chemotherapy agent. In one embodiment of this method, administration of pertuzumab does not increase cardiac toxicity compared to treatment without pertuzumab. Such treatment of early-stage HER2-positive breast cancer optionally comprises neoadjuvant or adjuvant therapy.

[0026] The present invention further relates to methods of treating HER2-positive cancer in a patient, comprising co-administering a mixture of pertuzumab and trastuzumab to the patient from the same intravenous infusion bag, optionally further comprising administering a chemotherapeutic agent to the patient.

[0027] In a related aspect, the present invention provides an intravenous infusion (IV) bag containing a stable mixture of pertuzumab and trastuzumab suitable for administration to cancer patients. The mixture is optionally a saline solution, e.g., containing about 0.9% NaCl or about 0.45% NaCl. The IV bag is optionally a polyolefin or polyvinyl chloride IV bag containing 250 mL of 0.9% saline. In one embodiment, the IV bag contains a mixture of about 420 mg or about 840 mg of pertuzumab and about 200 mg to about 1000 mg of trastuzumab. In one embodiment, the mixture is stable at 5°C or 30°C for up to 24 hours. The stability of the mixture may be assessed by one or more assays selected from the group consisting of color, appearance and clarity (CAC), concentration and turbidity analysis, particle analysis, size exclusion chromatography (SEC), ion exchange chromatography (IEC), capillary zone electrophoresis (CZE), image capillary isoelectric focusing (iCIEF), and potency assays.

[0028] The present invention provides a new treatment regimen for gastric cancer. In particular, the present invention relates to the treatment of HER2-positive gastric cancer in a human subject with a combination of trastuzumab, pertuzumab and at least one chemotherapeutic agent.

[0029] In one aspect, the present invention relates to a method of treating HER2-positive gastric cancer in a human subject, comprising administering pertuzumab, trastuzumab, and a chemotherapeutic agent to the subject.

[0030] In one aspect, the present invention relates to a method of treating gastric cancer in a human subject, comprising administering pertuzumab to a subject with gastric cancer, wherein pertuzumab is administered at a dose of 840 mg in all treatment cycles.

[0031] In another aspect, the present invention relates to a method of improving survival in a human subject with HER2-positive gastric cancer, comprising administering pertuzumab, trastuzumab, and a chemotherapeutic agent to the subject.

[0032] In yet another aspect, the invention relates to the combination of pertuzumab with trastuzumab and a chemotherapeutic agent for use in the treatment of HER2-positive gastric cancer in a human subject.

[0033] In a further aspect, the present invention relates to the use of pertuzumab in the preparation of a medicament for the treatment of HER2-positive gastric cancer, wherein the treatment comprises administering pertuzumab in combination with trastuzumab and a chemotherapeutic agent.

[0034] In a further aspect, the present invention relates to the use of trastuzumab in the preparation of a medicament for the treatment of HER2-positive gastric cancer, wherein the treatment comprises administering trastuzumab in combination with pertuzumab and a chemotherapeutic agent.

[0035] In another aspect, the invention relates to a kit comprising a container containing pertuzumab and instructions for use, for administering pertuzumab in combination with trastuzumab and a chemotherapeutic agent to treat HER2-positive gastric cancer in a subject.

[0036] In yet another aspect, the invention relates to a kit comprising a container containing trastuzumab and instructions for use, for administering trastuzumab in combination with pertuzumab and a chemotherapeutic agent to treat HER2-positive gastric cancer in a subject.

[0037] In all aspects, the gastric cancer may be, for example, unresectable locally advanced gastric cancer, metastatic gastric cancer, or progressive post-operative recurrent gastric cancer that may not be amenable to curative therapy by known methods. In all aspects, the gastric cancer includes adenocarcinoma of the stomach or gastroesophageal junction. In all aspects, in a specific embodiment, the patient has not received prior anti-cancer therapy for metastatic gastric cancer. In all aspects, in a specific embodiment, the chemotherapy includes administration of a platin and / or a fluoropyrimidine. In a specific embodiment, the platin is cisplatin. In other embodiments, the fluoropyrimidine includes capecitabine and / or 5-fluorouracil (5-FU). In all aspects, the patient's HER2-positive status may be, for example, IHC3+ or IHC2+ / ISH+. In all aspects, in certain embodiments, the treatment improves survival, including overall survival (OS) and / or progression-free survival (PFS), and / or response rate (RR). In all aspects, in certain embodiments, the patient has an ECOG PS of 0-1. In all embodiments, treatment cycles are generally separated from each other by no more than 4 weeks, or no more than 3 weeks, or no more than 2 weeks, or no more than 1 week.

[0038] In certain aspects, the invention relates to a method of treating HER2-positive unresectable or metastatic adenocarcinoma of the stomach or gastroesophageal junction in a human patient who has not been previously treated with chemotherapy for metastatic disease, except for previous adjuvant or neoadjuvant therapy completed more than 6 months prior to the current treatment, comprising administering to the patient pertuzumab, trastuzumab, cisplatin, and capecitabine and / or fluorouracil (5-FU) in amounts that improve progression-free survival (PFS) and / or overall survival (OS), wherein the patient has an Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) of 0 to 1. In certain embodiments, the patient has not been previously treated with platins.

[0039] In another aspect, the invention relates to a method of improving progression-free survival in patients with HER2-positive unresectable or metastatic adenocarcinoma of the stomach or gastroesophageal junction, comprising administering to the patient pertuzumab in combination with trastuzumab and a chemotherapeutic agent.

[0040] In a further aspect, the present invention relates to a method of treating HER2-positive breast cancer in a patient, comprising administering to the patient pertuzumab, trastuzumab, and vinorelbine. Optionally, pertuzumab and trastuzumab are co-administered to the patient from the same intravenous infusion bag. Optionally, the breast cancer is metastatic or locally advanced. In one embodiment, the patient has not been previously treated with systemic non-hormonal anti-cancer therapy in the metastatic setting.

[0041] In another aspect, the present invention relates to a method of treating HER2-positive breast cancer in a patient, comprising administering to the patient pertuzumab, trastuzumab, and an aromatase inhibitor (e.g., anastrozole or letrozole). Optionally, the breast cancer is hormone receptor-positive advanced breast cancer, where the hormone receptor is, for example, estrogen receptor (ER) and / or progesterone receptor (PgR). According to this embodiment of the present invention, the patient has not previously been treated with systemic non-hormonal anticancer therapy in the metastatic setting. Additionally, optionally, the patient in this case receives induction chemotherapy (e.g., including a taxane).

[0042] In an additional embodiment, the invention relates to a method of treating a cancer patient, comprising administering to the patient an initial dose of 840 mg of pertuzumab, followed by doses of 420 mg of pertuzumab every three weeks thereafter, and further comprising readministering to the patient an 840 mg dose of pertuzumab when the interval between two successive 420 mg doses is 6 weeks or more. Optionally, the method further comprises administering 420 mg of pertuzumab every three weeks after readministering the 840 mg dose. In one embodiment, the cancer patient has HER2-positive breast cancer.

[0043] In a further aspect, the invention relates to a method for treating HER2-positive metastatic or locally recurrent breast cancer in a patient, comprising administering to the patient pertuzumab, trastuzumab and a taxoid (e.g., docetaxel, paclitaxel or nab-paclitaxel), wherein the patient has been previously treated with trastuzumab and / or lapatinib as adjuvant or neoadjuvant therapy.

[0044] In a further aspect, the invention relates to a method for treating HER3-low ovarian, primary peritoneal or fallopian tube cancer in a patient, comprising administering to the patient pertuzumab and a chemotherapeutic agent, wherein the chemotherapeutic agent comprises a taxoid (e.g., paclitaxel) or topotecan.

[0045] In an additional aspect, the present invention relates to a method for treating HER3-low ovarian, primary peritoneal, or fallopian tube cancer in a patient, comprising administering to the patient pertuzumab and a chemotherapeutic agent, wherein the HER3-low cancer expresses HER3 mRNA at a concentration ratio of about 2.81 or less, as assessed by polymerase chain reaction (PCR). In one embodiment, the chemotherapeutic agent comprises gemcitabine, carboplatin, paclitaxel, docetaxel, topotecan, or pegylated liposomal doxorubicin (PLD). Optionally, the chemotherapeutic agent comprises paclitaxel or topotecan. In one embodiment, the cancer is platinum-resistant or platinum-refractory epithelial ovarian cancer. [Brief explanation of the drawings]

[0046] [Figure 1] FIG. 1 is a schematic diagram showing the structure of HER2 protein and the amino acid sequences of domains I to IV (SEQ ID NOs: 1 to 4, respectively) of its extracellular domain. [Figure 2A]Figure 1 shows an alignment of the amino acid sequences of the light chain variable (VL) domain of murine monoclonal antibody 2C4 (SEQ ID NO: 5); the VL domain of variant 574 / Pertuzumab (SEQ ID NO: 7); and the human VL consensus framework (hum κ1, light chain kappa subgroup I) (SEQ ID NO: 9). Asterisks identify differences between the variable domains of Pertuzumab and murine monoclonal antibody 2C4 and between the variable domains of Pertuzumab and the human framework. Complementarity-determining regions (CDRs) are in brackets. [Figure 2B] Figure 1 shows an alignment of the amino acid sequences of the heavy chain variable (VH) domain of murine monoclonal antibody 2C4 (SEQ ID NO: 6); the VH domain of variant 574 / Pertuzumab (SEQ ID NO: 8); and the human VH consensus framework (humIII, heavy chain subgroup III) (SEQ ID NO: 10). Asterisks identify differences between the variable domains of Pertuzumab and murine monoclonal antibody 2C4 and between the variable domains of Pertuzumab and the human framework. Complementarity-determining regions (CDRs) are in brackets. [Figure 3A] Figure 1 shows the amino acid sequence of the Pertuzumab light chain (SEQ ID NO: 11). The CDRs are shown in bold. The calculated molecular mass of the light chain is 23,526.22 Da (cysteines are reduced). [Figure 3B] Figure 1 shows the amino acid sequence of the Pertuzumab heavy chain (SEQ ID NO: 12). CDRs are shown in bold. The calculated molecular mass of the light chain is 49,216.56 Da (cysteines are reduced). Glycosylation is attached to Asn299 of the heavy chain. [Figure 4A] Figure 1 shows the amino acid sequence of the trastuzumab light chain (SEQ ID NO: 13), with the boundaries of the light chain variable domains indicated by arrows. [Figure 4B] Figure 1 shows the amino acid sequence of the trastuzumab heavy chain (SEQ ID NO: 14), with the boundaries of the heavy chain variable domains indicated by arrows. [Figure 5A] FIG. 1 shows the variant Pertuzumab light chain sequence (SEQ ID NO: 15). [Figure 5B]FIG. 1 shows the heavy chain sequence of variant Pertuzumab (SEQ ID NO: 16). [Figure 6] Figure 1 shows the study schema for Example 1. ECOG = Eastern Cooperative Oncology Group; PD = Progression. Note: Trastuzumab, pertuzumab, and cisplatin will be administered by IV infusion on day 1 of each 3-week cycle. Capecitabine will be administered orally twice daily from the evening of day 1 to the morning of day 15 of each 3-week cycle. (a) HER2-positive tumors defined as IHC3+ or a combination of IHC2+ and ISH+ (i.e., IHC3+ / ISH+ or IHC2+ / ISH+); (b) trastuzumab at a loading dose of 8 mg / kg in cycle 1 and a dose of 6 mg / kg in subsequent cycles; (c) pertuzumab at a loading dose of 840 mg in cycle 1 and a dose of 420 mg in cycles 2-6 on day 1 of each cycle. [Figure 7] FIG. 1 shows the enrollment, intent-to-treat and safety populations and patient dropouts in the study of Example 3. [Figure 8] 1 is a graph showing Kaplan-Meier curves of progression-free survival (PFS) assessed by an independent review facility (IRF) for the study of Example 3. [Figure 9] FIG. 1 shows PFS by patient subgroup for the study of Example 3. [Figure 10] 1 is a graph showing overall survival for the study of Example 3. [Figure 11] Figure 1 shows an overview of the dosing schedule in HER2-positive neoadjuvant breast cancer patients with low cardiac risk factors in Example 5. Additional radiation therapy, hormonal therapy, and chemotherapy were allowed after surgery and during adjuvant trastuzumab treatment if deemed necessary by the investigator. [Figure 12] 1 is a graph showing the mean change in LVEF (median reading) for the study of Example 5. [Figure 13] 1 is a graph showing pathological complete response (pCR) for the study of Example 5. [Figure 14] 1 is a graph showing pathologic complete response by hormone receptor status in the study of Example 5. [Figure 15] Figure 1 shows the pertuzumab SEC profile of the pertuzumab / trastuzumab blend (840 mg) in a 0.9% saline PO IV infusion bag at 30°C (1) Time = 0; (2) Time = 24 hours. Enlarged graph; full graph (inset). [Figure 16] Figure 1 shows the trastuzumab SEC profile of a pertuzumab / trastuzumab blend (840 mg) in a 0.9% saline PO IV infusion bag at 30°C (1) time = 0; (2) time = 24 hours. Enlarged graph; full graph (inset). [Figure 17] Figure 1 shows the Pertuzumab IEC profile of the Pertuzumab / Trastuzumab mixture at 30°C in 0.9% saline PO IV infusion bags (1) Time = 0; (2) Time = 24 hours. Full graph. [Figure 18] Figure 1 shows the trastuzumab IEC profile of the pertuzumab / trastuzumab mixture at 30°C in 0.9% saline PO IV infusion bags (1) Time = 0; (2) Time = 24 hours. Enlarged graph; full graph (inset). [Figure 19] Figure 1 shows the CE-SDS LIF non-reduced profile of Pertuzumab / Trastuzumab mixture at 30°C in 0.9% saline PO IV infusion bags (1) Time = 0; (2) Time = 24 hours. [Figure 20] Figure 1 shows the CE-SDS LIF reduction profile of Pertuzumab / Trastuzumab mixture at 30°C in 0.9% Saline PO IV infusion bags (1) Time = 0; (2) Time = 24 hours. [Figure 21] Figure 1 shows the CZE of Pertuzumab / Trastuzumab mixtures at 30°C in 0.9% saline PO IV infusion bags (1) Time = 0; (2) Time = 24 hours. Full graph. [Figure 22]Figure 1 shows the iCIEF of Pertuzumab / Trastuzumab mixtures at 30°C in 0.9% saline PO IV infusion bags (1) Time = 0; (2) Time = 24 hours. Full graph. [Figure 23] FIG. 1 shows the efficacy-dose response curves (μg / mL vs RFU) of pertuzumab / trastuzumab combination, pertuzumab alone, and trastuzumab alone in 0.9% saline PO IV infusion bags (1) time=0; (2) time=24 hours. [Figure 24] Figure 1 shows the pertuzumab SEC profile of the pertuzumab / trastuzumab blend (1560 mg) in 0.9% saline IV infusion bag: (1) PO 5°C T0; (2) PO 5°C T24 hr; (3) PO 30°C T0; (4) PO 30°C T24 hr; (5) PVC 5°C T0; (6) PVC 5°C T24 hr; (7) PVC 30°C T0; (8) PVC 30°C T24 hr. Enlarged graph; full graph (inset). [Figure 25] FIG. 10 shows the trastuzumab SEC profile of the pertuzumab / trastuzumab blend (1560 mg) in 0.9% saline IV infusion bag: (1) PO 5° C. T0; (2) PO 5° C. T24 hr; (3) PO 30° C. T0; (4) PO 30° C. T24 hr; (5) PVC 5° C. T0; (2) PVC 5° C. T24 hr; (7) PVC 30° C. T0; (8) PVC 30° C. T24 hr. Enlarged graph; full graph (inset). [Figure 26] Figure 1 shows the Pertuzumab IEC profile (Pertuzumab-Fast) of the Pertuzumab / Trastuzumab blend (1560 mg) in a 0.9% saline IV infusion bag: (1) PO 5°C T0; (2) PO 5°C T24 hr; (3) PO 30°C T0; (4) PO 30°C T24 hr; (5) PVC 5°C T0; (6) PVC 5°C T24 hr; (7) PVC 30°C T0; (8) PO 30°C T24 hr. Full graph. [Figure 27]Figure 1 shows the trastuzumab IEC profile of pertuzumab / trastuzumab blend (1560 mg) in a 0.9% saline IV infusion bag: (1) PO 5°C T0; (2) PO 5°C T24 hr; (3) PO 30°C T0; (4) PO 30°C T24 hr; (5) PVC 5°C T0; (6) PVC 5°C T24 hr; (7) PVC 30°C T0; (8) PO 30°C T24 hr. Full graph. [Figure 28] FIG. 1 shows the study schema for Example 7. [Figure 29] FIG. 1 shows the study design of Example 8. [Figure 30] FIG. 1 shows the study design for Part 1 of Example 11. [Figure 31] FIG. 1 shows the study design for Part 2 of Example 11. [Figure 32] 1 is a table showing the samples collected and time points for the Phase IIa Gastric Cancer (GC) study of Example 1. [Figure 33] 1 is a table showing the demographics of the patient populations in the two arms of the GC study treated with pertuzumab 420 mg (Arm A) or 840 mg (Arm B). [Figure 34] 1 is a table showing the GC history of patients in arms A and B, respectively. [Figure 35] 1 is a table showing the patient disposition of GC patients in arms A and B. [Figure 36] 1 is a table showing the overall response rate for each of arms A and B of the GC study. [Figure 37]Graphs and tables show the results of pertuzumab concentration assessment at day 42 in gastric cancer (GC) versus metastatic breast cancer (MBC). The C trough at day 42 was approximately 37% lower in GC (JOSHUA 840 / 420 mg) compared to MBC (CLEO 840 / 420 mg). Both the JOSHUA 840 / 420 mg and 840 / 840 mg regimens produced C troughs of 20 μg / mg or greater in 90% of patients. The JOSHUA 840 / 840 mg regimen produced C troughs in GC comparable to those observed in MBC (CLEO 840 / 420 mg). DETAILED DESCRIPTION OF THE INVENTION

[0047] Glossary of some abbreviations used herein: Adverse Drug Reaction (ADR), Adverse Event (AE), Alkaline Phosphatase (ALP), Absolute Neutrophil Count (ANC), Area Under the Concentration-Time Curve (AUC), Capillary Zone Electrophoresis (CZE), Color, Appearance, and Clarity (CAC), Clinical Evaluation of Pertuzumab and Trastuzumab (CLEOPATRA), Confidence Interval (CI), Chromogenic In Situ Hybridization (CISH), Maximum Concentration (C max), complete response (CR), case report form (CRF), computed tomography (CT), Common Terminology Criteria for Adverse Events (CTCAE), docetaxel (D), dose-limiting toxicity (DLT), ethics committee (EC), epirubicin, cisplatin, and 5-fluorouracil (ECF), echocardiogram (ECHO), epidermal growth factor receptor (EGFR), European Union (EU), estrogen receptor (ER), 5-fluorouracil, methotrexate, and doxorubicin (FAMTX), fluorescence in situ hybridization (FISH), 5-fluorouracil (5-FU), hazard ratio (HR), human epidermal growth factor receptor (EGFR), gastric cancer (GC), good clinical practice Good Clinical Practice (GCP), human epidermal growth factor receptor 2 (HER2), ion exchange chromatography (IEC), immunohistochemistry (IHC), independent review facility (IRF), institutional review board (IRB), in situ hybridization (ISH), intravenous or intravenous infusion (IV), image capillary isoelectric focusing (iCIEF), left ventricular ejection fraction (LVEF), mitomycin C, cisplatin, and 5-fluorouracil (MCF), magnetic resonance imaging (MRI), metastatic breast cancer (MBC), multiple gated acquisition (MUGA), not significant (NS), overall survival (OS), pathological complete response (pCR), polyolefin (PO), polyvinyl chloride (PVC), progression (PD), progression-free survival (PFS), pharmacokinetics (PK), partial response (PR), progesterone receptor (PgR), response evaluation criteria in solid tumors tumors; RECIST), serious adverse events (SAEs), size exclusion chromatography (SEC), stable disease (SD), study management team (SMT), sterile water for injection (SWFI), time to maximum plasma concentration (t max ), upper limit of normal (ULN).

[0048] I. Definition As used herein, the term "chemotherapy" refers to treatment that involves the administration of chemotherapeutic agents, as defined below.

[0049] "Survival" refers to the patient remaining alive, and includes overall survival and progression-free survival.

[0050] "Overall survival" or "OS" refers to a patient remaining alive for a period of time from the time of diagnosis or treatment, e.g., 1 year, 5 years, etc. For purposes of the clinical trials described in the Examples, overall survival (OS) is defined as the time from the date of randomization of a patient population to the date of death from any cause.

[0051] "Progression-free survival" or "PFS" refers to the duration of a patient's life without the progression or further deterioration of cancer. For purposes of the clinical trials described in the Examples, progression-free survival (PFS) is defined as the time from randomization of the study population to the first documented progression, unmanageable toxicity, or death from any cause, whichever occurs first. Progression can be documented by any clinically acceptable method, such as radiographic progression as determined by Response Evaluation Criteria in Solid Tumors (RECIST) (Therasse et al., J Natl Ca Inst 2000;92(3):205-216), carcinomatous meningitis diagnosed by cytological evaluation of cerebrospinal fluid, and / or medical photography to monitor chest wall recurrence of subcutaneous lesions.

[0052] "Prolonging survival" refers to increasing overall survival or progression-free survival in patients treated according to the present invention compared to untreated patients and / or compared to patients treated with one or more approved anti-tumor agents but not treated according to the present invention. In a specific example, "prolonging survival" refers to extending the progression-free survival (PFS) and / or overall survival (OS) of cancer patients receiving the combination therapy of the present invention (e.g., combination treatment with pertuzumab, trastuzumab, and a chemotherapy agent) compared to patients treated with trastuzumab and a chemotherapy agent alone. In another specific example, "prolonging survival" refers to extending the progression-free survival (PFS) and / or overall survival (OS) of cancer patients receiving the combination therapy of the present invention (e.g., combination treatment with pertuzumab, trastuzumab, and a chemotherapy agent) compared to patients treated with pertuzumab and a chemotherapy agent alone.

[0053] "Objective response" refers to a measurable response, including a complete response (CR) or a partial response (PR).

[0054] By "complete response" or "CR" is intended the disappearance of all signs of cancer in response to treatment. It does not necessarily mean that the cancer has been cured.

[0055] A "partial response" or "PR" refers to a decrease in the size of one or more tumors or lesions, or the extent of cancer in the body, in response to treatment.

[0056] A "HER receptor" is a receptor protein tyrosine kinase that belongs to the HER receptor family, which includes EGFR, HER2, HER3, and HER4 receptors. HER receptors generally contain an extracellular domain that can bind to a HER ligand and / or dimerize with another HER receptor molecule; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain with several tyrosine residues that can be phosphorylated. HER receptors can be "native sequence" HER receptors or "amino acid sequence variants" thereof. Preferably, the HER receptor is a native sequence human HER receptor.

[0057] The terms "ErbB2" and "HER2" are used interchangeably herein and refer to the human HER2 protein (Genebank Accession No. X03363) described, for example, in Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al., Nature 319:230-234 (1986). The term "erbB2" refers to the gene encoding human ErbB2, and "neu" refers to the gene encoding rat p185 neu The preferred HER2 is native sequence human HER2.

[0058] As used herein, "HER2 extracellular domain" or "HER2 ECD" refers to the domain of HER2 that is outside the cell, either anchored to the cell membrane or in circulation, including fragments thereof. The amino acid sequence of HER2 is shown in FIG. 1. In one embodiment, the extracellular domain of HER2 can comprise four domains: "Domain I" (approximately 1-195 amino acid residues; SEQ ID NO: 1), "Domain II" (approximately 196-319 amino acid residues; SEQ ID NO: 2), "Domain III" (320-488 amino acid residues; SEQ ID NO: 3), and "Domain IV" (approximately 489-630 amino acid residues; SEQ ID NO: 4) (residue numbering does not include the signal peptide). See Garrett et al. Mol. Cell. 11:495-505 (2003); Cho et al. Nature 421:756-760 (2003); Franklin et al. Cancer Cell 5:317-328 (2004); and Plowman et al. Proc. Natl. Acad. Sci. 90:1746-1750 (1993), and Figure 6 herein.

[0059] As used herein, "HER3" or "ErbB3" refers to a receptor as disclosed in, for example, U.S. Patent Nos. 5,183,884 and 5,480,968 and Kraus et al., PNAS (USA) 86:9193-9197 (1989).

[0060] A "HER3-low" cancer is a cancer type that expresses HER3 at a level lower than the median level of HER3 expression. In one embodiment, the HER3-low cancer is epithelial ovarian cancer, peritoneal cancer, or fallopian tube cancer. The HER3 DNA, protein, and / or mRNA levels of the cancer can be assessed to determine whether the cancer is a HER3-low cancer. For additional information regarding HER3-low cancers, see, e.g., U.S. Patent No. 7,981,418. In some cases, a HER3 mRNA expression assay is performed to determine whether the cancer is a HER3-low cancer. In one embodiment, the HER3 mRNA level of the cancer is assessed using, for example, polymerase chain reaction (PCR), e.g., quantitative reverse transcription PCR (qRT-PCR). In some cases, the cancer expresses HER3 at a concentration ratio of about 2.81 or less, as assessed by qRT-PCR, e.g., using a COBASz480® instrument.

[0061] As used herein, a "HER dimer" refers to a non-covalently bound dimer containing at least two HER receptors. Such complexes can be formed when cells expressing two or more HER receptors are exposed to a HER ligand, and can be isolated by immunoprecipitation and analyzed by SDS-PAGE (e.g., as described in Sliwkowski et al., J. Biol. Chem., 269 (20), 14661-14665 (1994)). Other proteins, such as cytokine receptor subunits (e.g., gp130), can bind to the dimer. Preferably, the HER dimer includes HER2.

[0062] As used herein, a "HER heterodimer" is a non-covalently linked heterodimer comprising at least two different HER receptors, for example, an EGFR-HER2, HER2-HER3, or HER2-HER4 heterodimer.

[0063] A "HER antibody" is an antibody that binds to a HER receptor. In some cases, the HER antibody also interferes with HER activation or function. Preferably, the HER antibody binds to the HER2 receptor. HER2 antibodies of interest herein are pertuzumab and trastuzumab.

[0064] "Activation of HER" refers to the activation or phosphorylation of any one or more HER receptors. Generally, HER activation results in signal transduction (e.g., caused by the intracellular kinase domain of a HER receptor phosphorylating a tyrosine residue in the HER receptor or a substrate polypeptide). HER activation can be mediated by a HER ligand binding to a HER dimer comprising the HER receptor of interest. A HER ligand binding to a HER dimer can 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 additional substrate polypeptide(s), such as Akt or MAPK intracellular kinases.

[0065] "Phosphorylation" refers to the addition of one or more phosphate groups to a protein, eg, a HER receptor or its substrate.

[0066] An antibody that "inhibits HER dimerization" is an antibody that inhibits or prevents the formation of HER dimers. Preferably, such an antibody binds to HER2 at its heterodimeric binding site. The most preferred dimerization-inhibiting antibody of the present invention is pertuzumab or MAb 2C4. Other examples of antibodies that inhibit HER dimerization include antibodies that bind to EGFR and inhibit its dimerization with one or more other HER receptors (e.g., EGFR monoclonal antibody 806, MAb 806, which binds to activated or "untethered" EGFR; see Johns et al., Biol. Chem. 279(29):30375-30384 (2004)); antibodies that bind to HER3 and inhibit its dimerization with one or more other HER receptors; and antibodies that bind to HER4 and inhibit its dimerization with one or more other HER receptors.

[0067] A "HER2 dimerization inhibitor" is a drug that inhibits the formation of dimers or heterodimers containing HER2.

[0068] The "heterodimer binding site" of HER2 refers to a region in the extracellular domain of HER2 that contacts or associates with a region in the extracellular domain of EGFR, HER3, or HER4 upon dimer formation therewith. This region is found in domain II of HER2 (SEQ ID NO: 15). Franklin et al., Cancer Cell 5:317-328 (2004).

[0069] HER2 antibodies that "bind to the heterodimeric binding site" of HER2 bind to residues in domain II (SEQ ID NO: 2) and, in some cases, may also bind to residues in other domains of the HER2 extracellular domain, such as domains I and III (SEQ ID NOs: 1 and 3), which may sterically hinder, at least to some extent, the formation of HER2-EGFR, HER2-HER3, or HER2-HER4 heterodimers. Franklin et al. (Cancer Cell 5:317-328 (2004)) identified the crystal structure of HER2-pertuzumab, which was deposited in the RCSB Protein Data Bank (ID code IS78), and show an exemplary antibody that binds to the heterodimeric binding site of HER2.

[0070] An antibody that "binds to domain II" of HER2 binds to residues in domain II (SEQ ID NO: 2) and optionally residues in other domain(s) of HER2, e.g., domains I and III (SEQ ID NOs: 1 and 3, respectively). Preferably, an antibody that binds to domain II binds to the junction between domains I, II, and III of HER2.

[0071] For purposes herein, "pertuzumab" and "rhuMAb 2C4", used interchangeably, refer to an antibody comprising the light chain variable amino acid sequence and the heavy chain variable amino acid sequence (SEQ ID NOs: 7 and 8, respectively). When pertuzumab is a whole antibody, it preferably comprises an IgG1 antibody; in one embodiment, it comprises the light chain amino acid sequence of SEQ ID NO: 11 or 15 and the heavy chain amino acid sequence of SEQ ID NO: 12 or 16. The antibody is optionally produced by recombinant Chinese hamster ovary (CHO) cells. As used herein, the terms "pertuzumab" and "rhuMAb 2C4" encompass biosimilar versions of the drug having the United States Adopted Name (USAN) or International Nonproprietary Name (INN): Pertuzumab.

[0072] For purposes herein, the interchangeable terms "trastuzumab" and "rhuMAb4D5" refer to an antibody comprising a light chain variable amino acid sequence and a heavy chain variable amino acid sequence (SEQ ID NOs: 13 and 14, respectively). When trastuzumab is a whole antibody, it preferably comprises an IgG1 antibody; in one embodiment, it comprises a light chain amino acid sequence of SEQ ID NO: 13 and a heavy chain amino acid sequence of SEQ ID NO: 14. The antibody is optionally produced in Chinese hamster ovary (CHO) cells. The terms "trastuzumab" and "rhuMAb4D5" herein encompass biosimilar versions of drugs having the United States Adopted Name (USAN) or International Nonproprietary Name (INN): trastuzumab.

[0073] The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.

[0074] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Most commonly, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all FRs are those of a human immunoglobulin sequence. Humanized antibodies also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see 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). Humanized HER2 antibodies specifically include trastuzumab (HERCEPTIN®), which is described in Table 3 of U.S. Patent No. 5,821,337, expressly incorporated herein by reference, and as defined herein; and humanized 2C4 antibodies, e.g., pertuzumab, as described and defined herein.

[0075] A "complete antibody" herein comprises two antigen-binding regions and an Fc region. Preferably, the complete antibody has a functional Fc region.

[0076] An "antibody fragment" is a portion of an intact antibody, preferably comprising the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragment(s).

[0077] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, 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, although the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ) followed by multiple constant domains. Each light chain has a variable domain (V L ) at one end and a constant domain at the other end. 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. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain.

[0078] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)), and / or those residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0079] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including the native sequence of an Fc region and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from position Pro230, to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering a nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations containing both antibodies with and without the K447 residue.

[0080] Unless otherwise specified, the numbering of residues in immunoglobulin heavy chains herein is that of the EU index as found in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), which is expressly incorporated herein by reference. "EU index as found in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0081] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors; BCRs), etc. Such effector functions generally require an Fc region in combination with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, using various assays such as those disclosed herein.

[0082] A "native-sequence Fc region" comprises an amino acid sequence identical to that found in nature. Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A allotypes and A allotypes), native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0083] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of 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, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide relative to the native-sequence Fc region or relative to the Fc region of the parent polypeptide. The variant Fc region herein will preferably have at least about 80%, most preferably at least about 90%, and more preferably at least 95% homology to the native-sequence Fc region and / or the Fc region of the parent polypeptide.

[0084] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0085] A "naked antibody" is an antibody that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or radiolabel.

[0086] An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions thereof that result in an improvement in the affinity of the antibody for antigen, compared to the parent antibody without the alterations. Preferred affinity matured antibodies will have nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by methods known in the art. Marks et al., Bio / Technology 10:779-783 (1992), describe affinity matured antibodies. H and V LAffinity maturation by domain shuffling has been described. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0087] A "deamidated" antibody is one in which one or more asparagine residues thereof has been derivatized, for example to an aspartic acid, a succinimide, or an isoaspartic acid.

[0088] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0089] "Gastric cancer" specifically includes metastatic or locally advanced unresectable gastric cancer, such as, but not limited to, histologically confirmed adenocarcinoma of the stomach or gastroesophageal junction with inoperable (unresectable) locally advanced or metastatic disease that is not amenable to curative therapy, and advanced gastric cancer that recurs after surgery when the intent of surgery was to cure the disease, e.g., adenocarcinoma of the stomach or gastroesophageal junction.

[0090] An "advanced" cancer is one that has spread outside the original site or organ, either by local invasion or metastasis. Thus, the term "advanced" cancer includes both locally advanced and metastatic disease.

[0091] A "refractory" cancer is a cancer that worsens even when an anti-tumor drug, such as a chemotherapeutic agent, is administered to the cancer patient. An example of a refractory cancer is a platinum-refractory cancer.

[0092] A "recurrent" cancer is one that has regrown at the original site or at a distant site after responding to initial therapy, eg, surgery.

[0093] A "locally recurrent" cancer is one that reappears after treatment in the same place as the previously treated cancer.

[0094] "Unresectable" or "unresectable" cancer cannot be removed (cut out) by surgery.

[0095] "Early stage breast cancer" herein refers to breast cancer that has not spread beyond the breast or axillary lymph nodes. Such cancers are generally treated with neoadjuvant or adjuvant therapy.

[0096] "Neoadjuvant therapy" refers to systemic therapy given before surgery.

[0097] "Adjuvant therapy" refers to systemic therapy given after surgery.

[0098] "Metastatic" cancer refers to cancer that has spread from one part of the body (eg, the breast) to another part of the body.

[0099] A "patient" or "subject" herein refers to a human patient. The patient may be a "cancer patient," i.e., a patient experiencing or at risk of experiencing one or more symptoms of cancer, particularly gastric or breast cancer.

[0100] A "patient population" refers to a group of cancer patients. Such a population can be used to demonstrate statistically significant efficacy and / or safety of a drug, such as pertuzumab.

[0101] A "relapsed" patient is one who has signs or symptoms of cancer after remission. In some cases, the patient has relapsed after adjuvant or neoadjuvant therapy.

[0102] A cancer or biological sample that "exhibits HER expression, amplification, or activation" is one that expresses (including overexpresses) a HER receptor, amplifies the HER gene, and / or otherwise exhibits activation or phosphorylation of a HER receptor in a diagnostic test.

[0103] A cancer or biological sample that "exhibits HER activation" is one that exhibits HER receptor activation or phosphorylation in a diagnostic test. Such activation can be determined directly (e.g., by measuring HER phosphorylation by ELISA) or indirectly (e.g., by gene expression profiling or by detecting HER heterodimers as described herein).

[0104] Cancer cells with "HER receptor overexpression or amplification" are those that have significantly higher levels of HER receptor protein or gene compared to noncancerous cells of the same tissue type. Such overexpression can be caused by gene amplification or by increased transcription or translation. HER receptor overexpression or amplification can be determined in diagnostic or prognostic assays by assessing increased levels of HER protein present on the surface of cells (e.g., by immunohistochemistry assay; IHC). Alternatively, or additionally, intracellular levels of nucleic acids encoding HER can be measured, for example, by in situ hybridization (ISH), such as fluorescent in situ hybridization (FISH; see WO 98 / 45479, published October 1998) and chromogenic in situ hybridization (CISH; see, e.g., Tanner et al., Am. J. Pathol. 157(5):1467-1472 (2000); Bella et al., J. Clin. Oncol. 26:(May 20 suppl; abstr 22147) (2008)), Southern blotting, or polymerase chain reaction (PCR) methods, 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 body fluids, such as serum (see, e.g., U.S. Pat. No. 4,933,294, issued June 12, 1990; WO 91 / 05264, published April 18, 1991; U.S. Pat. No. 5,401,638, issued March 28, 1995; and Siasr et al., J. Immunol. Methods 132:73-80 (1990)). In addition to the assays described above, various in vivo assays are available to those skilled in the art. For example, cells within a patient can be exposed to an antibody, optionally labeled with a detectable label, e.g., a radioisotope, and binding of the antibody to cells within the patient can be assessed, for example, by external scanning for radioactivity or by analyzing biopsies taken from patients previously exposed to the antibody.

[0105] "HER2-positive" cancers include cancer cells with higher-than-normal levels of HER2. Examples of HER2-positive cancers include HER2-positive breast cancer and HER2-positive gastric cancer. In some cases, HER2-positive cancers have an immunohistochemistry (IHC) score of 2+ or 3+ and / or an in situ hybridization (ISH) amplification ratio of 2.0 or greater.

[0106] "Antineoplastic agents" herein refer to drugs used to treat cancer. Non-limiting examples of anti-tumor agents herein include chemotherapeutic agents, HER dimerization inhibitors, HER antibodies, antibodies against tumor-associated antigens, antihormonal compounds, cytokines, EGFR-targeting drugs, anti-angiogenic agents, tyrosine kinase inhibitors, growth inhibitors and antibodies, cytotoxic drugs, antibodies that induce apoptosis, COX inhibitors, farnesyltransferase inhibitors, antibodies that bind to the oncofetal protein CA 125, HER2 vaccines, Raf or ras inhibitors, liposomal doxorubicin, topotecan, taxene, dual tyrosine kinase inhibitors, TLK286, EMD-7200, pertuzumab, trastuzumab, erlotinib, and bevacizumab.

[0107] "Epitope 2C4" refers to the region of the extracellular domain of HER2 to which antibody 2C4 binds. To screen for antibodies that essentially bind to the 2C4 epitope, routine cross-blocking assays, such as those described in *Antibodies, A Laboratory Manual*, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Preferably, the antibody blocks 2C4 binding to HER2 by about 50% or more. Alternatively, epitope mapping can be performed to assess whether an antibody essentially binds to the 2C4 epitope of HER2. Epitope 2C4 comprises residues from domain II (SEQ ID NO: 2) in the extracellular domain of HER2. 2C4 and pertuzumab bind to the extracellular domain of HER2 at the junction of domains I, II, and III (SEQ ID NOs: 1, 2, and 3, respectively). Franklin et al., Cancer Cell 5:317-328 (2004).

[0108] "Epitope 4D5" is the region of the extracellular domain of HER2 to which antibody 4D5 (ATCC CRL 10463) and trastuzumab bind. This epitope is located near the transmembrane domain of HER2, within domain IV (SEQ ID NO: 4) of HER2. To screen for antibodies that essentially bind to the 4D5 epitope, routine cross-blocking assays, such as those described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping can be performed to assess whether an antibody essentially binds to the 4D5 epitope of HER2 (e.g., any one or more residues in the region from about residue 625 to about residue 529, including the HER2 ECD (residue numbering includes the signal peptide)).

[0109] "Treatment" means therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with cancer and those in whom cancer is to be prevented. Thus, the patient being treated herein may be a patient diagnosed with cancer, or a patient who is susceptible or may be susceptible to cancer.

[0110] The term "effective amount" refers to an amount of drug effective to treat cancer in a patient. An effective amount of drug can reduce the number of cancer cells; shrink tumor size; inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into surrounding organs; inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with cancer. To the extent a drug can prevent growth and / or kill existing cancer cells, the drug can be cytostatic and / or cytotoxic. An effective amount can prolong progression-free survival (e.g., as measured by Response Evaluation Criteria in Solid Tumors (RECIST) or CA-125 change), result in an objective tumor regression response (including a partial response (PR) or complete response (CR)), increase overall survival, and / or improve one or more symptoms of cancer (e.g., as assessed by FOSI).

[0111] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. This term includes radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 and multiple 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).

[0112] "Chemotherapy" is the use of chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents used in chemotherapy include: alkylating agents, such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, such as altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; TLK286 (TELCYTA®); acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analogs Topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, including scopoletin and 9-aminocamptothecin; bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic analogs, KW-2189 and CB1 -TM1); eluterobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;Nitrosures, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; bisphosphonates, such as clodronate; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin γ1I and calicheamicin ω1I (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)) and anthracyclines, such as annamycin, AD 32, aclarubicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubicin, KRN5500, menogaril, dynemicins, e.g., dynemicin A, esperamicin, neocarzinostatin chromophores and related chromoprotein-based ediine antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (mono) morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; folic acid analogs such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine; androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane and testolactone; anti-adrenals such as aminoglutethimide, mitotane and trilostane; folic acid supplements such as folinic acid (leucovorin); aceglatone; antifolate antineoplastic agents such as ALIMTA (registered trademark) registered trademark), LY231514 pemetrexed, dihydrofolate reductase inhibitors, e.g., methotrexate, antimetabolites, e.g., 5-fluorouracil (5-FU) and its prodrugs, e.g., UFT, S-1, and capecitabine, as well as thymidylate synthase inhibitors and glycinamide ribonucleotide formyltransferase inhibitors, e.g., raltitrexed (TOMUDEX®, TDX); inhibitors of dihydropyrimidine dehydrogenase, e.g., eniluracil; aldophosphamide glycosides glycoside); aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziconazole; eflornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane;Vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes; chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; platinum; platinum analogs or platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine (VELBAN®); etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); vinca alkaloids; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing; and combinations of two or more of the foregoing, e.g., CHOP (an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone), and FOLFOX (an abbreviation for the treatment regimen of oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin);

[0113] Also included in this definition are antihormonal drugs that act to regulate or inhibit hormone action on tumors, e.g., antiestrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen (e.g., NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON® toremifene; aromatase inhibitors; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (1,3-dioxin). solan nucleoside cytosine analogs); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell growth, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, e.g., gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0114] "Taxanes" are chemotherapeutic agents that inhibit mitosis and interfere with microtubules. Examples of taxanes include paclitaxel (TAXOL®; Bristol-Myers Squibb Oncology, Princeton, NJ); cremophore-free, albumin-modified nanoparticle formulation of paclitaxel or nab-paclitaxel (ABRAXANE™; American Pharmaceutical Partners, Schaumberg, Illinois); and docetaxel (TAXOTERE®; Rhone-Poulenc Rorer, Antony, France).

[0115] "Anthracyclines" are a class of antibiotics derived from the fungus Streptococcus peucetius and include, for example, daunorubicin, doxorubicin, and epirubicin.

[0116] "Anthracycline chemotherapy" refers to a chemotherapy regimen consisting of or including one or more anthracyclines. Examples include 5-FU, epirubicin, and cyclophosphamide (FEC); 5-FU, doxorubicin, and cyclophosphamide (FAC); doxorubicin and cyclophosphamide (AC); epirubicin and cyclophosphamide (EC).

[0117] As used herein, "carboplatin-based chemotherapy" refers to a chemotherapy regimen consisting of or including one or more carboplatins. An example is TCH (docetaxel / TAXOL®, carboplatin, and trastuzumab / HERCEPTIN®).

[0118] An "aromatase inhibitor" inhibits the enzyme aromatase, which controls estrogen production in the adrenal gland. Examples of aromatase inhibitors include 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestane, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. In one embodiment, the aromatase inhibitor herein is letrozole or anastrozole.

[0119] "Antimetabolite chemotherapy" is the use of drugs that are structurally similar to metabolites but cannot be used proliferatively by the body. Many antimetabolite chemotherapeutics interfere with the production of nucleic acids, RNA, and DNA. Examples of chemotherapeutic agents that are antimetabolites include gemcitabine (GEMZAR®), 5-fluorouracil (5-FU), capecitabine (XELODA™), 6-mercaptopurine, methotrexate, 6-thioguanine, pemetrexed, raltitrexed, arabinosylcytosine ARA-C cytarabine (CYTOSAR U®), dacarbazine (DTIC-DOME®), azocytosine, deoxycytosine, pyridmidene, fludarabine (FLUDARA®), cladrabine, and 2-deoxy-D-glucose.

[0120] "Chemoresistant" cancer means that the cancer patient's disease worsens while undergoing a chemotherapy regimen (i.e., the patient is "chemotherapy refractory"), or that the cancer patient's disease worsens within 12 months (e.g., within 6 months) after completing a chemotherapy regimen.

[0121] As used herein, the term "platin" is used to refer to platinum-based chemotherapeutic agents, including, but not limited to, cisplatin, carboplatin, and oxaliplatin.

[0122] As used herein, the term "fluoropyrimidine" refers to chemotherapeutic agents that are antimetabolites, including, but not limited to, capecitabine, floxuridine, and fluorouracil (5-FU).

[0123] As used herein, a "fixed" or "constant" dose of a therapeutic agent refers to a dose administered to a human patient without regard to the patient's body weight (WT) or body surface area (BSA). Thus, a fixed or constant dose may be expressed as a mg / kg dose or a mg / m 2 It is not administered as a dose, but as an absolute amount of therapeutic agent.

[0124] A "loading" dose herein generally comprises an initial amount of a therapeutic agent administered to a patient, followed by one or more maintenance doses thereof. Generally, a single loading dose is administered, although multiple loading doses are also contemplated herein. Typically, the amount of the loading dose(s) administered exceeds the amount of the maintenance dose(s) administered, and / or the loading dose(s) are administered more frequently than the maintenance dose(s), thereby achieving a desired steady-state concentration of the therapeutic agent more quickly than can be achieved by the maintenance dose(s).

[0125] A "maintenance" dose herein refers to one or more doses of a therapeutic agent administered to a patient over a period of treatment. Typically, maintenance doses are administered at spaced treatment intervals, for example, about every week, about every two weeks, about every three weeks, or about every four weeks, preferably every three weeks.

[0126] "Infusion" or "infusing" refers to the introduction of a drug-containing solution into the body via a vein for therapeutic purposes. Typically, this is accomplished via an intravenous (IV) bag.

[0127] An "intravenous drip bag" or "IV bag" is a bag that can contain a solution that can be administered intravenously to a patient. In one embodiment, the solution is a saline solution (e.g., about 0.9% or about 0.45% NaCl). In some cases, IV bags are made from polyolefin or polyvinyl chloride.

[0128] By "co-administering" is meant administering two (or more) drugs intravenously during the same administration, rather than sequential infusion of the two or more drugs. Generally, this involves combining the two (or more) drugs in the same IV bag prior to simultaneous administration.

[0129] "Cardiotoxicity" refers to any toxic side effect resulting from the administration of a drug or drug combination. Cardiotoxicity may be assessed based on any one or more of the incidence of symptomatic left ventricular systolic dysfunction (LVSD) or congestive heart failure (CHF), or a decrease in left ventricular ejection fraction (LVEF).

[0130] The phrase "without increasing cardiac toxicity" in reference to a drug combination that includes pertuzumab means that the incidence of cardiac toxicity is equal to or less than that observed in patients treated with drugs other than pertuzumab in the drug combination (e.g., equal to or less than the incidence of cardiac toxicity resulting from the administration of trastuzumab and a chemotherapeutic agent, such as docetaxel).

[0131] A "vial" is a container suitable for holding a liquid or lyophilized formulation. In one embodiment, the vial is a single-use vial, for example, a stoppered 20 cc single-use vial.

[0132] A "package insert" is a leaflet that must be included in the package of every prescription drug by mandate of the Food and Drug Administration (FDA) or other regulatory agency. The leaflet generally includes the drug's trademark, its generic name, and its mechanism of action; describes its indications, contraindications, warnings, precautions, adverse effects, and dosage form; and includes instructions regarding the recommended dose, time, and route of administration.

[0133] The phrase "safety data" refers to data obtained from controlled clinical trials showing the incidence and severity of adverse events to instruct users regarding drug safety (including instruction on how to monitor and prevent adverse drug reactions). Tables 3 and 4 herein provide safety data for pertuzumab. The safety data may include any one or more (e.g., 2, 3, 4 or more) of the most common adverse events (AEs) or adverse drug reactions (ADRs) in Tables 3 and 4. For example, the safety data may include information regarding neutropenia, febrile neutropenia, diarrhea, and / or cardiac toxicity as disclosed herein.

[0134] "Efficacy data" refers to data obtained in controlled clinical trials that show that a drug effectively treats a disease, e.g., cancer. Efficacy data for pertuzumab are shown in the Examples herein. For HER2-positive metastatic or locally recurrent unresectable breast cancer, efficacy data for pertuzumab are found in Table 2, Table 5, Figures, and Figure 10 herein. Safety data include any one or more (e.g., 2, 3, 4, or more) of the primary endpoint (progression-free survival by IRF, PFS) and / or secondary endpoints (overall survival (OS); investigator-performed progression-free survival (PFS); objective response rate (ORR) including complete response (CR), partial response (PR), stable disease (SD), and progression (PD), and / or duration of response) in Table 2, Table 5, Figure 8, and Figure 10. For example, efficacy data includes information regarding progression-free survival (PFS) and / or overall survival (OS) as disclosed herein.

[0135] "Stable mixture," when referring to a mixture of two or more drugs, e.g., pertuzumab and trastuzumab, means that each drug in the mixture essentially retains its physical and chemical stability in the mixture as assessed by one or more analytical assays. Exemplary analytical assays for this purpose include color, appearance, and clarity (CAC), concentration and turbidity analysis, particle analysis, size exclusion chromatography (SEC), ion exchange chromatography (IEC), capillary zone electrophoresis (CZE), image capillary isoelectric focusing (iCIEF), and potency assays. In one embodiment, the mixture is stable at 5°C or 30°C for up to 24 hours.

[0136] A drug that is administered "concurrently" with one or more other drugs is administered during the same treatment cycle, on the same treatment day as the one or more other drugs, and optionally at the same time as the one or more other drugs. For example, in the case of a cancer therapy administered every three weeks, each of the concurrently administered drugs would be administered on day 1 of the three-week cycle.

[0137] II. Antibody and Chemotherapeutic Compositions The HER2 antigen used to produce antibodies can be, for example, a stable form of the extracellular domain of the HER2 receptor or a portion thereof containing the desired epitope. Alternatively, cells expressing HER2 on the cell surface (e.g., NIH-3T3 cells transformed to overexpress HER2; or cancer cell lines such as SK-Br-3 cells, see Stancovski et al., PNAS (USA) 88:8691-8695 (1991)) can be used to produce antibodies. Other forms of HER2 receptors useful for producing antibodies will be apparent to those skilled in the art.

[0138] Various methods for making the monoclonal antibodies herein are available in the art. For example, they may be made by recombinant DNA methods (U.S. Patent No. 4,816,567) using the hybridoma method first described by Kohler et al., Nature 256:495 (1975).

[0139] The anti-HER2 antibodies for use in accordance with the present invention, trastuzumab and pertuzumab, are commercially available.

[0140] (i) Humanized antibody Methods for humanizing non-human antibodies have been described in the art. Preferably, humanized antibodies have one or more amino acid residues introduced into them from a non-human source. These non-human amino acid residues are often referred to as "import" residues, typically from an "import" variable domain. Humanization can essentially be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact human variable domain has been substituted by 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 substituted by residues from analogous sites in rodent antibodies.

[0141] The selection of human variable domains, both light and heavy, used to generate humanized antibodies is crucial to reducing antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then accepted as the human framework region (FR) of the humanized antibody (Sim et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a specific framework region derived from the consensus sequence of all human antibodies of a particular light or heavy chain subgroup. 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)).

[0142] It is further important that antibodies be humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. By inspecting these displays, the possible role of the residues in the function of the candidate immunoglobulin sequence can be analyzed, i.e., residues that influence the ability of the candidate immunoglobulin to bind to its antigen can be analyzed. In this way, FR residues from the recipient and import sequences can be selected and matched to achieve the desired antibody characteristic, e.g., increased affinity for the target antigen(s). In general, hypervariable region residues directly affect antigen binding and are most substantially involved in influencing antibody binding.

[0143] US Pat. No. 6,949,245 describes the production of exemplary humanized HER2 antibodies that bind HER2 and block ligand activation of the HER receptor.

[0144] Humanized HER2 antibodies specifically include trastuzumab (HERCEPTIN®), as described in Table 3 of U.S. Pat. No. 5,821,337 (expressly incorporated herein by reference) and defined herein; and humanized 2C4 antibodies, such as pertuzumab, as described and defined herein.

[0145] The humanized antibodies herein can, for example, comprise non-human hypervariable region residues incorporated into a human heavy chain variable domain and can further comprise framework region (FR) substitutions at positions selected from the group consisting of 69H, 71H, and 73H (utilizing the variable domain numbering system described 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 of positions 69H, 71H, and 73H.

[0146] Exemplary humanized antibodies of interest herein comprise heavy chain variable domain complementarity-determining residues GFTFTDYTMX (SEQ ID NO: 17) [where X is preferably D or S]; DVNPNSGGSIYNQRFKG (SEQ ID NO: 18); and / or NLGPSFYFDY (SEQ ID NO: 19), and optionally include amino acid modifications of those CDR residues, e.g., where the modifications essentially maintain or improve the affinity of the antibody. For example, antibody variants for use in the methods of the invention can have from about one to about seven or about five amino acid substitutions in the heavy chain variable CDR sequences. Such antibody variants can be prepared, for example, by affinity maturation, as described below.

[0147] Humanized antibodies may, for example, contain, in addition to those heavy chain variable domain CDR residues of the immediately preceding paragraph, light chain variable domain complementarity determining residues KASQDVSIGVA (SEQ ID NO: 20); SASYX 1 X 2 X 3 [X 1 is preferably R or L, and X 2 is preferably Y or E, and X 3is preferably T or S] (SEQ ID NO: 21); and / or QQYYIYPYT (SEQ ID NO: 22). Such humanized antibodies optionally include amino acid modifications of the CDR residues, e.g., where the modifications essentially maintain or improve the affinity of the antibody. For example, the subject antibody variants can have from about 1 to about 7 or about 5 amino acid substitutions in the light chain variable CDR sequence. Such antibody variants can be prepared, for example, by affinity maturation, as described below.

[0148] The present application also contemplates affinity-matured antibodies that bind to HER2. The parent antibody can be a human antibody or a humanized antibody, for example, comprising the light chain variable sequences and / or heavy chain variable sequences of SEQ ID NOs: 7 and 8, respectively (i.e., comprising the VL and / or VH of pertuzumab). Affinity-matured variants of pertuzumab preferably bind to the HER2 receptor with an affinity superior to that of murine 2C4 or pertuzumab (e.g., about 2-fold or about 4-fold to about 10-fold or about 1000-fold improved affinity, as assessed using HER2-extracellular domain (ECD) ELISA). Exemplary heavy chain variable CDR residues for substitution include H28, H30, H34, H35, H64, H96, H99, or a combination of two or more (e.g., two, three, four, five, six, or seven) of these residues. Examples of light chain variable CDR residues for alteration include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97, or a combination of two or more (e.g., two, three, four, five, or up to about ten) of these residues.

[0149] Humanization of the murine 4D5 antibody to generate its humanized variants, including trastuzumab, is described in U.S. Patent Nos. 5,821,337, 6,054,297, 6,407,213, 6,639,055, 6,719,971, and 6,800,738, and Carter et al., PNAS (USA), 89:4285-4289 (1992). HuMAb4D5-8 (trastuzumab) binds to the HER2 antigen three times more tightly than the murine 4D5 antibody and possesses a secondary immune function (ADCC) that allows the humanized antibody to exert directed cytotoxic activity in the presence of human effector cells. HuMAb4D5-8 is a V L Light chain variable (V) sequences built into the subgroup I consensus framework L ) CDR residues, and V H Heavy chain variable (V) sequences built into the subgroup III consensus framework H ) CDR residues. H framework region (FR) substitutions at positions 71, 73, 78, and 93 (Kabat numbering of FR residues) of L It further contained a FR substitution at position 66 (Kabat numbering of FR residues). Trastuzumab contains a non-A allotype human γ1 Fc region.

[0150] Various forms of humanized or affinity matured antibodies are contemplated. For example, the humanized or affinity matured antibody can be an antibody fragment. Alternatively, the humanized or affinity matured antibody can be a complete antibody, e.g., a complete IgG1 antibody.

[0151] (ii) Pertuzumab Composition In one embodiment of the HER2 antibody composition, the composition comprises a mixture of a main species pertuzumab antibody and one or more variants thereof. A preferred embodiment of the pertuzumab main species antibody herein comprises the light chain variable amino acid sequences and heavy chain variable amino acid sequences of SEQ ID NOs: 7 and 8, and most preferably comprises the light chain amino acid sequence of SEQ ID NO: 11 and the heavy chain amino acid sequence of SEQ ID NO: 12 (including deamidated and / or oxidized variants of these sequences). In one embodiment, the composition comprises a mixture of a main species pertuzumab antibody and its amino acid sequence variants comprising an amino-terminal leader extension. Preferably, the amino-terminal leader extension is on the 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 can be a full-length antibody or an antibody fragment (e.g., the Fab of an R(ab')2 fragment), but preferably both are full-length antibodies. The antibody variant 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 on one or two light chains of the antibody. The amino-terminal leader extension preferably comprises or consists of VHS-. The presence of an amino-terminal leader extension in a composition can be detected by various analytical techniques, including, but not limited to, N-terminal sequence analysis, charge heterogeneity assays (e.g., cation exchange chromatography or capillary zone electrophoresis), mass spectrometry, etc. The amount of antibody variant in a composition generally ranges from an amount that constitutes the detection limit of any assay used to detect the variant (preferably, N-terminal sequence analysis) to an amount less than the amount of the predominant species antibody. Generally, about 20% or less (e.g., about 1% to about 15%, e.g., 5% to about 15%) of the antibody molecules in a composition contain 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, e.g., a PROPAC WCX 10™ cation exchange column).In addition to amino-terminal leader extension variants, further amino acid sequence alterations of the primary species antibody and / or variants are contemplated, including, but not limited to, antibodies containing a C-terminal lysine residue in one or both of their heavy chains, deamidated antibody variants, etc.

[0152] Furthermore, the major species antibody or variant may further comprise glycosylation variations, non-limiting examples of which include an antibody comprising a G1 or G2 oligosaccharide structure attached to its Fc region, an antibody comprising a carbohydrate chain attached to its light chain (e.g., one or two carbohydrate chains, e.g., glucose or galactose, attached to one or more lysine residues, attached to one or two light chains of the antibody), an antibody comprising one or two non-glycosylated heavy chains, or an antibody comprising sialidated oligosaccharides attached to one or two heavy chains.

[0153] The composition may be recovered from a genetically engineered cell line, for example a Chinese Hamster Ovary (CHO) cell line expressing a HER2 antibody, or may be prepared by peptide synthesis.

[0154] For more detailed information regarding exemplary pertuzumab compositions, see U.S. Patent Nos. 7,560,111 and 7,879,325 and US2009 / 0202546A1.

[0155] (iii) Trastuzumab Composition Trastuzumab compositions generally comprise a mixture of the major species antibody (containing 13 and 14 light and heavy chain sequences, respectively) and its variant forms, particularly acidic variants (including deamidated variants). Preferably, the amount of such acidic variants in the composition is less than about 25%, or less than about 20%, or less than about 15%. See U.S. Patent No. 6,339,142. See also Harris et al., J. Chromatography, B752:233-245 (2001), regarding forms of trastuzumab resolvable by cation exchange chromatography, e.g., Peak A (Asn30 deamidated to Asp in both light chains); Peak B (Asn55 deamidated to isoAsp in one heavy chain); Peak 1 (Asn30 deamidated to Asp in one light chain); Peak 2 (Asn30 deamidated to Asp in one light chain and Asp102 isomerized to isoAsp in one heavy chain); Peak 3 (major peak form, or major species antibody); Peak 4 (Asp102 isomerized to isoAsp in one heavy chain); and Peak C (Asp102 succinimide (Asu) in one heavy chain). Such variant forms and compositions are encompassed by the invention herein.

[0156] (iv) 5-FU and cisplatin Although there is no single, standard, globally accepted chemotherapy regimen for advanced gastric cancer, 5-fluorouracil (5-FU) plus cisplatin is widely used for its efficacy. In phase II studies in chemotherapy-naive patients, 5-FU plus cisplatin resulted in a response rate of approximately 40% and a median overall survival of 7 to 10.6 months (Lacave AJ, Baron FJ, Anton LM, et al. Ann Oncol 1991; 2: 751-754; Rougier P, Ducreux M, Mahjoubi M, et al. Eur J Cancer 1994; 30A: 1263-1269; Vanhoefer U, Wagner T, Lutz M, et al. Eur J Cancer 2001; 37 Suppl 6: abstract S27).

[0157] (v) capecitabine Capecitabine has been extensively tested in patients with advanced gastric cancer. Phase II efficacy results of capecitabine monotherapy in a 2003 study by Koizumi et al. (Koizumi W, Kurihara M, Sasai T, et al. Cancer 1993;72:658-62; Sakamoto J, Chin K, Kondo K, et al. Anti-Cancer Drugs 2006;17:2331-6) showed response rates of 19% and 26%, and overall survival times of 8.1 and 10.0 months. Regarding capecitabine in combination with platinum, multiple studies have shown response rates ranging from 28% to 65%, time to progression ranging from 5.8 to 9 months, and overall survival ranging from 10.1 to 12 months (Kang Y, Kang WK, Shin DB, et al. J Clin Oncology 2006;24 Suppl 18:abstract LBA4018; Park Y, Kim B, Ryoo B, et al. Proc Am Soc Clin Oncol 2006;24 Suppl 18:abstract 4079; Kim TW, Kang YK, Ahn JH, et al. Ann Oncol 2002;13:1893-1898; Park YH, Kim BS, Ryoo BY, et al. Br J Cancer 2006;94:959-63).

[0158] III. Patient Selection for Treatment Detection of HER2 can be used to select patients for treatment according to the present invention. Several FDA-approved commercial assays are available for identifying patients with HER2-positive cancer. These methods include HERCEPTEST® (Dako) and PATHWAY® HER2 (immunohistochemistry (IHC) assays) and PathVysion® and HER2 FISH pharmDx™ (FISH assays). Users should refer to the package insert of the specific assay kit for information regarding the validation and performance capabilities of each assay.

[0159] For example, HER2 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 meet the following HER2 protein staining intensity criteria: Score 0: no staining is observed or membranous staining is observed in less than 10% of the tumor cells. Score 1+: Faint / barely perceptible membrane staining is detected in more than 10% of the tumor cells. Cells are only stained in part of their membrane. Score 2+: Weak to moderate complete membrane staining is observed in more than 10% of the tumor cells. Score 3+: Moderate to strong complete membrane staining is observed in more than 10% of the tumor cells.

[0160] Those tumors that score 0 or 1+ on the HER2 overexpression assessment can be characterized as HER2 negative, and those tumors that score 2+ or 3+ can be characterized as HER2 positive.

[0161] HER2-overexpressing tumors can be graded by an immunohistochemical score, which corresponds to the number of copies of the HER2 molecule expressed per cell, and can be determined biochemically: 0 = 0–10,000 copies / cell; 1+ = at least approximately 200,000 copies / cell; 2+ = at least approximately 500,000 copies / cell; 3+ = at least approximately 2,000,000 copies / cell.

[0162] Overexpression of HER2 at a 3+ level, which results in ligand-independent activation of tyrosine kinase (Hudziak et al., Proc. Natl. Acad. Sci. USA 84:7159-7163 (1987)), occurs in approximately 30% of breast cancers and is associated with decreased recurrence-free and overall survival in these patients (Slamon et al., Science 244:707-712 (1989); Slamon et al., Science 235:177-182 (1987)).

[0163] The presence of HER2 protein overexpression is highly correlated with gene amplification, and therefore, alternatively or additionally, the use of an in situ hybridization (ISH) assay to detect gene amplification, such as a fluorescent in situ hybridization (FISH) assay, can be used to select patients suitable for treatment according to the present invention. FISH assays, such as INFORM™ (sold by Ventana, Arizona) or PathVysion® (Vysis, Illinois), can be performed on formalin-fixed, paraffin-embedded tumor tissue to determine the extent, if any, of HER2 amplification in the tumor.

[0164] Most commonly, HER2-positive status is confirmed using archival paraffin-embedded tumor tissue using any of the methods described above.

[0165] Preferably, HER2-positive patients with a 2+ or 3+ IHC score or who are FISH or ISH positive are selected for treatment according to the present invention.

[0166] See also US Pat. No. 7,981,418 and Example 11 for an alternative assay for screening patients for therapy with pertuzumab.

[0167] IV. Pharmaceutical Preparations Therapeutic formulations of HER2 antibodies used in accordance with the present invention are prepared for storage, typically in the form of a lyophilized formulation or aqueous solution, by combining the antibody of the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A., ed. (1980)). Crystals of the antibody are also contemplated (see U.S. Patent Application No. 2002 / 0136719). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and examples include: buffers, such as phosphate, citrate, and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polymers, The lyophilized antibody formulation may be selected from the group consisting of peptides, 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 such as 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 non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Lyophilized antibody formulations are described in WO 97 / 04801, which is expressly incorporated herein by reference.

[0168] Lyophilized antibody formulations are described in U.S. Patent Nos. 6,267,958, 6,685,940, and 6,821,515, which are expressly incorporated herein by reference. A preferred HERCEPTIN® (trastuzumab) formulation is a preservative-free, white to pale yellow, sterile, lyophilized powder formulation for intravenous (IV) administration containing 440 mg of trastuzumab, 400 mg of α,α-trehalose dehydrate, 9.9 mg of L-histidine-HCl, 6.4 mg of L-histidine, and 1.8 mg of polysorbate 20 (USP). Reconstitution with 20 mL of bacteriostatic water for injection (BWFI) containing 1.1% benzyl alcohol as a preservative yields a multi-dose solution containing 21 mg / mL trastuzumab at a pH of approximately 6.0. For further details, see the trastuzumab prescribing information.

[0169] A preferred pertuzumab formulation for therapeutic use contains 30 mg / mL pertuzumab in 20 mM histidine acetate, 120 mM sucrose, 0.02% polysorbate 20, pH 6.0. An alternative pertuzumab formulation contains 25 mg / mL pertuzumab in 10 mM histidine-HCl buffer, 240 mM sucrose, 0.02% polysorbate 20, pH 6.0.

[0170] The placebo formulation used in the clinical trials described in the Examples is equivalent to pertuzumab without the active agent.

[0171] The formulations of the present invention may also contain two or more active compounds as needed for the particular indication being treated, preferably compounds with complementary activities that do not adversely affect each other.Various drugs that can be used in combination with HER dimerization inhibitors are described in the "Methods" section below.Such molecules are preferably present together in amounts effective for the intended purpose.

[0172] The formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0173] V. Treatment method In a first aspect of the methods of treatment herein, there is provided a method for extending progression-free survival by 6 months or more in a population of patients with HER2-positive breast cancer, the method comprising administering pertuzumab, trastuzumab, and a chemotherapeutic agent (e.g., a taxane, e.g., docetaxel) to patients in said population. The patient population optionally includes a suitable number of patients (e.g., 200 or more, 300 or more, or 400 or more patients) so that a statistically significant extension of PFS in the population can be assessed.

[0174] The Phase III CLEOPATRA clinical data in Example 3 below show that the median investigator-assessed PFS was 12.4 months for placebo + trastuzumab + docetaxel and 18.5 months for pertuzumab + trastuzumab + docetaxel, thus representing an improvement in median PFS of more than 6 months (e.g., 6.1 months) compared to patients who did not receive pertuzumab (i.e., patients who received trastuzumab and docetaxel only).

[0175] In further or alternative embodiments, a method is provided for achieving a response rate of 80% or greater in a population of HER2-positive breast cancer patients, comprising administering to patients in said population pertuzumab, trastuzumab and a chemotherapeutic agent (e.g., a taxane, e.g., docetaxel).

[0176] In a related embodiment, a method of combining two HER2 antibodies to treat HER2-positive cancer without increasing cardiotoxicity in a HER2-positive cancer patient population is provided, comprising administering pertuzumab, trastuzumab, and a chemotherapeutic agent to patients in the population. The patient population optionally includes a suitable number of patients (e.g., 200 or more, 300 or more, or 400 or more patients) to allow for a statistically significant assessment of the lack of cardiotoxicity resulting from the combination. Phase III CLEOPATRA clinical data in Example 3 below demonstrate that the combination of pertuzumab and trastuzumab does not exacerbate cardiotoxicity. Cardiotoxicity can be monitored, for example, by the incidence of symptomatic left ventricular systolic dysfunction (LVSD) or congestive heart failure (CHF), or a decline in left ventricular ejection fraction (LVEF), as disclosed in Example 3 below.

[0177] In some cases, the breast cancer is metastatic or locally recurrent unresectable breast cancer or de novo stage IV disease, defined as immunohistochemistry (IHC) 3+ and / or a fluorescent in situ hybridization (FISH) amplification ratio of 2.0 or greater.

[0178] In some cases, patients in the population are previously untreated or have relapsed after adjuvant therapy, have a left ventricular ejection fraction (LVEF) of 50% or greater at baseline, and / or have an Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 or 1.

[0179] In an alternative embodiment, the present invention relates to a method of treating early-stage HER2-positive breast cancer, comprising administering to a breast cancer patient pertuzumab, trastuzumab, and a chemotherapeutic agent, wherein the chemotherapeutic agent comprises an anthracycline chemotherapeutic agent or a carboplatin chemotherapeutic agent. This aspect of the present invention is supported by the clinical data in Example 5. In one embodiment, the chemotherapeutic agent comprises an anthracycline chemotherapeutic agent, e.g., 5-FU, epirubicin, and cyclophosphamide (FEC). In an alternative embodiment, the chemotherapeutic agent comprises, in addition to HERCEPTIN® / trastuzumab, a carboplatin chemotherapeutic agent, e.g., a taxane (e.g., docetaxel), carboplatin (e.g., TCH regimen). In one embodiment, pertuzumab is administered simultaneously with the anthracycline chemotherapeutic agent or carboplatin chemotherapeutic agent. For example, pertuzumab, trastuzumab, and a chemotherapy agent are administered in a 3-week cycle, with pertuzumab, trastuzumab, and a chemotherapy agent being administered on day 1 of each cycle. The data in the Examples herein demonstrate that administration of pertuzumab does not increase cardiac toxicity compared to treatment without pertuzumab (i.e., compared to treatment including trastuzumab and an anthrycline-based chemotherapy agent but not pertuzumab (e.g., FEC)); or compared to treatment including trastuzumab and a carboplatin-based chemotherapy agent but not pertuzumab (i.e., TCH). The early stage HER2-positive breast cancer therapies contemplated herein include neoadjuvant therapy and adjuvant therapy.

[0180] The invention herein also relates to a method of treating HER2-positive cancer in a patient, comprising co-administering a mixture of pertuzumab and trastuzumab to the patient from the same intravenous infusion bag. This embodiment is applicable to the treatment of any HER2-positive cancer, including HER2-positive breast cancer, HER2-positive gastric cancer, HER2-positive metastatic or locally recurrent unresectable breast cancer, or de novo stage IV disease, early stage HER2-positive breast cancer, etc. Optionally, the method further comprises administering a chemotherapeutic agent to the patient.

[0181] In yet another embodiment, the therapeutic method of the present invention comprises, consists essentially of, or consists of the administration of pertuzumab, trastuzumab and a chemotherapeutic agent, such as a platin (e.g., cisplatin) and / or a fluoropurimidine (e.g., capecitabine and / or 5-fluorouracil (5-FU)), to treat HER2-positive gastric cancer.

[0182] In particular, the therapeutic methods of the present invention comprise, consist essentially of, or consist of the administration of pertuzumab and trastuzumab and a chemotherapeutic agent, such as a platin and / or a fluoroprimidine, such as cisplatin and / or capecitabine and / or 5-fluorouracil (5-FU), to a human patient with metastatic gastric cancer, unresectable locally advanced gastric cancer, or post-operative recurrent gastric cancer. In certain embodiments, the gastric cancer is not amenable to curative therapy.

[0183] In an alternative embodiment, a method of treating HER2-positive breast cancer in a patient is provided, comprising administering pertuzumab, trastuzumab, and vinorelbine to the patient. The breast cancer according to this embodiment is optionally metastatic or locally advanced. Optionally, the patient has not previously been treated with systemic non-hormonal anti-cancer therapy in the metastatic setting.

[0184] In another aspect, the present invention provides a method of treating HER2-positive breast cancer in a patient, comprising administering to the patient pertuzumab, trastuzumab, and an aromatase inhibitor (e.g., anastrozole or letrozole). According to this embodiment, the breast cancer is advanced breast cancer, e.g., hormone receptor-positive breast cancer, e.g., estrogen receptor (ER)-positive and / or progesterone receptor (PgR)-positive breast cancer. Optionally, the patient has not been previously treated with systemic non-hormonal anticancer therapy in the metastatic setting. Optionally, the treatment method further comprises administering to the patient an induction chemotherapy agent (e.g., comprising a taxane).

[0185] Therapies according to the present invention extend progression-free survival (PFS) and / or overall survival (OS) of treated patients.

[0186] The antibodies and chemotherapeutic agents are administered to human patients according to known methods, with particular administration schedules and formulations described in the Examples herein.

[0187] According to one embodiment, pertuzumab has a steady-state C of 20 μg / mL in 90% of patients receiving pertuzumab and trastuzumab. min The dose is administered at a level that results in

[0188] According to one particular embodiment of the invention, about 840 mg (loading dose) of pertuzumab is administered, followed by one or more doses of about 420 mg (maintenance dose) of the antibody. The maintenance doses are preferably administered about every three weeks for a total of at least two doses, preferably no more than about 6 doses, or 7 doses, or 8 doses, or 9 doses, or 10 doses, or 11 doses, or 12 doses, or 13 doses, or 14 doses, or 15 doses, or 16 doses, or 17 doses or more, until clinical progression or unmanageable toxicity. Longer treatment periods involving more treatment cycles are also contemplated.

[0189] According to another particular embodiment, pertuzumab is administered at a dose of 840 mg for the entire treatment cycle.

[0190] Trastuzumab is typically administered as an intravenous loading dose of about 8 mg / kg, followed by doses of 6 mg / kg in subsequent cycles. Trastuzumab is typically administered every 3 weeks, preferably for up to 17 doses or more, until clinical progression or unmanageable toxicity.

[0191] In one particular embodiment, trastuzumab is administered as an intravenous (IV) infusion on day 1 of each treatment cycle at a loading dose of 8 mg / kg in cycle 1 and at doses of 6 mg / kg in subsequent cycles until investigator-assessed progression or unmanageable toxicity.

[0192] In another specific embodiment, pertuzumab is administered as an IV infusion on day 1 of each cycle at either an 840 mg loading dose on cycle 1 and a 420 mg dose on subsequent cycles, or an 840 mg loading dose on cycle 1 and an 840 mg dose on subsequent cycles for a total of 6 cycles or until investigator-assessed progression or unmanageable toxicity, whichever occurs first.

[0193] To treat gastric cancer, cisplatin 80 mg / m 2 is typically administered as an IV infusion on day 1 of each cycle for a total of at least 6 cycles.

[0194] To treat gastric cancer, capecitabine 1000 mg / m 2 is typically administered orally twice daily, from the evening of day 1 to the morning of day 15 of each cycle, for a total of at least six cycles. Capecitabine administration may be extended at the discretion of the treating physician after a careful risk-benefit assessment for each individual patient.

[0195] Doses and schedules of chemotherapeutic agents used to treat HER2-positive breast cancer are disclosed in the Examples below, although other doses and schedules are known and are contemplated by the invention herein.

[0196] VI. Manufactured products One embodiment of the article of manufacture herein includes an intravenous (IV) bag containing a stable mixture of pertuzumab and trastuzumab suitable for administration to cancer patients. In some cases, the mixture is a saline solution containing about 0.9% NaCl or about 0.45% NaCl. An exemplary IV bag is a polyolefin or polyvinyl chloride IV bag, e.g., a 250 mL IV bag. According to one embodiment of the present invention, the mixture contains about 420 mg or about 840 mg of pertuzumab and about 200 mg to about 1000 mg of trastuzumab (e.g., about 400 mg to about 900 mg of trastuzumab).

[0197] In some cases, the mixture in the IV bag is stable for up to 24 hours at 5° C. or 30° C. The stability of the mixture can be assessed by one or more assays selected from the group consisting of color, appearance and clarity (CAC), concentration and turbidity analysis, particle analysis, size exclusion chromatography (SEC), ion exchange chromatography (IEC), capillary zone electrophoresis (CZE), image capillary isoelectric focusing (iCIEF), and potency assays.

[0198] In an alternative embodiment, the invention provides an article of manufacture comprising a vial containing pertuzumab and a package insert, wherein the package insert provides the safety data in Table 3 or Table 4 and / or the efficacy data in Table 2, Table 5, Figure 8 or Figure 10. Optionally, the vial is a single dose vial containing about 420 mg of pertuzumab. In one embodiment, the vial is placed inside a cardboard carton.

[0199] In a related aspect, the invention relates to a method for producing an article of manufacture comprising packaging a vial containing pertuzumab together with a package insert, wherein the package insert provides the safety data in Table 3 or Table 4 and / or the efficacy data in Table 2, Table 5, Figure 8 or Figure 10.

[0200] In a further related aspect, the invention provides a method for ensuring the safe and effective use of pertuzumab, comprising packaging a vial containing pertuzumab together with a package insert, wherein the package insert provides the safety data in Table 3 or Table 4 and / or the efficacy data in Table 2, Table 5, Figure 8 or Figure 10.

[0201] VII. Deposit of Biological Materials The following hybridoma cell lines have been deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA (ATCC): Antibody designation ATCC No. Deposit date 4D5 ATCC CRL 10463 May 24, 1990 2C4 ATCC HB-12697 April 8, 1999

[0202] Further details of the present invention are illustrated by the following non-limiting examples, the disclosures of all citations throughout the specification are expressly incorporated herein by reference. [Example]

[0203] A phase IIa study evaluating pertuzumab in combination with trastuzumab and chemotherapy in patients with HER2-positive advanced gastric cancer Despite a dramatic global decline in incidence and mortality rates in the second half of the 20th century, gastric cancer remains the second leading cause of cancer death worldwide, after lung cancer (Parkin, D. Oncogene 23:6329-40 (2004)). Incidence rates of gastric cancer vary significantly by geographic region (Kelley et al. J Clin Epidemiol 56:1-9 (2003); Plummer et al. Epidemiology of gastric cancer. Butlet et al., eds. Mechanisms of carcinogenesis: contribution of molecular epidemiology. Lyon: IARC Scientific Publications No. 157, IARC (2004)). In Japan, Korea, China, and certain countries in Latin America, incidence rates range from 20 to 95 cases per 100,000 males. In contrast, in the United States, India, and Thailand, incidence rates range from 4 to 8 cases per 100,000 males. Incidence rates in Western Europe range from 37 cases per 100,000 men in some parts of Italy to 12 cases per 100,000 men in France. Incidence rates in women follow a similar geographic pattern but are approximately 50% lower than those in men. There is a clear epidemiological divide between cancers confined to the gastric cardia (gastroesophageal junction) and those confined to the remainder of the stomach. Cardiac cancer accounts for 39% of gastric cancer cases in white U.S. men but only 4% of gastric cancers in Japanese men. For unknown reasons, cancers of the gastric cardia and lower esophagus have increased sharply in developed countries since the 1970s.

[0204] To date, the only potentially curative treatment for gastric cancer is surgery. Survival rates for gastric cancer have improved significantly in Japan in recent years as a result of earlier detection and better surgical techniques (Inoue et al., Postgrad Med J 81:419-24 (2005)). However, in Western Europe and North America, gastric cancer is often diagnosed at a later stage when resection is no longer possible. Consequently, the 5-year overall survival rate in these populations is less than 25% (Ajani, J., The Oncologist 10 Suppl 3:49-58 (2005); Catalano et al., Clin Rev Oncol / Hematol 54:209-41 (2005)).

[0205] Regardless of their geographic region, patients with unresectable disease due to locally advanced growth or metastatic spread have a poor prognosis, with 5-year overall survival rates ranging from 5% to 15% (Cunningham et al., Annals of Oncology 16 Suppl 1:i22-3 (2005)). For patients with unresectable disease at diagnosis and for patients with recurrent disease after surgery, the primary treatment option is chemotherapy (National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncology. Gastric cancer. Version 1. National Comprehensive Cancer Network (2006)). Chemotherapy administered with palliative intent has been shown to be superior to best supportive care in patients with advanced gastric cancer (Wagner et al., J Clin Oncol 24:2903-9 (2006)).

[0206] The BO18255 (ToGA) study was an international, phase III, multicenter, randomized, open-label, comparative trial designed to evaluate the efficacy and safety of trastuzumab in combination with chemotherapy compared with chemotherapy alone as first-line treatment in patients with inoperable, locally advanced or recurrent and / or metastatic HER2-positive adenocarcinoma of the stomach or gastroesophageal junction. The primary objective of the study was to compare overall survival (OS) in patients treated with trastuzumab in combination with a fluoropyrimidine (5-FU or capecitabine) plus cisplatin. Results of the BO18255 study demonstrated a significant clinical benefit when trastuzumab was combined with chemotherapy in patients with gastric cancer. The primary endpoint of OS was significantly improved in the trastuzumab plus chemotherapy arm compared with the chemotherapy alone arm (p=0.0045, log-rank test; hazard ratio 0.74). Median survival was 13.8 months in the trastuzumab plus chemotherapy arm and 11.1 months in the chemotherapy alone arm, and the risk of death was reduced by 26% for patients in the trastuzumab plus chemotherapy arm. All other secondary endpoints demonstrated clinical significance with similar hazard ratios and odds ratios (see, e.g., Bang et al., Lancet 28;376(9742):687-97 (2010)).

[0207] As a result of this study, trastuzumab is now indicated in combination with cisplatin plus capecitabine or 5-FU for the treatment of patients with HER2-positive metastatic gastric or gastroesophageal junction adenocarcinoma who have not received prior treatment for metastatic disease, including in the EU and the US.

[0208] Currently, there is no single, standard, globally accepted chemotherapy regimen for advanced gastric cancer. Despite the success of the ToGA trial, new, effective treatment options for this serious condition are greatly needed. In particular, novel therapeutic approaches aimed at avoiding treatment-related morbidity and / or increasing survival in patients with gastric cancer are needed. Therefore, this example is a multicenter, randomized, open-label study evaluating two different doses of pertuzumab in patients with HER2-positive adenocarcinoma of the stomach or gastroesophageal junction. Patients are randomized 1:1 into two treatment arms. Patients in arm A receive a pertuzumab loading dose of 840 mg for cycle 1 and a 420 mg dose for cycles 2-6, while patients in arm B receive 840 mg of pertuzumab for all six cycles. Patients in both treatment arms receive trastuzumab, cisplatin, and capecitabine. The study schema is shown in Figure 6. The study will last approximately 24 months (4 months for recruitment and 20 months for follow-up after the last patient recruitment). The study will end when all patients have progressed or have withdrawn or discontinued from the study, whichever comes first.

[0209] Target population The study will include approximately 30 patients.

[0210] Patients must meet the following study entry criteria: Histologically confirmed chronic adenocarcinoma of the stomach or gastroesophageal junction with inoperable locally advanced or metastatic disease not amenable to curative therapy. · Patients with progressive disease who demonstrate recurrence after surgery (when the surgical intent was curative) are also eligible for entry. Measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST), v1.1, assessed using imaging (computed tomography (CT) or magnetic resonance imaging (MRI)) or non-measurable disease with follow-up. HER2-positive tumors, defined as either IHC3+ or a combination of IHC2+ and ISH+ when assessed by a central laboratory on primary or metastatic tumors. ISH positivity is defined as a ratio of HER2 gene copy number to CEP17 signal number of 2.0. · Availability of formalin-fixed, paraffin-embedded (FFPE) tissue containing at least 5 mm of invasive tumor for central laboratory confirmation of HER2 eligibility is mandatory. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Baseline left ventricular ejection fraction (LVEF) ≥ 55% (measured by echocardiogram (ECHO) or multi-gated acquisition (MUGA) scan). · Life expectancy of at least three months. Male or female. · Age 18 or older. · Have signed informed consent. For women of childbearing potential and male participants with female partners of childbearing potential: A highly effective non-hormonal form of contraception or agreement by the patient and / or partner to use two effective forms of non-hormonal contraception. Contraceptive use must be continued throughout the study treatment period and for at least 6 months after the last dose of study drug.

[0211] Patients who meet any of the following criteria will be excluded from study entry: Prior chemotherapy for advanced or metastatic disease, except that prior adjuvant or neoadjuvant therapy is permitted if at least 6 months elapse between completion of adjuvant or neoadjuvant therapy and study entry. Platinum-based adjuvant or neoadjuvant therapy is not permitted. Lack of physical integrity of the upper gastrointestinal tract or malabsorption syndrome (e.g., patients who have undergone partial or total gastrectomy may be enrolled in the study, but patients with a percutaneous jejunostomy probe may not). Active (significant or uncontrollable) gastrointestinal bleeding. Residual related toxicity resulting from previous therapy (e.g., grade 2 or higher neurological toxicity (NCICTCAE)), excluding alopecia. Other malignancies within the past five years, excluding cervical intraepithelial neoplasia or basal cell carcinoma. Any of the following abnormal laboratory tests immediately prior to randomization: Serum total bilirubin >1.5 times the upper limit of normal (ULN) or, in patients with known Gilbert's syndrome, serum total bilirubin >2 × ULN For patients without liver or bone metastases: AST or ALT > 2.5 x ULN, and alkaline phosphatase (ALP) > 2.5 x ULN For patients with liver metastases but no bone metastases: AST or ALT > 5 x ULN, and ALP > 2.5 x ULN For patients with both liver and bone metastases: AST or ALT > 5 x ULN, and ALP > 10 x ULN For patients with bone metastases but no liver metastases: AST or ALT > 2.5 x ULN, and ALP > 10 x ULN Albumin < 25 g / L Creatinine clearance <60 mL / min Total white blood cell (WBC) count <2500 / μL (<2.5 × 10 9 / L) Absolute neutrophil count (ANC) <1500 / μL (<1.5 × 10 9 / L) Platelets <100,000 / μL (<100×10 9 / L) Serious heart disease or medical conditions, including but not limited to: history of documented heart failure or systolic dysfunction (LVEF <50%); High-risk uncontrolled arrhythmias, e.g., atrial tachycardia with a resting heart rate of 100 beats per minute; Severe ventricular arrhythmia (ventricular tachycardia) or high-degree AV block (second-degree AV block type 2 (Mobitz type II) or third-degree AV block); angina requiring antianginal medication; clinically significant valvular heart disease; Evidence of transmural infarction on ECG; poorly controlled hypertension (e.g., systolic pressure >180 mmHg or diastolic pressure >100 mmHg); Dyspnea at rest or need for supportive oxygen therapy due to progressive malignant disease or complications of other diseases; Treatment with long-term or high-dose corticosteroid therapy; Inhaled steroids and short-term oral steroids for antiemetics or as appetite stimulants are permitted; Clinically significant hearing abnormality; known dihydropyrimidine dehydrogenase deficiency; History or clinical evidence of brain metastases; severe, poorly controlled systemic intercurrent illness (e.g., infection or poorly controlled diabetes). · Pregnant or breastfeeding Women of childbearing potential must have a negative serum pregnancy test within 7 days prior to randomization, regardless of the method of contraception used. Radiation therapy within 4 weeks before initiation of study treatment, or within 2 weeks before initiation of study treatment if palliative radiation therapy was administered peripherally to the site of bone metastases and the patient had recovered from any acute toxicity. Major surgery within 4 weeks before starting study treatment, unless fully recovered. Known active infection with HIV, hepatitis B virus or hepatitis C virus. Known hypersensitivity to any of the study drugs. Failure to comply with follow-up tests or procedures, as determined by the investigator.

[0212] Investigational Drug Products: Dosage, Route and Regimen The duration of a treatment cycle is 3 weeks. Trastuzumab will be administered as an intravenous (IV) infusion on Day 1 of each cycle at a loading dose of 8 mg / kg in Cycle 1 and 6 mg / kg in subsequent cycles until progression or unmanageable toxicity as assessed by the investigator. Pertuzumab will be administered as an IV infusion on Day 1 of each cycle for a total of 6 cycles or until investigator-assessed progression or unmanageable toxicity (whichever occurs first), as follows for each arm: Arm A: Patients will receive pertuzumab at a loading dose of 840 mg in cycle 1 and at a dose of 420 mg in cycles 2-6. Arm B: Patients will receive pertuzumab at 840 mg in cycles 1-6.

[0213] Non-clinical drug products The duration of a treatment cycle is 3 weeks. Cisplatin 80mg / m 2 will be administered as an IV infusion on day 1 of each cycle for a total of 6 cycles. Capecitabine 1000mg / m 2 will be administered orally twice daily, from the evening of day 1 to the morning of day 15 of each cycle, for a total of six cycles. (Capecitabine may be extended at the investigator's discretion after a careful risk-benefit assessment for each individual patient.) ·

[0214] formulation Pertuzumab formulations Each lot of recombinant antibody produced for clinical use meets the viral safety requirements and sterility requirements of the United States Pharmacopoeia and the European Pharmacopoeia. Each lot meets the required specifications for identity, purity, and potency.

[0215] Pertuzumab is provided as a single-use formulation containing 30 mg / mL pertuzumab formulated in 20 mM L-histidine-acetate (pH 6.0), 120 mM sucrose, and 0.02% polysorbate 20. Each 20 cc vial (14.0 mL of solution per vial) contains approximately 420 mg of pertuzumab.

[0216] Trastuzumab formulations Investigational trastuzumab is supplied in most countries as a lyophilized preparation with a nominal content of 150 mg per vial (vial size varies by country).

[0217] Trastuzumab is formulated in histidine, trehalose, and polysorbate 20. After reconstitution, each solution contains 21 mg / mL of active drug at a pH of approximately 6.0.

[0218] evaluation Effectiveness Investigator-assessed tumor response will be used to summarize best overall response, defined as patients with a complete or partial response as determined by RECIST, for each treatment arm at the end of cycles 3 and 6.

[0219] safety Safety will be assessed based on a summary of adverse events, laboratory changes, and vital sign changes.

[0220] Pharmacokinetics / Pharmacodynamics Minimum (trough) serum concentration (C) for pertuzumab on day 43 min ) are evaluated. In addition, PK parameters such as CL, Vss, AUC, and half-life are estimated. Evaluation of PK parameters from data collected through Day 43 allows for modeling and simulation of an estimated dose that predicts a steady-state trough of ≥20 μg / mL in 90% of patients.

[0221] statistical analysis Pharmacokinetic analysis Individual and mean serum pertuzumab concentration-time data are tabulated and plotted by dose level. Serum pharmacokinetics of pertuzumab were assessed by total exposure (area under the curve (AUC)), maximum serum concentration (C max ), minimum serum concentration (C min ), steady state C max and C mintime to elimination, total serum clearance, volume of distribution and elimination half-life (t 1 / 2 ) are summarized by estimating the PK parameters. Estimates of these parameters are tabulated and summarized by descriptive statistics (mean, standard deviation, minimum and maximum). Depending on the observed pertuzumab serum concentration-time data, a population PK approach can be used to estimate the dose that will achieve the PK target concentration.

[0222] C observed for trastuzumab max and C min will be tabulated and summarized by descriptive statistics for each designated PK sampling time point. For all PK analyses, actual time points of sample collection (not scheduled time points) will be used.

[0223] PK parameters of pertuzumab (AUC, C max , t 1 / 2 ) is calculated using non-compartmental methods, and total body clearance is derived from plasma concentrations using standard methods.

[0224] Analysis population Intent-to-treat population All randomized patients who receive at least one dose of study drug will be included in the intention-to-treat population (patients are assigned to the treatment group to which they were randomized for analytical purposes).

[0225] Safety Group All patients who receive at least one dose of study drug will be included in the safety-evaluable population (patients will be assigned to the arm in which they are treated).

[0226] Sample size: The purpose of this study was to assess the C value of pertuzumab at day 43 in patients receiving two different pertuzumab dose regimens. minThese data are then analyzed using a population PK model to identify a dose of pertuzumab that achieves a PK target steady-state trough concentration of 20 μg / mL or greater in approximately 90% of patients with advanced gastric cancer. Analysis using the assumption that pertuzumab behaves similarly to trastuzumab in advanced gastric cancer suggests that for a sample size of 15 patients per arm (total number of patients in this study was 30), the dose achieving the desired target concentration can be estimated with acceptable precision (coefficient of variation <15%).

[0227] clinical results The clinical results of the Phase IIa gastric cancer (GC) study are shown in Figures 32-37.

[0228] Figure 32 shows the samples and time points taken.

[0229] Figure 33 shows the demographics of the patient population in the two arms of the GC study, treated with pertuzumab 420 mg (Arm A) or 840 mg (Arm B).

[0230] FIG. 34 shows the GC history of patients in arms A and B, respectively.

[0231] Figure 35 shows the patient breakdown for Arms A and B.

[0232] Figure 36 shows the overall response rates for arms A and B, respectively.

[0233] Safety Data Diarrhea was the most common event, occurring in 90% of subjects, typically grade 1 and 2 with onset in cycle 1. No patients discontinued treatment due to diarrhea.

[0234] Grade 3 or higher adverse events (AEs) (>13%) included diarrhea, stomatitis, fatigue / asthenia, decreased appetite, hyponatremia, anemia, and neutopenia. With the exception of neutropenia and hyponatremia (higher in Arm A) and decreased appetite (higher in Arm B), the incidence of these events was similar in the standard and high-dose pertuzumab arms.

[0235] Asymptomatic changes in ejection fraction (EF), neutropenic fever, rash, and drug hypersensitivity reactions were not associated with higher doses of pertuzumab.

[0236] Serious adverse events (SAEs) occurred in 60% of patients and the incidence was not associated with high-dose pertuzumab.

[0237] More patients withdrew from treatment in arm B, but it is unclear whether this was due to the higher pertuzumab dose, as events leading to treatment discontinuation were not uniform.

[0238] Pharmacokinetic (PK) results Figure 37 shows the results of pertuzumab concentration assessment at day 42 in gastric cancer (GC) (JOSHUA) versus metastatic breast cancer (MBC) (CLEOPATRA).

[0239] Summary of results - Pertuzumab trough concentrations are lower in GC compared with MBC. Cyclic concentrations (i.e., days 7, 14) are consistent with expected MBC concentrations because clearance is linear at these higher concentrations. Trough concentrations for the 840 / 420 mg dose were approximately 37% lower compared to the CLEOPATRA study (Example 3), likely due to non-linear clearance (incomplete receptor saturation) at lower concentrations. - The 840 / 840 mg dose in GC produces similar trough concentrations as the 840 / 420 mg dose in MBC. - Covariates do not affect PK.

[0240] conclusion Based on pertuzumab PK in GC, the 840 / 840 mg dose is used for the treatment of gastric cancer. This dose is expected to maintain trough concentrations above target levels of >20 μg / mL in 90% of patients, resulting in trough levels similar to those observed in MBC. [Example]

[0241] A phase III study evaluating pertuzumab in combination with trastuzumab and chemotherapy in patients with HER2-positive advanced gastric cancer This is a Phase III, multicenter, randomized, open-label clinical study designed to evaluate the efficacy and safety of pertuzumab in combination with trastuzumab and chemotherapy in patients with HER2-positive locally advanced or metastatic gastric cancer.

[0242] Patients in the treatment arm will receive trastuzumab, cisplatin, and capecitabine and / or 5-fluorouracil. In the other arms, patients will receive either placebo or pertuzumab.

[0243] Treatment regimen: Pertuzumab: 840mg for cycles 1-6. Trastuzumab 8 mg / kg loading dose, followed by 6 mg / kg every 3 weeks Capecitabine 1000 mg / m twice daily on days 1 through 14 every 3 weeks 2 , 6 cycles 5-fluorouracil, 800 mg / m on days 1 through 5 every 3 weeks 2 / day continuous IV infusion, 6 cycles Cisplatin 800 mg / m every 3 weeks 2 , 6 cycles

[0244] Primary endpoint: Overall survival (OS)

[0245] Secondary endpoints: Progression-free survival (PFS), time to disease progression (TTP), objective response rate (ORR), clinical benefit rate, duration of response, quality of life (QoL), safety, pain intensity, analgesic consumption, weight change, and pharmacokinetics.

[0246] Main patient selection criteria Inclusion Criteria: Adenocarcinoma of the stomach or GEJ Inoperable locally advanced and / or metastatic disease Measurable (RECIST) or non-measurable evaluable disease HER2-positive tumors: IHC 2+ or 3+ and / or ISH+ Adequate organ function and ECOG performance status ≤ 2 Written informed consent

[0247] Exclusion criteria Prior adjuvant chemotherapy within 6 months Chemotherapy for advanced disease Congestive heart failure or baseline LVEF < 50% Creatinine clearance <60 mL / min

[0248] The treatment methods described herein, including administration of pertuzumab, trastuzumab, and chemotherapeutic agent(s) (e.g., cisplatin and capecitabine), are expected to meet the primary endpoint (OS). In particular, the treatment methods described herein are expected to be therapeutically effective in treated gastric cancer patients, e.g., by prolonging survival, e.g., overall survival (OS) and / or progression-free survival (PFS) and / or time to progression (TTP), and / or increasing objective response rate (ORR), compared to treatment with trastuzumab and a chemotherapeutic agent alone. [Example]

[0249] Results of a Phase III, Randomized, Double-Blind, Placebo-Controlled Registration Trial (CLEOPATRA) to Evaluate the Efficacy and Safety of Placebo, Trastuzumab, and Docetaxel Versus Pertuzumab, Trastuzumab, and Docetaxel in Patients with Previously Untreated HER2-Positive Metastatic Breast Cancer Protocols for evaluating pertuzumab in HER2-positive metastatic breast cancer can be found at http: / / clinicaltrials.gov / ct2 / show / NCT00567190 and US2009 / 0137387 and WO2009 / 154651.

[0250] This example presents clinical data from a Phase III, randomized, double-blind, placebo-controlled trial in patients with HER2-positive MBC who had not previously been treated with chemotherapy or biologic therapy for metastatic disease. Patients were randomized 1:1 to receive placebo, trastuzumab, and docetaxel or pertuzumab, trastuzumab, and docetaxel. The primary endpoint was progression-free survival (PFS) based on tumor assessment. PFS was defined as the time from randomization to first documented radiographic progression (PD) according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.0 (Therasser et al., Cancer Inst. 92:205-16 (2000)), or death from any cause if the patient died within 18 weeks of their last tumor assessment. Secondary endpoints included overall survival (OS), investigator-assessed PFS, objective response rate (ORR), and safety.

[0251] Patients: Eligible patients had centrally confirmed, locally recurrent, unresectable, or metastatic breast cancer or de novo Stage IV disease that was HER2-positive (defined as immunohistochemistry (IHC) 3+ and / or fluorescence in situ hybridization (FISH) amplification ratio ≥ 2.0) (Carlson et al. J Natl Compr Canc Netw 4 Suppl 3:S1-22 (2006)). Patients were 18 years of age or older, had a baseline left ventricular ejection fraction (LVEF) ≥ 50% (determined by echocardiogram or multi-gated acquisition), and an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1. Patients may have received one prior hormonal therapy for MBC prior to randomization, or may have received neoadjuvant or adjuvant systemic breast cancer therapy including trastuzumab and / or taxanes, provided they experienced a disease-free interval of 12 months or more between the completion of neoadjuvant or adjuvant therapy and the diagnosis of metastatic disease. Exclusion criteria included the following: therapy for MBC (other than those listed above); central nervous system metastases; 2 history of exposure to > 1000 cumulative doses of doxorubicin or its equivalent; history of a decrease in LVEF to < 50% during or after previous trastuzumab therapy; current uncontrolled hypertension; history of cardiac dysfunction; bone marrow, renal, or hepatic dysfunction; currently known HIV, HBV, or HCV infection; pregnancy; lactation; and refusal to use non-hormonal contraception.

[0252] Procedure: Patients received trastuzumab at a loading dose of 8 mg / kg followed by a maintenance dose of 6 mg / kg every 3 weeks until investigator-assessed radiographic or clinical PD or unmanageable toxicity. Docetaxel was administered at a starting dose of 75 mg / m 2 and 100 mg / m if tolerated. 2 Per protocol, investigators continued to administer 55 mg / m every 3 weeks to address tolerability. 2 Up to 25% or 75 mg / m 2The dose could be reduced up to 100 mg (if the patient had a dose increase). Patients were recommended to receive at least six cycles of docetaxel. Pertuzumab or placebo was administered at a flat loading dose of 840 mg, followed by 420 mg every three weeks until investigator-assessed radiographic or clinical PD or unmanageable toxicity. If chemotherapy was discontinued due to cumulative toxicity, antibody therapy was continued until PD, unacceptable toxicity, or withdrawal of consent. All drugs were administered intravenously.

[0253] Assessments: PFS was assessed by standard RECIST-approved methods every 9 weeks by each center and by the IRF until IRF-assessed PD. LVEF assessments were performed at baseline, every 9 weeks during treatment, at treatment discontinuation, every 6 months for the first year after treatment discontinuation, and then annually for up to 3 years during follow-up. Laboratory parameters and ECOG status were assessed every cycle. Adverse events (AEs) were monitored serially and graded according to NCI-CTCAE version 3.0. All cardiac events and serious adverse events (SAEs) ongoing at the time of treatment discontinuation were followed until resolution or stabilization, up to 1 year after the last dose. Treatment-emergent cardiac events and treatment-related SAEs

[0254] result Study Population: A total of 808 patients were enrolled and randomized to receive placebo, trastuzumab, and docetaxel (n=406) or pertuzumab, trastuzumab, and docetaxel (n=402) (Figure 7). Baseline characteristics were similar between treatment arms (Table 1). TIFF2026028254000001.tif244170

[0255] Progression-Free Survival: Treatment with pertuzumab + trastuzumab + docetaxel significantly improved PFS-IRF stratified by prior treatment status and region compared with placebo + trastuzumab + docetaxel (HR = 0.62; 95% CI 0.51-0.75; p < 0.0001) (Figure 8). The median PFS-IRF was extended by 6.1 months with pertuzumab + trastuzumab + docetaxel, from 12.4 months with placebo + trastuzumab + docetaxel to 18.5 months. The PFS benefit of pertuzumab + trastuzumab + docetaxel treatment was observed across all predefined subgroups (Figure 9).

[0256] Investigator-assessed PFS closely matched the PFS-IRF. Median investigator-assessed PFS was 12.4 months for placebo plus trastuzumab plus docetaxel and 18.5 months for pertuzumab plus trastuzumab plus docetaxel (HR=0.65; 95% CI 0.54-0.78; p<0.0001).

[0257] Key secondary efficacy endpoint: The interim analysis of OS was performed when 43% (n = 165) of events planned for the final OS analysis had occurred. More deaths occurred in the placebo plus trastuzumab plus docetaxel arm (n = 96; 23.6%) than in the pertuzumab plus trastuzumab plus docetaxel arm (n = 69; 17.2%) (Figure 10). The HR for OS (0.64; 95% CI 0.47-0.88; p = 0.0053) was not statistically significant because it did not meet the O'Brien Fleming stopping boundary of the Lan DeMets α-spending function (HR ≤ 0.603, p ≤ 0.0012). However, the data showed a strong trend suggesting a survival benefit in favor of pertuzumab plus trastuzumab plus docetaxel. At data cutoff, patients in both treatment arms were followed for a median OS of 19.3 months (Kaplan-Meier estimate). ORRs were 69.3% and 80.2% in the placebo plus trastuzumab plus docetaxel arm and the pertuzumab plus trastuzumab plus docetaxel arm, respectively. The difference in response rate between treatment arms was 10.8% (95% CI 4.2-17.5; p=0.0011) (Table 2). TIFF2026028254000002.tif71170

[0258] Treatment Exposure: The median number of dosing cycles per patient was 15 and 18, and the median time on treatment was estimated to be 11.8 and 18.1 months for placebo + trastuzumab + docetaxel and pertuzumab + trastuzumab + docetaxel, respectively. No dose reductions were permitted for placebo, pertuzumab, or trastuzumab. Patients received a median of 8 cycles of docetaxel in each arm. Based on the safety population, 61 (15.4%) patients in the placebo + trastuzumab + docetaxel arm received 100 mg / m or ... 2The docetaxel dose was increased in the placebo + trastuzumab + docetaxel arm. The median docetaxel dose intensity was 24.8 mg / m 2 / week and 24.6 mg / m in the pertuzumab + trastuzumab + docetaxel arm. 2 The reasons for permanent discontinuation of all study treatments are shown in Figure 7.

[0259] Tolerability and Cardiac Safety: AE profiles during treatment were generally balanced between treatment arms (Table 3). The incidence of the following AEs (all grades) was >5% higher with pertuzumab + trastuzumab + docetaxel than with docetaxel: diarrhea, rash, mucosal inflammation, febrile neutropenia, and dry skin. TIFF2026028254000003.tif163170

[0260] The incidence of the following AEs of grade 3 or higher was >2% higher with pertuzumab + trastuzumab + docetaxel: neutropenia, febrile neutropenia, and diarrhea (Table 3). The incidence of febrile neutropenia of grade 3 or higher in patients from Asia was 12% in the placebo + trastuzumab + docetaxel arm and 26% in the pertuzumab + trastuzumab + docetaxel arm; in all other geographic regions, the incidence was 10% or less in both arms.

[0261] LVSD (all grades) was reported more frequently in the placebo + trastuzumab + docetaxel arm compared with the pertuzumab + trastuzumab + docetaxel arm (8.3% and 4.4%, respectively). Grade 3 or higher LVSD was reported in 2.8% of patients receiving placebo + trastuzumab + docetaxel and 1.2% of patients receiving pertuzumab + trastuzumab + docetaxel. Among patients who underwent post-baseline LVEF assessment, a decline in LVEF of 10 percentage points or more from baseline to less than 50% at any stage during treatment was reported in 6.6% and 3.8% of patients in the placebo + trastuzumab + docetaxel arm and the pertuzumab + trastuzumab + docetaxel arm, respectively.

[0262] In the safety population, the majority of deaths in both treatment arms were due to PD (81 (20.4%) in the placebo arm and 57 (14.0%) in the pertuzumab arm). Deaths due to causes other than PD were generally balanced, and similar numbers of patients died from AEs (10 (2.5%) in the placebo arm and 8 (2.0%) in the pertuzumab arm), with infection being the most common cause of death due to an AE.

[0263] Consideration These data demonstrate that the anti-HER2 monoclonal antibodies pertuzumab and trastuzumab, in combination with docetaxel, extend PFS in patients with HER2-positive MBC in the first-line setting. Treatment with pertuzumab + trastuzumab + docetaxel exceeded expectations by resulting in a statistically significant reduction in PFS risk (HR=0.62) and an improvement in median PFS of 6.1 months.

[0264] The combination was well tolerated, and pertuzumab did not increase the incidence of symptomatic or asymptomatic cardiac dysfunction. Prior to the data described here, treatment with two HER2 antibodies was expected to exacerbate cardiac toxicity. However, these data indicate that this was not the case based on the studies herein to assess cardiac toxicity: incidence of symptomatic left ventricular systolic dysfunction (LVSD), including congestive heart failure (CHF), and reduced left ventricular ejection fraction (LVEF).

[0265] Pertuzumab-related AEs, including skin rash, mucosal inflammation, and dry skin, were mostly mild. Treatment with pertuzumab, trastuzumab, and docetaxel was associated with increased rates of grade 3 or higher diarrhea and febrile neutropenia. The control arm of CLEOPATRA had a PFS similar to that of a previous randomized trial (Marty et al., J Clin Oncol 23:4265-74 (2005)), which showed that the combination of trastuzumab and docetaxel in HER2-positive MBC had a median PFS of 11.7 months.

[0266] Without being bound to any one theory, these data demonstrate that targeting HER2-positive tumors with two anti-HER2 monoclonal antibodies with complementary mechanisms of action results in more comprehensive HER2 blockade and highlight the clinical importance of preventing the ligand-dependent formation of HER2 dimers to optimally suppress HER2 signaling. This study demonstrates that HER2 blockade with the combination of trastuzumab and pertuzumab improves outcomes for patients with advanced HER2-positive disease in the first-line setting. These data are important in supporting the first approved use of a HER2 dimerization inhibitor for the treatment of patients with HER2-positive cancers. [Example]

[0267] Products containing pertuzumab The Phase III clinical data of Example 3 was used to develop an article of manufacture comprising a vial (e.g., a single-dose vial) containing pertuzumab and a package insert providing information regarding its safety and / or efficacy, as well as a method of manufacturing an article of manufacture comprising packaging pertuzumab in a vial (e.g., a single-dose vial) together with a package insert providing prescribing information for pertuzumab, as described herein below.

[0268] Pertuzumab is a sterile, clear to slightly opalescent, colorless to pale yellow liquid for IV infusion. Each single-use vial contains 420 mg of pertuzumab at a concentration of 30 mg / mL in 20 mM L-histidine acetate (pH 6.0), 120 mM sucrose, and 0.02% polysorbate 20.

[0269] Pertuzumab is supplied in single-dose vials containing a ready-to-inject, preservative-free liquid concentrate at a concentration of 30 mg / mL. Each vial of pertuzumab drug product contains a total of 420 mg of pertuzumab. Vials should be stored in a refrigerator at 2°C to 8°C (36°F to 46°F) until time of use. Vials should be kept in the outer carton to protect from light.

[0270] TIFF2026028254000004.tif40170

[0271] 1. Efficacy and usage Pertuzumab, in combination with trastuzumab and docetaxel, is indicated for the treatment of patients with HER2-positive metastatic breast cancer who have not received prior anti-HER2 therapy or chemotherapy for metastatic disease.

[0272] 2. Dosage and Administration 2.1 Recommended Dosage and Schedule Pertuzumab is administered at a loading dose of 840 mg as a 60-minute intravenous infusion, followed by 420 mg every 3 weeks as a 30- to 60-minute intravenous infusion. When administered in combination with pertuzumab, the recommended loading dose of trastuzumab is 8 mg / kg administered as a 90-minute intravenous infusion, followed by 6 mg / kg every 3 weeks as a 30- to 90-minute intravenous infusion. When administered in combination with pertuzumab, the recommended loading dose of docetaxel is 75 mg / m 2 is administered as an intravenous infusion. If the initial dose is well tolerated, the dose can be increased to 100 mg / m 2 The dose can be increased to .

[0273] 2.2 Dose Modification For delayed or missed doses, if there is less than 6 weeks between two sequential infusions, a 420 mg dose of pertuzumab should be administered. Do not wait until the next scheduled dose. If there is more than 6 weeks between two sequential infusions, the initial dose of 840 mg pertuzumab should be re-administered as a 60-minute intravenous infusion, followed by 420 mg doses administered intravenously over 30 to 60 minutes every 3 weeks thereafter. If a patient experiences an infusion-related reaction, the pertuzumab infusion rate may be slowed or interrupted. If a patient experiences a serious hypersensitivity reaction, the infusion should be stopped immediately [see Warnings and Precautions (5.2)].

[0274] Left ventricular ejection fraction (LVEF): Pertuzumab and trastuzumab should be withheld for at least 3 weeks if any of the following occurs: A decrease in LVEF to less than 40%, or LVEF 40%-45% with an absolute decrease of 10% or more from the baseline value [see Warnings and Precautions (5.2)]

[0275] Pertuzumab may be restarted when LVEF recovers to >45% or to 40%-45% with an absolute decrease of less than 10% from pretreatment values.

[0276] If LVEF has not improved or has further deteriorated after repeat assessment within approximately 3 weeks, discontinuation of pertuzumab and trastuzumab should be strongly considered unless the benefit to the individual patient is judged to outweigh the risk [see Warnings and Precautions (5.2)]. If trastuzumab treatment is withheld or discontinued, pertuzumab should be withheld or discontinued. If docetaxel is discontinued, treatment with pertuzumab and trastuzumab may be continued. Dose reductions are not recommended for pertuzumab. For docetaxel dose modifications, see the docetaxel prescribing information.

[0277] 2.3 Preparation for Administration Administer as an intravenous infusion only. Do not administer as an intravenous injection or bolus. Do not mix pertuzumab with other drugs.

[0278] Preparation: Solutions for infusion are prepared using aseptic technique as follows: Parenteral drug products should be visually inspected for particulates and discoloration prior to administration. Withdraw the appropriate volume of pertuzumab solution from the vial(s). Dilute in a PVC or non-PVC polyolefin infusion bag containing 250 mL of 0.9% sodium chloride solution. Mix the diluted solution gently by inversion, do not shake. Administer immediately after preparation. If not used immediately, the diluted infusion solution may be stored at 2°C to 8°C for up to 24 hours. Dilute only with 0.9% sodium chloride injection. Do not use dextrose (5%) solution.

[0279] 3. Dosage form and strength Pertuzumab 420 mg / 14 mL (30 mg / mL) in a single-use vial

[0280] 4 Contraindications none

[0281] 5. Warnings and Precautions 5.1 Embryo-fetal toxicity Pertuzumab can cause fetal harm when administered to pregnant women. Treatment of pregnant cynomolgus monkeys with pertuzumab resulted in oligohydramnios, delayed fetal renal development, and embryo-fetal death. If pertuzumab is administered during pregnancy or if a patient becomes pregnant while receiving this drug, the patient should be informed of the potential hazard to the fetus [see Use in Specific Populations (8.1)]. Confirm pregnancy status before initiating pertuzumab. Advise patients about the risk of embryo-fetal death and congenital anomalies and the need for contraception during and after treatment. Advise patients to contact their health care provider immediately if they suspect they are pregnant. If pertuzumab is administered during pregnancy or if a patient becomes pregnant while receiving pertuzumab, report the exposure immediately to the Genentech Adverse Event Line (1-888-835-2555). Encourage women potentially exposed during pregnancy to enroll in the MotHER Pregnancy Registry by contacting 1-800-690-6720 [see Patient Counseling Information (17)]. Monitor patients who become pregnant during pertuzumab therapy for oligohydramnios. If oligohydramnios occurs, perform fetal testing appropriate for gestational age and consistent with local standard of care. The effectiveness of intravenous hydration in managing oligohydramnios due to pertuzumab exposure is unknown.

[0282] 5.2 Left ventricular dysfunction LVEF decline has been reported with drugs that block HER2 activity, including pertuzumab. In a randomized trial, pertuzumab in combination with trastuzumab and docetaxel was not associated with an increased incidence of symptomatic left ventricular systolic dysfunction (LVSD) or a decline in LVEF compared with placebo in combination with trastuzumab and docetaxel [see Clinical Studies (14.1)]. LVSD occurred in 4.4% of patients in the pertuzumab group and 8.3% in the placebo group. Symptomatic left ventricular systolic dysfunction (congestive heart failure) occurred in 1.0% of patients in the pertuzumab group and 1.8% in the placebo group [see Adverse Reactions (6.1)]. Patients with prior anthracycline therapy or prior chest radiation therapy may be at higher risk for LVEF decline. Pertuzumab has not been studied in patients with the following: a pretreatment LVEF of 50% or less, a history of CHF, a decrease in LVEF to <50% during previous trastuzumab therapy, or conditions that may impair left ventricular function, such as uncontrolled hypertension, recent myocardial infarction, serious cardiac arrhythmias requiring treatment, or a 360 mg / m 2 Cumulative exposure to doxorubicin or its equivalent prior to anthracycline therapy should be >100 mg / kg. LVEF assessment should be performed prior to initiating pertuzumab and periodically (e.g., every 3 months) during treatment to ensure LVEF is within the normal range for the local institution. If LVEF is <40% or LVEF is 40-45% with an absolute decrease of ≥10% from baseline, withhold pertuzumab and trastuzumab and repeat LVEF assessment in approximately 3 weeks. If LVEF does not improve or further declines, discontinue pertuzumab and trastuzumab unless the benefit outweighs the risk to the individual patient [see Dosage and Administration (2.2)].

[0283] 5.3 Infusion-related reactions, hypersensitivity reactions / anaphylaxis Pertuzumab has been associated with infusion and hypersensitivity reactions [see Adverse Reactions (6.1)]. Infusion reactions were defined in randomized trials as any event described as hypersensitivity, anaphylactic reaction, acute infusion reaction, or cytokine release syndrome occurring during or on the same day as an infusion. The first dose of pertuzumab was administered the day before trastuzumab and docetaxel to allow for investigation of pertuzumab-related reactions. On Day 1, when pertuzumab alone was administered, the overall incidence of infusion reactions was 13.0% in the pertuzumab-treated group and 9.8% in the placebo-treated group. Grade 3 or 4 reactions occurred in less than 1% of patients. The most common infusion reactions (≥1.0%) were fever, chills, fatigue, headache, asthenia, hyperirritability, and vomiting. During the second cycle, in which all drugs were administered on the same day, the most common infusion reactions (≥1.0%) in the pertuzumab group were fatigue, dysgeusia, hypersensitivity, myalgia, and vomiting. In this randomized trial, the overall incidence of hypersensitivity / anaphylactic reactions was 10.8% in the pertuzumab group and 9.1% in the placebo group. The incidence of grade 3-4 hypersensitivity / anaphylactic reactions according to the National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI CTCAE) (version 3) was 2% in the pertuzumab group and 2.5% in the placebo group. Overall, four patients in the pertuzumab group and two in the placebo group experienced anaphylaxis. Patients should be closely observed for 60 minutes after the first pertuzumab infusion and for 30 minutes after each subsequent infusion. If a significant infusion-related reaction occurs, slow or interrupt the infusion and administer appropriate medical treatment. Monitor patients closely until signs and symptoms have completely resolved. Consider permanent discontinuation in patients experiencing severe infusion reactions [see Dosage and Administration (2.2)].

[0284] 5.4 HER2 testing Only patients who overexpress the HER2 protein have been studied and shown to benefit. Therefore, detection of HER2 protein overexpression is necessary to select appropriate patients for pertuzumab therapy [see Indications and Dosage (1) and Clinical Studies (14)]. In randomized trials, breast cancer patients were required to have evidence of HER2 overexpression, defined as IHC 3+ using the Dako HERCEPTEST® or a FISH amplification ratio of 2.0 or greater using the Dako HER2 FISH PHARMDX™ test kit. Limited data are available for patients with breast cancers that are FISH-positive but do not demonstrate protein overexpression by IHC. Assessment of HER2 status should be performed in laboratories skilled in the specific techniques utilized. Improper assay performance, such as the use of suboptimally fixed tissue, failure to utilize specified reagents, deviations from specific assay instructions, and failure to include appropriate controls in assay validation, may result in unreliable results.

[0285] 6 Adverse Reactions The following adverse reactions are described in more detail in other sections of the label: Embryo-fetal toxicity [see Warnings and Precautions (5.1)] Left ventricular dysfunction [see Warnings and Precautions (5.2)] Infusion-related reactions, hypersensitivity reactions / anaphylaxis [see Warnings and Precautions (5.3)]

[0286] 6.1 Clinical Trial Experience Because clinical trials are conducted under a wide variety of conditions, the adverse reaction rates observed in clinical trials of a drug cannot be directly compared to rates in clinical trials of another drug and may not reflect the rates observed in clinical practice. In clinical trials, pertuzumab has been evaluated in over 1,400 patients for a variety of malignancies, although treatment with pertuzumab has mostly been in combination with other antineoplastic agents.

[0287] The adverse reactions listed in Table 4 were observed in 804 patients with HER2-positive metastatic breast cancer treated in the randomized trial. Patients were randomized to receive either pertuzumab in combination with trastuzumab and docetaxel or placebo in combination with trastuzumab and docetaxel. The median duration of study treatment was 18.1 months for patients in the pertuzumab group and 11.8 months for patients in the placebo group. No dose adjustments were permitted for pertuzumab or trastuzumab. The rate of adverse events leading to permanent discontinuation of all study treatments was 6.1% for patients in the pertuzumab group and 5.3% for patients in the placebo group. Adverse events leading to discontinuation of docetaxel alone occurred in 23.6% of patients in the pertuzumab group and 23.2% of patients in the placebo group. Table 4 reports adverse reactions that occurred in at least 10% of patients in the pertuzumab treatment group. The most common adverse reactions (30%) with pertuzumab in combination with trastuzumab and docetaxel were diarrhea, alopecia, neutropenia, nausea, fatigue, rash, and peripheral neuropathy.

[0288] The most common NCI CTCAE (version 3) grade 3-4 adverse reactions (>2%) were neutropenia, febrile neutropenia, leukopenia, diarrhea, peripheral neuropathy, anemia, asthenia, and fatigue. A higher incidence of febrile neutropenia was observed in Asian patients compared with patients of other ethnicities and other geographic regions in both treatment arms. Among Asian patients, the incidence of febrile neutropenia was higher in the pertuzumab-treated group (26%) compared with the placebo-treated group (12%). TIFF2026028254000005.tif203170TIFF2026028254000006.tif77170

[0289] The following clinically relevant adverse reactions were reported in less than 10% of patients in the pertuzumab-treated groups: Skin and subcutaneous tissue disorders: paronychia (7.1% in the pertuzumab group vs. 3.5% in the placebo group); respiratory, thoracic, and mediastinal disorders: pleural effusion (5.2% in the pertuzumab group vs. 5.8% in the placebo group); cardiac disorders: left ventricular dysfunction (4.4% in the pertuzumab group vs. 8.3% in the placebo group), including symptomatic left ventricular systolic dysfunction (CHF) (1.0% in the pertuzumab group vs. 1.8% in the placebo group); immune system disorders: hypersensitivity (10.1% in the pertuzumab group vs. 8.6% in the placebo group).

[0290] Adverse reactions reported in patients receiving pertuzumab and trastuzumab after discontinuation of docetaxel In randomized trials, adverse reactions were reported less frequently after discontinuation of docetaxel treatment. All adverse reactions in the pertuzumab and trastuzumab treatment groups, except for diarrhea (19.1%), upper respiratory tract infection (12.8%), rash (11.7%), headache (11.4%), and fatigue (11.1%), occurred in less than 10% of patients.

[0291] 6.2 Immunogenicity As with all therapeutic proteins, there is a possibility of an immune response to pertuzumab. Patients in the randomized trial were tested for antibodies to pertuzumab at multiple time points. Approximately 2.8% (11 / 386) of patients in the pertuzumab treatment group and 6.2% (23 / 372) of patients in the placebo treatment group tested positive for anti-pertuzumab antibodies. Of these 34 patients, none experienced an anaphylactic / hypersensitivity reaction clearly related to anti-therapeutic antibodies (ATA). Pertuzumab, present in patient serum at expected levels at the time of ATA sample collection, may interfere with the assay's ability to detect anti-pertuzumab antibodies. In addition, the assay may have detected antibodies to trastuzumab. As a result, the data may not accurately reflect the true incidence of anti-pertuzumab antibodies. Immunogenicity data are highly dependent on the sensitivity and specificity of the testing method used. Furthermore, the incidence of positive results observed in a test method can be influenced by several factors, such as sample handling, timing of sample collection, drug interference, concomitant medications, and underlying diseases. For these reasons, comparison of the incidence of antibodies to pertuzumab with that to other products can be misleading.

[0292] 7. Drug Interactions No drug-drug interactions were observed between pertuzumab and trastuzumab or between pertuzumab and docetaxel.

[0293] 8 Use in specific populations 8.1 Pregnancy Pregnancy Category D Risk Overview There are no adequate, well-controlled studies of pertuzumab in pregnant women. Findings from animal studies indicate that pertuzumab may cause fetal harm when administered to pregnant women. The effects of pertuzumab are likely to be present during all trimesters of pregnancy. Administration of pertuzumab to pregnant cynomolgus monkeys has been shown to maxClinically significant exposures at 2.5 to 20 times the recommended human dose based on the FDA guidelines have resulted in oligohydramnios, delayed fetal renal development, and embryofetal death. If pertuzumab is administered during pregnancy or if a patient becomes pregnant while receiving pertuzumab, the patient should be informed of the potential risk to the fetus. If pertuzumab is administered during pregnancy or if a patient becomes pregnant while receiving pertuzumab, the exposure should be reported immediately to the Genentech Adverse Event Line (1-888-835-2555). Women potentially exposed during pregnancy should be encouraged to contact 1-800-690-6720 to enroll in the MotHER Pregnancy Registry [see Patient Counseling Information (17)].

[0294] Animal Data Reproductive toxicity studies were conducted in cynomolgus monkeys. Pregnant monkeys were treated with a pertuzumab loading dose of 30–150 mg / kg on gestational day 19 (GD19), followed by doses of 10–100 mg / kg every other week. These dose levels were max This resulted in clinically relevant exposures 2.5 to 20 times the recommended human dose based on the FDA guidelines. Intravenous administration of pertuzumab between GD19 and GD50 (during organogenesis) was embryotoxic, with a dose-dependent increase in embryofetal loss between GD70 and GD25. The incidence of embryofetal loss was 33, 50, and 85% in dams treated with pertuzumab doses of 10, 30, and 100 mg / kg every other week, respectively (C max (2.5-20 times the recommended human dose based on the FDA guidelines.) At cesarean section on GD100, oligohydramnios, reduced relative lung and kidney weights, and microscopic evidence of renal hypoplasia consistent with renal developmental delay were observed in all pertuzumab dose groups. Pertuzumab exposure was reported in liveborn infants from all treatment groups, with exposures ranging from 29% to 40% of maternal serum levels on GD100.

[0295] 8.3 Nursing mothers It is unknown whether pertuzumab is excreted in human milk, but human IgG is excreted in human milk. Because many drugs are excreted in human milk and because pertuzumab can cause serious adverse reactions in nursing infants, the elimination half-life of pertuzumab and the importance of the drug to the mother should be considered when deciding whether to discontinue nursing or the drug [see Warnings and Precautions (5.1) and Clinical Pharmacology (12.3)].

[0296] 8.4 Pediatric Use The safety and effectiveness of pertuzumab in pediatric patients have not been established.

[0297] 8.5 Elderly Use Of 402 patients who received pertuzumab in randomized trials, 60 (15%) patients were 65 years of age or older and 5 patients were 75 years of age or older. No overall differences in the efficacy or safety of pertuzumab were observed between these patients and patients under 65 years of age. In a population pharmacokinetic analysis, no significant differences in the pharmacokinetics of pertuzumab were observed between patients under 65 years of age (n=306) and patients 65 years of age or older (n=175).

[0298] 8.6 Females of reproductive potential Pertuzumab may cause embryo-fetal harm when administered during pregnancy. Counseling patients regarding pregnancy prevention and planning. Advise females of reproductive potential to use effective contraception during pertuzumab treatment and for 6 months after the final dose of pertuzumab. If pertuzumab is administered during pregnancy or if a patient becomes pregnant while receiving pertuzumab, report the exposure immediately to the Genentech Adverse Event Line (1-888-835-2555). Encourage women potentially exposed during pregnancy to enroll in the MotHER Pregnancy Registry by contacting 1-800-690-6720 [see Patient Counseling Information (17)].

[0299] 8.7 Renal dysfunction No pertuzumab dose adjustment is necessary in patients with mild (creatinine clearance [CLcr] 60-90 mL / min) or moderate (CLcr 30-60 mL / min) renal impairment. Dose adjustment is not recommended for patients with severe renal impairment (CLcr <30 mL / min) due to limited availability of pharmacokinetic data [see Clinical Pharmacology (12.3)].

[0300] 8.8 Liver dysfunction No clinical studies have been conducted to evaluate the effects of pertuzumab pharmacokinetics in patients with hepatic impairment.

[0301] 10 Overdose To date, no drug overdose has been reported with pertuzumab.

[0302] 11. Description Pertuzumab is a recombinant humanized monoclonal antibody that targets the extracellular dimerization domain (subdomain II) of the human epidermal growth factor receptor 2 protein (HER2). Pertuzumab is produced by recombinant DNA technology in mammalian cell (Chinese hamster ovary) cultures containing the antibiotic gentamicin. Gentamicin is undetectable in the final product. Pertuzumab has an estimated molecular weight of 148 kDa. Pertuzumab is a sterile, clear to slightly opalescent, colorless to light brown liquid for intravenous infusion. Each single-use vial contains 420 mg of pertuzumab at a concentration of 30 mg / mL in 20 mM L-histidine acetate (pH 6.0), 120 mM sucrose, and 0.02% polysorbate 20.

[0303] 12 Clinical Pharmacology 12.1 Mechanism of Action Pertuzumab targets the extracellular dimerization domain (subdomain II) of the human epidermal growth factor receptor 2 (HER2) protein, thereby preventing ligand-dependent heterodimerization of HER2 with other HER family members, including EGFR, HER3, and HER4. Consequently, pertuzumab inhibits ligand-triggered intracellular signaling via two major signaling pathways: mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3K). Inhibition of these signaling pathways can lead to cell growth arrest and apoptosis, respectively. Additionally, pertuzumab mediates antibody-dependent cell-mediated cytotoxicity (ADCC). While pertuzumab alone inhibits the growth of human tumor cells, the combination of pertuzumab and trastuzumab significantly enhanced antitumor activity in HER2-overexpressing xenograft models.

[0304] 12.2 Pharmacokinetics Pertuzumab demonstrated linear pharmacokinetics over the dose range of 2 to 25 mg / kg. A population pharmacokinetic analysis including 481 patients revealed that the median clearance (CL) of pertuzumab was 0.24 L / day, and the median half-life was 18 days. Using an initial dose of 840 mg followed by a maintenance dose of 420 mg every 3 weeks, pertuzumab reached steady-state concentrations after the first maintenance dose. Population pharmacokinetic analysis suggested no pharmacokinetic differences based on age, sex, or ethnicity (Japanese vs. non-Japanese). Only baseline serum albumin level and lean body mass as covariates had a minimal effect on PK parameters. Therefore, dose adjustments based on body weight or baseline albumin level are not necessary. In a substudy of 37 patients from the randomized trial, no drug-drug interactions were observed between pertuzumab and trastuzumab or between pertuzumab and docetaxel. No renal impairment studies were conducted with pertuzumab alone. Population pharmacokinetic analysis showed that pertuzumab exposure in patients with mild (CLcr 60-90 mL / min, n=200) and moderate (CLcr 30-60 mL / min, n=71) renal impairment was similar to that in patients with normal renal function (CLcr >90 mL / min, n=200). No relationship between CLcr and pertuzumab exposure was observed across the observed CLcr range (27-244 mL / min).

[0305] 12.3 Cardiac Electrophysiology The effect of pertuzumab on the QTc interval at an initial dose of 840 mg followed by a maintenance dose of 420 mg every 3 weeks thereafter was evaluated in a subgroup of 20 patients with HER2-positive breast cancer in a randomized trial. According to the Fridericia correction method, large changes in the mean QT interval from placebo (i.e., greater than 20 ms) were not detected in this study. Small increases in the mean QTc interval (i.e., less than 10 ms) cannot be excluded due to limitations in the study design.

[0306] 13 Non-clinical toxicity 13.1 Carcinogenicity, Mutagenicity, and Impairment of Fertility No long-term studies have been conducted in animals to evaluate the carcinogenic potential of pertuzumab. No studies have been conducted to evaluate the mutagenic potential of pertuzumab. No specific fertility studies have been conducted in animals to evaluate the effects of pertuzumab. No adverse effects on male or female reproductive organs were observed in repeat-dose toxicity studies in cynomolgus monkeys of up to 6 months.

[0307] 14 Clinical Trials 14.1 Metastatic breast cancer The randomized trial was a multicenter, double-blind, placebo-controlled study of 808 patients with HER2-positive metastatic breast cancer. Breast tumor specimens were required to demonstrate HER2 overexpression, defined as IHC 3+ or a FISH amplification ratio of 2.0 as determined by a central laboratory. Patients were randomized 1:1 to receive placebo plus trastuzumab and docetaxel or pertuzumab plus trastuzumab and docetaxel. Randomization was stratified by prior treatment history (whether or not they had received prior adjuvant / neoadjuvant anti-HER2 therapy or chemotherapy) and geographic region (Europe, North America, South America, and Asia). Patients previously treated with adjuvant or neoadjuvant therapy were required to have a disease-free interval of more than 12 months before study enrollment. Pertuzumab was administered intravenously at a loading dose of 840 mg, followed by 420 mg every 3 weeks. Trastuzumab was administered intravenously at an initial dose of 8 mg / kg, followed by 6 mg / kg every 3 weeks thereafter. Patients were treated with pertuzumab and trastuzumab until progression, withdrawal of consent, or unacceptable toxicity. Docetaxel was administered at an initial dose of 75 mg / m 2 Docetaxel was administered intravenously every 3 weeks for at least 6 cycles at a dose of 100 mg / m² at the investigator's discretion, provided the initial dose was well tolerated. 2 I was able to increase it to.

[0308] The mean number of cycles of study treatment administered at the time of the primary analysis was 16.2 in the placebo group and 19.9 in the pertuzumab group.

[0309] The primary endpoint of the randomized trial was progression-free survival (PFS) assessed by an independent review facility (IRF). PFS was defined as the time from the date of randomization to the date of progression or death (all-cause death) if death occurred within 18 weeks of the last tumor assessment. Additional endpoints included overall survival (OS), PFS (investigator-assessed), objective response rate (ORR), and duration of response.

[0310] Patient demographic and baseline characteristics were balanced across treatment arms. The median age was 54 years (range, 22-89 years). 59% were white, 32% were Asian, and 4% were black. All but two patients were women. 17% of patients were enrolled in North America, 14% in South America, 38% in Europe, and 31% in Asia. Tumor prognostic characteristics, including hormone receptor status (48% positive, 50% negative), presence of visceral disease (78%), and non-visceral disease only (22%), were similar across study arms. Approximately one-half of patients had received prior adjuvant or neoadjuvant anti-HER2 therapy or chemotherapy (placebo 47%, pertuzumab 46%). Among patients with hormone receptor-positive tumors, 45% had received prior adjuvant hormone therapy, and 11% had received hormone therapy for metastatic disease. Eleven percent of patients had received prior adjuvant or neoadjuvant trastuzumab.

[0311] The randomized trial demonstrated a statistically significant improvement in IRF-assessed PFS in the pertuzumab arm compared to the placebo arm [hazard ratio (HR) = 0.62 (95% CI: 0.51, 0.75), p < 0.0001], with a 6.1-month increase in median PFS (median PFS 18.5 months in the pertuzumab arm vs. 12.4 months in the placebo arm) (see Figure 8). Investigator-assessed PFS results were comparable to those observed for IRF-assessed PFS. Consistent results were observed across several patient subgroups, including age (< 65 years or ≥ 65 years), race, geographic region, prior treatment with adjuvant / neoadjuvant anti-HER2 therapy or chemotherapy (yes or no), and prior treatment with adjuvant / neoadjuvant trastuzumab (yes or no). In the subgroup of patients with hormone receptor-negative disease (n = 408), the hazard ratio was 0.55 (95% CI: 0.42, 0.72). In the subgroup of patients with hormone receptor-positive disease (n = 388), the hazard ratio was 0.72 (95% CI: 0.55, 0.95). In the subgroup of patients with disease limited to nonvisceral metastases (n = 178), the hazard ratio was 0.96 (95% CI: 0.61, 1.52).

[0312] At the time of PFS analysis, 165 patients had died. More deaths occurred in the placebo group (23.6%) compared with the pertuzumab group (17.2%). For the interim OS analysis, results were not complete and the pre-specified stopping limit for statistical significance was not met. See Table 5 and Figure 10. TIFF2026028254000007.tif121170

[0313] 16. Supply method / storage and handling 16.1 Supply method Pertuzumab is supplied as a preservative-free 420 mg / 14 mL (30 mg / mL) single-use vial. NDC 50242 145 01. Vials should be stored in a refrigerator at 2°C to 8°C (36°F to 46°F) until time of use. Vials should be kept in the outer carton to protect from light. Do not freeze. Do not shake.

[0314] 17 Patient Counseling Information Advise pregnant women and women of reproductive potential that exposure to pertuzumab may result in harm to the fetus, including embryo-fetal death or congenital anomalies [see Warnings and Precautions (5.1) and Use in Specific Populations (8.1)].

[0315] Advise females of reproductive potential to use effective contraception during treatment with pertuzumab and for 6 months after the final dose of pertuzumab [see Warnings and Precautions (5.1) and Use in Specific Populations (8.6)].

[0316] Nursing mothers treated with pertuzumab should be advised to discontinue breastfeeding or discontinue pertuzumab, taking into account the importance of the drug to the mother [see Use in Specific Populations (8.3)].

[0317] Women exposed to pertuzumab during pregnancy should be encouraged to enroll in the MotHER Pregnancy Registry by contacting 1-800-690-6720 [see Warnings and Precautions (5.1) and Use in Specific Populations (8.1)].

[0318] Thus, the comprehensive Phase III safety and efficacy data for pertuzumab, as seen in Example 3, enables an article of manufacture in this example that can be used in methods to ensure the safe and effective use of pertuzumab to treat patients. [Example]

[0319] Early breast cancer treatment with pertuzumab Anthracyclines, commonly in combination with 5-FU and cyclophosphamide, play a central role in the management of breast cancer. Romond et al., NEJM 353(16):1673-1684 (2005) and Poole et al., NEJM 355(18):1851-1852 (2006).

[0320] Taxanes are also an integral part of standard regimens for treating breast cancer, either combined with anthracyclines in a regimen known as TAC (Martin et al., NEJM 352(22):2302-2313 (2005)) or used sequentially with anthracyclines in a regimen known as AC→T (Romond et al., supra; Joensuu et al., NEJM 354(8):809-820 (2006)).

[0321] Carboplatin is an active and well-tolerated chemotherapy agent, and breast cancer studies have shown clear efficacy in combination with taxanes and trastuzumab in a regimen known as TCH (Slamon et al. BCIRG 006. SABS (2007); Robert et al. J. Clin. Oncol. 24:2786-2792 (2006)). However, there are negative data in metastatic breast cancer (Forbes et al. BCIRG 007 Proc. Am. Soc. Clin. Oncol. Abstract No. LBA516 (2006)).

[0322] Previous neoadjuvant studies of pertuzumab (NeoSphere) evaluated it in combination with docetaxel and trastuzumab, but not in combination with anthracycline or carboplatin-based chemotherapy (Gianni et al., Cancer Research 70(24)(Suppl. 2) (December 2010)).

[0323] In this example, the following chemotherapy regimens were evaluated: TIFF2026028254000008.tif36170

[0324] The treatment arms of the study were as follows: Arm A Trastuzumab and pertuzumab are administered from the beginning of the chemotherapy regimen (i.e., given simultaneously with the anthracycline) in a course of 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) followed by a course of docetaxel (T) (FEC→T). 3 cycles were administered with 5-fluorouracil (500 mg / m 2 ), epirubicin (100 mg / m 2 ), followed by cyclophosphamide (600 mg / m 2 ), followed by three cycles of docetaxel in combination with trastuzumab (8 mg / kg with epirubicin on day 1 of initial treatment, then 6 mg / kg every 3 weeks) and pertuzumab (840 mg with FEC on day 1 of treatment, then 420 mg every 3 weeks). The starting dose of docetaxel was 75 mg / m in cycle 4 (first docetaxel cycle). 2 Then, if no dose-limiting toxicity occurs, 100 mg / m 2 All drugs are administered by the IV route. or Arm B In FEC→T, trastuzumab and pertuzumab are administered from the beginning of taxane treatment (i.e., after the anthracycline). 3 cycles were administered with 5-fluorouracil (500 mg / m 2 ), epirubicin (100 mg / m 2 ), followed by cyclophosphamide (600 mg / m 2 ), followed by three cycles of docetaxel in combination with trastuzumab (8 mg / kg on day 1 of initial docetaxel treatment, then 6 mg / kg every 3 weeks) and pertuzumab (840 mg on day 1 of docetaxel treatment, then 420 mg every 3 weeks). The starting dose of docetaxel was 75 mg / m in cycle 4 (first docetaxel cycle). 2Then, if no dose-limiting toxicity occurs, 100 mg / m 2 All drugs are administered by the IV route. or Arm C Taxane (docetaxel), carboplatin and trastuzumab (TCH) and pertuzumab (both antibodies administered from the start of chemotherapy). Carboplatin (AUC6 using the Calvert formula) on day 1, followed by docetaxel and trastuzumab (8 mg / kg on day 1 of initial treatment with carboplatin and docetaxel, then 6 mg / kg every 3 weeks) and pertuzumab (840 mg on day 1, then 420 mg every 3 weeks) for 6 cycles. The docetaxel dose was 75 mg / m for all cycles. 2 All drugs are administered by the IV route.

[0325] Regardless of whether they receive additional chemotherapy, all patients will receive trastuzumab every 3 weeks from the start of treatment for a total of 1 year (cycles 1 to 17 for patients in arms A and C and cycles 4 to 20 for patients in arm B).

[0326] Main purpose The primary objective was to assess all patients once they had received six cycles of neoadjuvant treatment, had undergone surgery and had all necessary samples taken, or had withdrawn from the study (whichever came first).

[0327] Secondary Objectives To preliminarily assess the activity associated with each regimen, as indicated by pathologic complete response rates. To assess the safety profile of each treatment regimen, including preoperative (neoadjuvant) and postoperative (adjuvant) treatment. For each treatment arm, overall survival, time to clinical response, time to response, disease-free survival, and progression-free survival will be examined. To examine biomarkers that may be associated with the primary and secondary efficacy endpoints according to each treatment arm. To examine the rate of breast-conserving surgery for all patients with T2-3 tumors for whom mastectomy was planned at diagnosis. A comprehensive assessment of the risks and benefits of each regimen will be performed.

[0328] Overview of study design This was a Phase II, multicenter, randomized, open-label study evaluating the tolerability and activity associated with trastuzumab and pertuzumab when used in addition to neoadjuvant anthracycline- or carboplatin-based chemotherapy regimens in patients with early-stage, greater than 2 cm in diameter, locally advanced, or inflammatory HER2-positive breast cancer (see Figure 11).

[0329] Six cycles of active chemotherapy were administered. However, if further treatment was deemed necessary for the patient after surgery, those who received FEC→T were offered CMF (cyclophosphamide, methotrexate, and 5-fluorouracil), and those who received TCH but were deemed to require further treatment were offered FEC (5-fluorouracil, epirubicin, and cyclophosphamide).

[0330] After completion of surgery (and postoperative chemotherapy, if necessary), patients received radiotherapy according to local clinical standards, and patients whose tumors were estrogen receptor positive received hormonal manipulation according to local clinical standards.

[0331] In summary, patients received at least six cycles of active chemotherapy and two antibodies, pertuzumab and trastuzumab, plus surgery and radiotherapy (according to local standards) plus any hormonal manipulations indicated (according to local standards), and continued trastuzumab for up to a total of one year.

[0332] Patients whose neoadjuvant study treatment was discontinued before surgery were managed according to local practice. Approximately 28 days after the last dose of study medication, patients were asked to undergo a final safety assessment (referred to as the Final Visit).

[0333] Study population overview Female patients aged 18 years or older with early-stage HER2-positive breast cancer with primary tumors larger than 2 cm and no metastases.

[0334] Inclusion criteria 1. Female patients with locally advanced, inflammatory, or early-stage, histologically confirmed unilateral invasive breast cancer. Initial evaluation of breast cancer should be performed by a physician experienced in breast cancer surgery. Patients with inflammatory breast cancer must be able to undergo core needle biopsy. 2. Primary tumor >2cm in diameter 3. Central laboratory confirmed HER2 positive breast cancer. Tumors must be HER2 3+ or FISH / CISH+ by IHC (FISH / CISH positivity is mandatory for HER2 2+ tumors). 4. Availability of FFPE tissue (buffered formalin fixation is acceptable) for central laboratory confirmation of HER2 eligibility (FFPE tumor tissue will be subsequently used for assessment of biomarker status). 5. Female patients, aged 18 years or older. 6. Baseline LVEF ≥ 55% (measured by echocardiography or MUGA). 7. Performance status ECOG 1 or less. 8. At least 4 weeks have passed since any unrelated major surgery and you have fully recovered.

[0335] Concomitant medications and treatments Acceptable treatments Concomitant treatment is any prescription drug, over-the-counter medication, herbal medicine, or radiation therapy used by the patient during the period beginning 7 days before the patient is recruited into the study and continuing throughout the study.

[0336] The following treatments are permitted during this study: 1. If the female patient or male partner is not surgically sterilized or does not meet the study definition of postmenopausal (12 months of amenorrhea), they must use an acceptable method of contraception. 2. H1 and H2 antagonists (e.g., diphenhydramine, cimetidine). 3. Pain medications (e.g., paracetamol / acetaminophen, meperidine, opioids) 4. Short-term use of corticosteroids to treat or prevent allergic or infusion reactions. 5. Antiemetics (approved prophylactic serotonin antagonists, benzodiazepines, ondansetron, etc.) 6. Medications for treating diarrhea (e.g., loperamide) 7. Colony-stimulating factors (e.g., G-CSF) 8. Estrogen receptor antagonists (e.g., tamoxifen) or aromatase inhibitors (e.g., anastrozole, exemestane) after completion of adjuvant chemotherapy according to local practice.

[0337] Excluded treatments During the treatment period of this study, the following therapies will be excluded: 9. Anticancer therapy other than that administered in this study, including cytotoxic chemotherapy, radiation therapy, immunotherapy (excluding adjuvant radiation therapy for breast cancer after completion of chemotherapy or additional adjuvant chemotherapy immediately after surgery, if deemed necessary), and biological anticancer therapy. 10. Any targeted therapy. 11. Treatment with steroids, other than thyroid hormone replacement therapy and short-term corticosteroids, to treat or prevent allergic or infusion reactions. 12. High-dose systemic corticosteroids. A high dose is considered greater than 20 mg of dexamethasone (or equivalent) per day for more than 7 consecutive days. 13. Any investigational drug other than that used in this study. 14. Initiation of herbal medications. Herbal medications initiated prior to study entry and continued throughout the study are permitted and must be reported on the appropriate eCRF. 15. Any oral, injected or implanted hormonal contraceptive method.

[0338] result The baseline characteristics of patients with HER2-positive early breast cancer are shown in Table 6 below. TIFF2026028254000009.tif172170

[0339] Safety data are shown in Figure 12 and Tables 7 and 8 below. TIFF2026028254000010.tif111170TIFF2026028254000011.tif171170

[0340] Efficacy data are shown in Figures 13 and 14 and Tables 9 and 10 below. TIFF2026028254000012.tif123170TIFF2026028254000013.tif79170

[0341] conclusion The results of this study show that the incidence of symptomatic and asymptomatic LVSD was low across all arms. - Concomitant administration of pertuzumab and trastuzumab with epirubicin resulted in similar cardiac tolerability compared with sequential administration or anthracycline-free regimens. Neutropenia, febrile neutropenia, leukopenia, and diarrhea were the most frequently reported adverse events (grade 3 or higher) across all arms. · The combination of pertuzumab and trastuzumab in the neoadjuvant setting resulted in high pathologic complete response (pCR) rates (57-66%), regardless of the chemotherapy chosen. TRYPHAENA supports the use of pertuzumab and trastuzumab plus anthracycline or carboplatin chemotherapy in the neoadjuvant and adjuvant settings for early-stage breast cancer. [Example]

[0342] Concurrent administration of pertuzumab and trastuzumab In the Phase III clinical trial, pertuzumab was administered to patients with HER2-positive metastatic breast cancer via intravenous (IV) infusion in a saline IV bag, followed by trastuzumab and the chemotherapy drug docetaxel, also administered via IV saline infusion. The pertuzumab and trastuzumab IV infusion processes each took approximately 60 to 90 minutes, with approximately 30 to 60 minutes of patient observation time following each drug infusion. A total visit for this treatment regimen per patient could take up to 7.5 hours. In recent years, as healthcare costs for both drugs and drug administration services have come under increased scrutiny, business practices that reduce time and increase the utilization of medical resources have become increasingly important in clinical and hospital settings. Patient care, compliance, and treatment efficiency are expected to increase by reducing the amount of time patients spend in the clinic during each treatment cycle.

[0343] As part of a Phase III pertuzumab clinical trial, pertuzumab and trastuzumab are administered sequentially to patients via intravenous (IV) infusion, i.e., one drug is administered after the other. Pertuzumab is administered at a fixed dose (420 mg maintenance dose, 840 mg loading dose), while trastuzumab is administered based on weight (6 mg / kg maintenance dose). To improve convenience and minimize patient time in the clinic, the feasibility of co-administering pertuzumab and trastuzumab in a single IV infusion bag made of polyolefin (PO) or polyvinyl chloride (PVC) containing 250 mL of 0.9% saline was evaluated. The individual monoclonal antibodies have been demonstrated to be stable in the infusion bags (PO and / or PVC) at 5°C and 30°C for 24 hours. In this study, the compatibility and stability of pertuzumab (420 mg and 840 mg) mixed with either 420 mg (6 mg / kg dose for a 70 kg patient) or 720 mg (6 mg / kg dose for a 120 kg patient) trastuzumab in IV bags was evaluated for up to 24 hours at 5° C. or 30° C. Samples of the controls (i.e., pertuzumab alone in IV bags, trastuzumab alone in IV bags) and monoclonal antibody (mAb) mixtures were evaluated using existing pertuzumab and trastuzumab analytical methods, including color, appearance and clarity (CAC), concentration and turbidity by UV-spec scan, particle analysis by HIAC-Royco, size exclusion chromatography (SEC), and ion exchange chromatography (IEC). Additionally, capillary zone electrophoresis (CZE), image capillary isoelectric focusing (iCIEF) and potency assays (pertuzumab antiproliferative assay only) were used to measure only the mixtures containing 1:1 pertuzumab:trastuzumab and their respective controls (pertuzumab 420 mg and trastuzumab 420 mg only) as representative cases.

[0344] The results showed that for the pertuzumab / trastuzumab mixture, no differences were observed by the assays between the time zero (T0) control and samples stored at either 5°C or 30°C for up to 24 hours. The physicochemical assays listed above were able to detect both molecules and minor variants in the drug mixture, although some overlap of monoclonal antibody species was observed chromatographically. Furthermore, the drug mixture, tested by pertuzumab-specific inhibition of cell proliferation assay, showed equivalent potency before and after storage. The results of this study demonstrated that the pertuzumab and trastuzumab mixture is physically and chemically stable in an IV infusion bag for up to 24 hours at 5°C or 30°C and can be used for clinical administration if necessary.

[0345] Dose I: 840 mg of pertuzumab / trastuzumab mixture (420 mg pertuzumab and 420 mg trastuzumab) Sample preparation: All procedures were performed aseptically under a laminar flow hood. For this study, three types of PO IV infusion bag samples containing drug combinations were prepared: 1) a mixture of pertuzumab 420 mg / trastuzumab 420 mg, 2) pertuzumab 420 mg alone, and 3) trastuzumab 420 mg alone. Pertuzumab alone and trastuzumab alone samples served as controls.

[0346] Trastuzumab was reconstituted with 20 mL of bacteriostatic water for injection (BWFI) and allowed to stand on the benchtop for approximately 15 minutes before use. To prepare the pertuzumab / trastuzumab sample dose, 14 mL (420 mg) of pertuzumab was diluted directly into an IV infusion bag containing a nominal 250 mL (±25 mL excess) of 0.9% saline at room temperature using an 18-gauge needle, without removing an equal volume of saline, followed by 20 mL (420 mg) of reconstituted trastuzumab. The total concentration of the two proteins combined in the 250 mL IV bag was expected to be approximately 3 mg / mL. Similarly, an IV bag of pertuzumab (420 mg) alone was prepared using 14 mL of the 30 mg / mL drug product diluted directly into the IV infusion bag. The final expected concentration was approximately 1 mg / mL. IV bags of trastuzumab (420 mg) alone were prepared in a similar manner, except that 20 mL of the 21 mg / mL drug product was added to the bag, resulting in a final expected concentration of approximately 1 mg / mL.

[0347] The PO IV bags were thoroughly mixed manually by gently rocking back and forth several times to ensure homogeneity. After mixing, a 10 mL sample was removed from each bag with a syringe and stored in a sterile 15 cc Falcon tube to serve as the diluted sample control at time zero (T0). The IV bags were then wrapped in foil and stored at 30°C for 24 hours (T24). Immediately after storage, the remaining sample was removed from each bag with a syringe and placed in a sterile 250 mL PETG container. The T0 and T24 samples were held at 5°C for up to 24 hours or immediately analyzed by CAC, UV-spec scan (concentration and turbidity), SEC, IEC, CZE, iCIEF, HIAC-Royco, and potency assays. The product quality of the samples was tested by SEC and IEC methods specific to pertuzumab and trastuzumab products, while only the specific potency assay specific to pertuzumab was performed. Other assays utilized were non-product specific. All assays were suitable for their intended testing of their respective molecules and were used without further method optimization.

[0348] Dose II: 1560 mg of pertuzumab / trastuzumab mixture (840 mg pertuzumab and 720 mg trastuzumab) Sample Preparation: The upper end of the mAb co-administration dose range was investigated in PO and PVC IV infusion bag samples (1560 mg total mixture: 840 mg pertuzumab and 720 mg trastuzumab). If increased protein aggregation is observed, the upper end dose of 1560 mg total mAb would likely have a higher propensity to form high molecular weight species (HMWS) than the mixture containing 840 mg. To mitigate risk during in-use conditions at the higher dose range, both PO and PVC IV infusion bags were studied to ensure no interactions were observed.

[0349] Three types of drug combinations (mixture, pertuzumab 840 mg alone, and trastuzumab 720 mg alone) were prepared and handled similarly to the Dose I study. The pertuzumab / trastuzumab mixture contained pertuzumab (840 mg) diluted directly into a PO or PVC IV infusion bag at room temperature using an 18-gauge needle, followed by reconstituted trastuzumab (720 mg). The total concentration of the two mAbs combined in the 250 mL IV bag was expected to be approximately 5 mg / mL. As controls, pertuzumab-alone and trastuzumab-alone IV infusion bag samples were prepared and handled similarly to the Dose I study, except that 28 mL of 30 mg / mL pertuzumab and 34 mL of 21 mg / mL trastuzumab were diluted directly into the respective PO or PVC IV infusion bags. The final expected concentration was approximately 3 mg / mL for the pertuzumab (840 mg) and trastuzumab (720 mg) alone samples. Bags were stored uncovered at either 5°C or 30°C for up to 24 hours. TO and T24 samples were analyzed immediately or held at 5°C for 24-48 hours by CAC, UV-spec scan (concentration and turbidity), SEC, IEC, and HIAC-Royco.

[0350] Dose type, IV infusion bag, dose & preparation, storage temperature and assay details are summarized in Table 11. TIFF2026028254000014.tif112170

[0351] Assay All samples were held at 5°C or analyzed immediately. Typically, samples were analyzed within 24-48 hours after preparation and storage. The following assays were performed to confirm product quality and short-term stability of the pertuzumab / trastuzumab mixture, pertuzumab alone, and trastuzumab alone samples diluted in saline IV infusion bags. Because some assays, i.e., SEC, IEC, CZE, iCIEF, and potency assays, were not optimized for quantitative evaluation of the mAb mixture, chromatographic or electrophoretic overlays of these samples and their respective controls before and after storage at 5°C or 30°C are shown here. For consistency, values, e.g., peak area percent, were not calculated for all three sample types from the liquid chromatography and electrophoresis assays performed.

[0352] Color, Appearance and Clarity (CAC) The color, appearance, and clarity of the samples were determined by visual inspection under white fluorescent light with a black and white background at room temperature. For CAC testing, 3 cc glass vials were filled with 1 mL of each sample. A negative control (purified water) with the corresponding sample volume was used for comparison.

[0353] UV-Vis spectrophotometer scan for concentration measurement Concentrations were determined by UV-absorbance measurements on an HP8453 spectrophotometer after volumetric sample preparation. The instrument was blanked with 0.9% saline. A was measured in a quartz cuvette with a path length of 1 cm. max The absorbance at 278 nm or 279 nm and 320 nm was measured for each sample. The absorbance at 320 nm was used to correct for background light scattering in the solution. The concentration was determined to be 1.50 (mg / mL) for both pertuzumab and trastuzumab molecules. -1 cm -1 The calculation was performed using the absorption rate of TIFF2026028254000015.tif14170

[0354] Size Exclusion Chromatography (SEC: Pertuzumab-specific and Trastuzumab-specific) Each sample was injected onto a TOSOHAAS® G3000SWXL (7.8 × 300 mm) column on an AGILENT 1100® HPLC at ambient temperature. Elution peaks were monitored at 280 nm. Chromatographic integration was analyzed using CHROMELEON® software. The autosampler temperature was maintained at 2-8°C throughout the run, and 0.2 M potassium phosphate, 0.25 mM potassium chloride (pH 6.2), and 100 mM potassium phosphate (pH 6.8) were used as mobile phases for the pertuzumab and trastuzumab assays, respectively. The recommended injection load specified by the test procedure was 200 μg in a 20 μL injection volume. Due to the low protein concentration after dilution in the IV bag, the diluted 420 mg sample was injected with a load less than the recommended amount. The maximum injection volume of the HPLC sample loop was 100 μL, limiting the volume that could be injected at one time. Consequently, the injection volumes were changed to 100 μL with 160 μg protein for the pertuzumab and trastuzumab alone samples (420 mg dose group) and 73 μL with 200 μg protein for the pertuzumab / trastuzumab mix (840 mg dose group). The change in injection volume has been utilized in previous studies with IV bags and is necessary when working with low concentration samples.

[0355] Ion Exchange Chromatography (IEC) Analysis of charge heterogeneity of Pertuzumab and Trastuzumab digested with Carboxypeptidase B (CpB) by IEC was used for each sample. For Pertuzumab-specific IEC, samples were examined using either a regular IEC ("Pertuzumab-Regular IEC") or a "fast" version of IEC ("Pertuzumab-IEC-Fast") that was modified for high-throughput methods for the purposes of these experiments. The IEC assay utilized a DIONEX® WCX weak cation exchange column equilibrated with solvent A (20 mM MES, 1 mM NaEDTA pH 6.00) and solvent B (250 mM sodium chloride in solvent A) monitored at 280 nm for Pertuzumab-regular IEC and Pertuzumab-IEC-Fast, and for Trastuzumab, an AGILENT 1100® HPLC was used with solvent A (10 mM sodium phosphate, pH 7.5) and solvent B (100 mM sodium chloride in solvent A) monitored at 214 nm. Peaks were eluted with a gradient of solvent B increasing from 18% to 100% over 35 and 90 minutes for Pertuzumab-regular IEC and Pertuzumab-IEC-Fast, respectively, and from 15% to 100% over 55 minutes for Trastuzumab-IEC, at a flow rate of 0.8 mL / min. The column temperature was maintained at either 34°C or 42°C and ambient temperature for Pertuzumab-regular IEC or Pertuzumab-IEC-fast and Trastuzumab-IEC, respectively, and the autosampler temperature was maintained between 2 and 8°C throughout the run.

[0356] HIAC-ROYCO™ light obscuration for sub-visible particles Particulate counts in diluted drug products were performed using a HIAC-ROYCO™ Liquid Particulate Counting System Model 9703. The average cumulative number of particles ≥10 μm and ≥25 μm per milliliter in each sample was compiled using PHARMSPEC v2.0™. The test procedure was modified for a small-volume method, using either four 1 mL or four 0.4 mL readings per test session, with the first reading of each sample discarded. Each HIAC-ROYCO™ sample was degassed under vacuum for approximately 10-15 minutes. Sizes below 10 μm were not collected for this sample set.

[0357] UV-Vis Spectrophotometer Scan for Turbidity Measurement The optical density of samples (1 mg / mL or 3 mg / mL) from the IV bag was measured in a quartz cuvette with a 1 cm path length on an HP8453 spectrophotometer. Purified water was used as a blank for sample readings. Absorbance measurements were recorded at 340 nm, 345 nm, 350 nm, 355 nm, and 360 nm, and turbidity was expressed as the average value at these wavelengths.

[0358] Capillary Zone Electrophoresis, CZE CZE was performed using a PROTEOMELABPA800™ capillary electrophoresis system (Beckman Coulter) equipped with a neutral-coated capillary (50 μm × 50 cm). The buffer consisted of 40 mM ε-amine caproic acid / acetic acid, pH 4.5, 0.2% hydroxypropylmethylcellulose (HPMC). Samples were diluted to 0.5 mg / mL in water and injected into the capillary at 1 psi for 10 seconds. Separation was performed for 15 minutes using a voltage of 30 kV, and species detection was performed by UV at 214 nm.

[0359] CE-SDS-LIF (reduced or non-reduced) Each sample was derivatized with the fluorescent dye 5-carboxytetramethylrhodamine succinimidyl ester. After removal of free dye by gel filtration (using a NAP column), non-reduced samples were prepared by adding 40 mM iodoacetamide and heating at 70 °C for 5 min. For analysis of reduced samples, the derivatized samples were mixed with 10 mL of a solution containing SDS to a final concentration of 1% (v / v) and 1 M DTT and heated at 70 °C for 20 min. The prepared samples were analyzed on a Beckman Coulter ProteomeLab PA800 system using a 50 mm diameter, 31.2 cm fused silica capillary maintained at 20 °C throughout the analysis. The sample was introduced into the capillary by electrokinetic injection at 10 kV for 40 s. Separation was performed in polarity reversal (positive-negative) mode at a constant voltage of 15 kV using CE-SDS running buffer as the sieving medium. An argon ion laser operating at 488 nm was used for fluorescence excitation, and the resulting emission signal was monitored at 560 nm.

[0360] iCIEF Charge variant distributions of pertuzumab / trastuzumab mixtures, pertuzumab alone, and trastuzumab alone were assessed by iCIEF using an iCE280™ Analyzer (Convergent Bioscience) equipped with a fluorocarbon-coated capillary cartridge (100 μm x 5 cm). The ampholyte solution consisted of a mixture of 0.35% methylcellulose (MC), 0.47% Pharmalyte 3-10 carrier ampholyte, 2.66% Pharmalyte 8-10.5 carrier ampholyte, and 0.20% pI markers 7.05 and 9.77 in purified water. The anolyte was 80 mM phosphoric acid, and the catholyte was 100 mM sodium hydroxide, both in 0.10% methylcellulose. Samples were diluted in purified water, and CpB was added to each diluted sample at an enzyme-to-substrate ratio of 1:100, followed by incubation at 37°C for 20 minutes. The CpB-treated sample was mixed with an ampholyte solution and then focused by introducing a potential of 1500 V for 1 minute, followed by a potential of 3000 V for 10 minutes. An image of the focused charge variants was obtained by passing 280 nm ultraviolet light through the capillary into the lens of a charge-coupled device digital camera. This image was then analyzed to determine the distribution of the various charge variants.

[0361] Antiproliferative potency assay This test procedure is based on the ability of pertuzumab to inhibit the growth of MDA MB 175 VII human breast cancer cells. Briefly, cells were seeded into 96-well tissue culture microtiter plates and incubated overnight at 37°C under 5% CO2 to allow cells to attach. The following day, the medium was removed, and serial dilutions of each standard, control, and sample(s) were added to the plates. The plates were then incubated at 37°C under 5% CO2 for 4 days, and the relative number of viable cells was indirectly quantified using the redox dye, ALAMARBLUE®, according to the manufacturer's protocol. Each sample was assayed in triplicate. The color change, measured by fluorescence, was directly proportional to the number of viable cells in the culture. The absorbance of each well was then measured using a fluorescence 96-well plate reader. The results, expressed in relative fluorescence units (RFU), were plotted against the antibody concentration. Because a pertuzumab / trastuzumab mixture reference was not available, quantitative measurements were not and could not be performed. Therefore, the results represent a comparison of dose-response curves only.

[0362] Results and Discussion Dose I: 840 mg total pertuzumab / trastuzumab mixture (420 mg pertuzumab and 420 mg trastuzumab) The product quality (n=1) of a total of 840 mg of Pertuzumab / Trastuzumab mixture (Pertuzumab 420 mg and Trastuzumab 420 mg), Pertuzumab alone (420 mg), and Trastuzumab alone (420 mg) in IV infusion bags was evaluated by CAC, concentration measurement by UV-pec scan, turbidity, and HIAC Royco before and after storage at 30°C for up to 24 hours (Table 12). Pertuzumab alone and Trastuzumab alone IV infusion bags served as controls, which were also prepared to evaluate the ability of the assay to select for the appropriate product attributes. TIFF2026028254000016.tif92170

[0363] After storage, the Pertuzumab / Trastuzumab mixture, Pertuzumab alone, and Trastuzumab alone samples appeared as clear, colorless liquids, and no visible particles were observed by CAC. Concentration and turbidity measurements showed no measurable changes in any of the three sample types after 24 hours at 30°C. Particulate analysis by HIAC Royco detected no more than six particles ≥10 μm in size, and no particles >25 μm in the Pertuzumab / Trastuzumab mixture, Pertuzumab alone, and Trastuzumab alone samples after storage. These results are comparable to a 0.9% saline-only solution. The absence of visible precipitates or particulates indicates that the mixture and control are sufficiently stable upon dilution in a 0.9% saline IV infusion bag. SEC (using both Pertuzumab-specific and Trastuzumab-specific methods) of the Pertuzumab / Trastuzumab mixture diluted in saline showed comparable peak profiles from T0 to T24 (Figures 15 and 16). No increase in high molecular weight species (HMWS) or low molecular weight species (LMWS) was observed. Similarly, no changes in the main peaks were observed in any of the samples. The main peaks and peak areas of HMWS and LMWS are overlaid and indistinguishable in the Pertuzumab / Trastuzumab mixture due to the similar size of Pertuzumab and Trastuzumab (molecular weight approximately 150 kD). Furthermore, comparison of T0 and T24 for both Pertuzumab-alone and Trastuzumab-alone samples showed no observable changes in the peak areas or profiles detected by the two SEC methods.

[0364] Two product-specific methods for pertuzumab or trastuzumab IEC were used to analyze the pertuzumab / trastuzumab mixture (Figures 17 and 18). In cation exchange chromatography assays, each molecule typically contains three distinct regions eluted based on relative charge: an early-eluting acidic variant, followed by a main peak, and finally a late-eluting basic variant. In the chromatograms of pertuzumab alone and trastuzumab alone, profiles showing the acidic variant, main peak, and basic variant were observed and were judged to be comparable between the starting material and after storage at 30°C. These results are also consistent with previous studies performed in saline IV infusion bags with either pertuzumab alone or trastuzumab alone. In the chromatogram of the pertuzumab / trastuzumab mixture, the pertuzumab peak elutes first, followed by the trastuzumab peak. Due to the nature of the cation exchange separation and the difference in net charge between Pertuzumab (pI approx. 8.7) and Trastuzumab (pI approx. 8.9), two main peaks, or major charged species, are observed in the Pertuzumab / Trastuzumab mixture. In contrast, the SEC assay separates based on the hydrodynamic size of the molecules and shows only one main peak due to the similar size of Pertuzumab and Trastuzumab. The charged regions of each molecule appear to overlap with each other in the Pertuzumab / Trastuzumab mixture. In particular, the basic variant of Pertuzumab, predicted to elute at approximately 32 and 35 minutes, appears to overlap with the main Trastuzumab peak (Figures 17 and 18). Furthermore, the acidic variant of Trastuzumab, predicted to elute before the main Trastuzumab peak, coelutes with the basic variant of Pertuzumab and the main peak. Despite overlapping peak areas, the pertuzumab / trastuzumab mixture exhibited comparable chromatographic peak profiles before and after storage in IV saline bags at 30° C. for 24 hours.

[0365] The Pertuzumab / Trastuzumab mixture, Pertuzumab alone, and Trastuzumab alone samples were also assayed under non-reducing conditions by CE-SDS LIF after 24 hours of storage at 30°C. The Pertuzumab / Trastuzumab mixture showed a consistent peak profile with no observable changes after storage compared to the starting material (Figures 19 and 20). Very slight baseline level fluctuations due to noise were also observed, but this did not affect the peak areas. Similar to SEC, the non-reduced Pertuzumab / Trastuzumab mixture showed only overlapping monomers comprising both the major Pertuzumab and Trastuzumab species. The Pertuzumab alone and Trastuzumab alone samples showed no change at T0 compared to T24. However, individual molecular attributes, such as peak fragment levels and species, between the Pertuzumab / Trastuzumab mixture, Pertuzumab alone and Trastuzumab alone were observed as expected.

[0366] When the Pertuzumab / Trastuzumab mixture, Pertuzumab alone, and Trastuzumab alone were subjected to DTT-reduced CE-SDS LIF, two major peaks known as the light chain (LC) and heavy chain (HC) were detected at 17 and 21.5 minutes, respectively (Figure 20). The Pertuzumab / Trastuzumab mixture did not show an increase in fragmentation and a concomitant decrease in LC and HC after storage at 30°C. Furthermore, there were no detectable differences in the peak profiles between the Pertuzumab alone and Trastuzumab alone samples after storage.

[0367] The charge separation assays CZE and iCIEF show comparable peak profiles for the Pertuzumab / Trastuzumab mixture after storage at 30°C (Figures 21 and 22). Pertuzumab alone and Trastuzumab alone also showed consistent peak profiles when compared to their respective TOs, with no changes after storage. Furthermore, the presence of various minor species was observed, but no new peaks were detected upon dilution into IV bag saline solution. As seen in the charge-based IEC assay, two main peaks adjacent to smaller overlapping peaks could be detected, which was due to the pI differences of the molecules.

[0368] The results of the potency assay based on a comparison of dose-response curves showed no effect on the potency of the Pertuzumab / Trastuzumab mixture stored at 30°C for 24 hours compared to its corresponding TO dose-response curve. Trastuzumab alone showed little activity in the Pertuzumab potency assay. Comparing the dose-response curve of the Pertuzumab / Trastuzumab mixture with that of Pertuzumab alone or Trastuzumab alone showed that the Pertuzumab / Trastuzumab mixture required a lower dose to inhibit cell growth compared to Pertuzumab alone, suggesting that the mixture may have an additive or synergistic effect on inhibiting cell proliferation.

[0369] Dose II: 1560 mg total pertuzumab / trastuzumab mixture (840 mg pertuzumab and 720 mg trastuzumab) In addition to the Dose I study with 840 mg total mAb, a higher dose of 1560 mg of the mixture (840 mg pertuzumab and 720 mg trastuzumab) and their individual drug product controls (840 mg pertuzumab alone and 720 mg trastuzumab alone) were selected to investigate the effect of dilution of these three mAb types in PO or PVC IV infusion bags for up to 24 hours at 5° C. or 30° C. Product quality of these IV infusion bags before and after storage was assessed by CAC, UV-spec scan (concentration and turbidity) and HIAC-ROYCO™ (summarized in Table 13), as well as SEC and IEC (shown in Figures 24-27). TIFF2026028254000017.tif198170

[0370] Two PO or PVC IV infusion bags were prepared for each of the pertuzumab / trastuzumab mixture conditions, and only one IV infusion bag was prepared for the pertuzumab alone and trastuzumab alone samples.

[0371] Particulates from these bags were determined by visual observation, turbidity, and HIAC-Royco measurements. All samples appeared clear and colorless after up to 24 hours of storage at 5°C or 30°C. No visible particulates were observed, and no significant change in turbidity was noted after storage. For the pertuzumab / trastuzumab mixture, pertuzumab alone, and trastuzumab alone, the HIAC-Royco showed comparable particle values ​​before and after storage for both PO and PVC IV infusion bags stored at 5°C or 30°C, with an increase in particles from zero to 10 per milliliter above 10 μm and no increase in particles per milliliter above 25 μm. For all three sample types, UV-spec scans showed no change in protein concentration beyond the standard assay variation, indicating no protein adsorption or precipitation in the IV infusion bags between TO and T24 hours of storage at 5°C or 30°C.

[0372] Using pertuzumab-specific or trastuzumab-specific SEC and IEC methods, samples of the pertuzumab / trastuzumab mixture, pertuzumab alone, and trastuzumab alone were analyzed to evaluate their physical and chemical stability, respectively, as described above. For the pertuzumab / trastuzumab mixture, similar to the results for Dose I of the 840 mg mixture, no SEC changes were observed in the chromatographic profile between the TO and T24 time samples at 5°C or 30°C in PO or PVC IV infusion bags (Figures 24 and 25). In addition, no increase or decrease in high molecular weight species (HMWS), main peak, or low molecular weight species (LMWS) was observed. This indicates a stable dosing solution at the upper range of protein content in 0.9% saline. Similarly, samples of pertuzumab alone and trastuzumab alone showed no change after storage in IV infusion bags.

[0373] IEC analysis of Pertuzumab / Trastuzumab mixtures using both Pertuzumab-specific and Trastuzumab-specific methods, used to assess chemical stability, showed comparable charge variant peak profiles, with no changes observed compared to the initial time point after exposure to 5°C or 30°C in PO or PVC IV infusion bags (Figures 26 and 27). Although significant overlap of charge variant species of the two mAbs was observed, these peak species were not affected by increasing mAb content in the IV infusion bags. Samples of Pertuzumab alone or Trastuzumab alone in PO or PVC IV infusion bags showed no change before or after exposure to 5°C or 30°C. These results are consistent with the 840 mg dose I study.

[0374] conclusion All physicochemical assays showed no significant changes in IV infusion bags (PO or PVC) containing the mixture (≤840 mg pertuzumab and ≤720 mg trastuzumab for a total dose of 1560 mg) or the individual pertuzumab (≤840 mg) and trastuzumab (≤720 mg) from T0 to T24 hours at 5°C or 30°C. Furthermore, the potency of the mixture (≤840 mg) and the individual mAbs was comparable before and after storage. No differences were observed throughout the study in IV bags containing the mixture of pertuzumab and trastuzumab compared to the individual mAb components in the IV bag. This study also demonstrates that many of the assays used to measure the individual mAbs are sufficient for qualitative characterization of the mixture. [Example]

[0375] Concurrent administration of pertuzumab and trastuzumab and combination therapy with vinorelbine This is a two-arm, phase II, multicenter, randomized, open-label study evaluating pertuzumab in patients with HER2-positive advanced breast cancer (metastatic or locally advanced) who have not received prior systemic non-hormonal anticancer therapy in the metastatic setting. The study design is shown in Figure 28.

[0376] Patients will be randomized in a 2:1 ratio to one of two treatment arms: Pertuzumab in combination with trastuzumab and vinorelbine (Arm A) Trastuzumab and vinorelbine (control arm, arm B)

[0377] Arm A will consist of two cohorts as follows: Cohort 1: (First 95 patients): Pertuzumab and trastuzumab are administered sequentially in separate infusion bags, followed by vinorelbine. Patients will receive pertuzumab, then trastuzumab, sequentially in separate infusion bags, followed by vinorelbine.

[0378] Pertuzumab (IV infusion) A loading dose of 840 mg was administered on day 1 of the first treatment cycle, followed by 420 mg on day 1 of each subsequent 3-week cycle.

[0379] The first infusion of pertuzumab is administered over 90 (±) minutes, and the patient is observed for infusion-related symptoms, e.g., fever, chills, etc., for at least 30 minutes after the end of the infusion. Interrupting or delaying the infusion may reduce such symptoms. If the infusion is well tolerated, subsequent infusions may be administered over 30 (± 10) minutes, with the patient observed for an additional 30 minutes.

[0380] Trastuzumab (IV infusion) A loading dose of 8 mg / kg on Day 1 of the first treatment cycle, followed by 6 mg / kg on Day 1 of each 3-week cycle thereafter; administered according to product labeling.

[0381] Vinorelbine (IV infusion after trastuzumab) 25 mg / m on days 1 and 8 of the first treatment cycle 2 followed by 30-35 mg / m on days 1 and 8 of each subsequent 3-week cycle 2 ;Administer according to product label.

[0382] Cohort 2: The second 95 patients will receive pertuzumab and trastuzumab together from a single infusion bag, followed by vinorelbine, from cycle 2 onwards.

[0383] Cycle 1 Dosage In the first treatment cycle, pertuzumab and trastuzumab will be administered in separate infusion bags as described for Cohort 1.

[0384] Vinorelbine will be administered after pertuzumab and trastuzumab as described in Cohort 1.

[0385] Subsequent cycle dosing If administration of all three drugs in Cycle 1 is well tolerated, pertuzumab 420 mg and trastuzumab 6 mg / kg will be administered together in a single infusion bag on Day 1 of each subsequent 3-week treatment cycle.

[0386] The first combination infusion of pertuzumab and trastuzumab should be administered over 90 (± 10) minutes with cardiac monitoring throughout the procedure and close observation for infusion-related reactions, followed by a 60-minute observation period. If this first combination infusion is well tolerated, subsequent combination infusions may be administered over 60 (± 10) minutes, followed by a 30-minute observation period with cardiac monitoring.

[0387] Vinorelbine will be administered after pertuzumab and trastuzumab as described in Cohort 1.

[0388] Control arm - Arm B A total of 95 patients will be randomized to Arm B.

[0389] Trastuzumab (IV infusion) 8 mg / kg loading dose on Day 1 of the first treatment cycle, followed by 6 mg / kg on Day 1 of each 3-week cycle thereafter; administered according to product labeling.

[0390] Vinorelbine (IV infusion after trastuzumab) 25 mg / m on days 1 and 8 of the first treatment cycle 2 followed by 30-35 mg / m on days 1 and 8 of each subsequent 3-week cycle 2 ;Administer according to product label.

[0391] Efficacy outcomes: Primary efficacy outcome To compare the objective overall response rate (ORR) assessed by a blinded independent review committee (IRC) between (pertuzumab in combination with trastuzumab and vinorelbine) versus (rastuzumab and vinorelbine).

[0392] Secondary efficacy outcomes Within the pertuzumab treatment group, to compare the efficacy and safety of pertuzumab and trastuzumab administered together in a single infusion bag versus traditional sequential administration in separate infusion bags. To compare (pertuzumab in combination with trastuzumab and vinorelbine) versus (rastuzumab and vinorelbine) for: Investigator-assessed ORR ○ Time to response assessed by IRC and investigator Duration of response assessed by IRC and investigator Progression-free survival (PFS) Time to progression (TTP) ○ Overall survival (OS) Safety and tolerability Quality of life (EQ-5D and FACT-B questionnaires)

[0393] Inclusion criteria Patients must meet the following criteria to be eligible for the study according to the timing of the assessment schedule: 1. Female or male patients aged 18 or over 2. Histologically or cytologically confirmed and documented breast adenocarcinoma with metastatic or locally advanced disease not amenable to curative resection 3. HER2 positivity (defined as either immunohistochemistry (IHC) 3+ or in situ hybridization (ISH) positive) when evaluated by a local laboratory for primary or metastatic tumors (ISH positivity is defined as a HER2 gene copy number to CEP17 signal ratio of 2.0 or a HER2 gene count >4 for single-probe testing). 4. At least one measurable lesion and / or non-measurable disease evaluable according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 5.ECOG performance status 0 or 1 6. Left ventricular ejection fraction (LVEF) of at least 50% 7. Negative pregnancy test in women of childbearing potential (premenopausal or postmenopausal amenorrhea for less than 12 months and never undergone sterilization) 8. For sexually active women of childbearing potential, a highly effective form of non-hormonal contraception or agreement to use two effective forms of non-hormonal contraception 9. Males of reproductive potential who are willing and able to use effective non-hormonal contraception (barrier contraception combined with spermicidal jelly, or surgical sterilization) during and for at least 6 months after study treatment. 10. Life expectancy of at least 12 weeks.

[0394] Exclusion criteria Patients who meet any of the following exclusion criteria are not eligible for this study: 1. Prior systemic non-hormonal anti-cancer therapy in the setting of metastatic or locally advanced breast cancer 2. Approved or investigational anti-HER2 agents in any breast cancer treatment setting (excluding trastuzumab in the adjuvant or neoadjuvant setting) 3. Progression during trastuzumab treatment in the adjuvant or neoadjuvant setting 4. A disease-free interval of less than 6 months from the completion of adjuvant or neoadjuvant systemic non-hormonal treatment to disease recurrence 5. History of persistent Grade 2 or higher (NCI-CTC, Version 4.0) hematologic toxicity resulting from previous adjuvant or neoadjuvant therapy 6. Radiographic evidence of central nervous system (CNS) metastases assessed by CT or MRI 7. Current peripheral neuropathy of grade 3 or higher (NCI-CTC, Version 4.0) 8. History of other malignancies within the past 5 years, except for cervical intraepithelial neoplasia or basal cell carcinoma. 9. Any serious uncontrolled comorbidity that contraindicates the use of any of the investigational drugs used in this study or that places the patient at high risk for treatment-related complications. 10. Inadequate organ function as evidenced by the following tests: Absolute neutrophil count <1,500 cells / mm 3 · Platelet count <100,000 cells / mm 3 Hemoglobin <9g / dL Total bilirubin above the upper limit of normal (ULN) (unless the patient has documented Gilbert syndrome) AST (SGOT) or ALT (SGPT) > 2.5 × ULN Serum alkaline phosphatase >2.5 x ULN with AST (SGOT) or ALT (SGPT) >1.5 x ULN; serum alkaline phosphatase may be >2.5 x ULN only if bone metastases are present and AST (SGOT) and ALT (SGPT) <1.5 x ULN Serum creatinine >2.0 mg / dL or 177 μmol / L International normalized ratio (INR) and activated partial thromboplastin time or partial thromboplastin time (aPTT or PTT) > 1.5 x ULN (when therapeutic coagulation is not being performed) 11. Uncontrolled hypertension (systolic pressure >150 mmHg and / or diastolic pressure >100 mmHg) or clinically significant (i.e., active) cardiovascular disease: cerebrovascular accident (CVA) / stroke or myocardial infarction within 6 months prior to administration of the first study drug, unstable angina, New York Heart Association (NYHA) grade II or greater congestive heart failure (CHF), or serious cardiac arrhythmia requiring medication 12.Currently known infection with HIV, HBV, or HCV 13. Dyspnea at rest due to complications of progressive malignant disease or other diseases requiring continuous oxygen therapy 14. Major surgery or significant trauma within 28 days prior to randomization, or anticipated need for major surgery during study treatment. 15. Receipt of intravenous (IV) antibiotics for infection within 14 days prior to randomization 16. Current chronic daily treatment with corticosteroids (equivalent to methylprednisolone at a dose of 10 mg / day or more) (excluding inhaled steroids) 17. Known hypersensitivity to any of the study drugs or to any excipients of the recombinant human or humanized antibody 18. History of any investigational treatment within 28 days prior to randomization 19. Concurrent participation in any clinical trial

[0395] It is anticipated that the treatments described herein will demonstrate the safety and effectiveness of simultaneous administration of pertuzumab and trastuzumab from the same intravenous (IV) bag to patients with HER2-positive cancer (exemplified by HER2-positive breast cancer), as well as the safety and effectiveness of the combination of pertuzumab and vinorelbine according to any one or more of the primary or secondary efficacy outcomes described above. [Example]

[0396] Pertuzumab plus aromatase inhibitor This example is a two-arm, phase II, multicenter, randomized, open-label study demonstrating the efficacy and safety of pertuzumab in combination with trastuzumab plus an aromatase inhibitor in first-line patients with HER2-positive and hormone receptor-positive advanced (metastatic or locally advanced) breast cancer. The study design is shown in Figure 29.

[0397] Main purpose To compare progression-free survival with pertuzumab plus trastuzumab plus aromatase inhibitor (AI) versus trastuzumab plus AI.

[0398] Secondary Objectives To compare pertuzumab and trastuzumab plus an aromatase inhibitor (AI) versus trastuzumab plus an AI in terms of: - Overall survival (OS) - Objective response rate (ORR) - Clinical Benefit Rate (CBR) - Duration of response - Time to response - Safety and tolerability - Quality of life (EQ-5D questionnaire)

[0399] Study design Patients will be randomized in a 1:1 ratio to one of two treatment arms: - Pertuzumab in combination with trastuzumab + AI (Arm A). - trastuzumab + AI (control arm, arm B).

[0400] At the investigator's discretion, patients may receive induction chemotherapy (either taxane, docetaxel, or paclitaxel) in combination with their assigned monoclonal antibody treatment arm for up to the first 18 weeks of the treatment period. In patients receiving induction chemotherapy, treatment with the AI ​​will begin after the chemotherapy induction phase.

[0401] The stratification factors for the analysis were as follows: - Deciding whether to receive induction chemotherapy (yes / no). - Time since adjuvant hormone therapy (<12 months, >12 months, or no prior hormone therapy). Patients with HER2-positive and hormone receptor-positive (estrogen receptor (ER)-positive and / or progesterone receptor (PgR)-positive) advanced breast cancer (metastatic or locally advanced) who have not received prior systemic non-hormonal anticancer therapy in the metastatic setting.

[0402] Inclusion criteria 1. Age 18 or older. 2. Postmenopausal status for more than 1 year (meeting the National Comprehensive Cancer Network (NCCN) guideline criteria, Version 2.2011). 3. Histologically or cytologically confirmed and documented breast adenocarcinoma with metastatic or locally advanced disease not amenable to curative resection. 4. HER2 positivity (defined as either IHC3+ or ISH positive) when evaluated by a local laboratory for primary or metastatic tumors (ISH positivity defined as a HER2 gene copy number to CEP17 signal count ratio of 2.0 or a HER2 gene count >4 for single-probe testing). 5. Hormone receptor positivity defined as ER positivity and / or PgR positivity assessed locally as defined by institutional criteria. 6. At least one measurable lesion and / or non-measurable disease evaluable according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. 7.ECOG performance status 0 or 1. 8. Left ventricular ejection fraction (LVEF) of at least 50%. 9. Life expectancy of at least 12 weeks.

[0403] Exclusion criteria 1. Prior systemic non-hormonal anticancer therapy in the setting of metastatic or locally advanced breast cancer. 2. Disease-free interval of 6 months or less from completion of adjuvant or neoadjuvant systemic non-hormonal treatment to recurrence. 3. Approved or investigational anti-HER2 agents in any breast cancer treatment setting, except trastuzumab and / or lapatinib in the adjuvant or neoadjuvant setting. 4. Progression during treatment with trastuzumab and / or lapatinib in the adjuvant setting. 5. History of persistent Grade 2 or higher (NCI-CTC, Version 4.0) hematologic toxicity resulting from previous adjuvant or neoadjuvant therapy. 6. Radiographic evidence of central nervous system (CNS) metastases assessed by CT or MRI. 7. Current peripheral neuropathy of grade 3 or higher (NCI-CTC, Version 4.0). 8. History of other malignancies within the past 5 years, except for cervical intraepithelial neoplasia or basal cell carcinoma. 9. Serious uncontrolled comorbidities that contraindicate the use of any of the investigational drugs used in this study or that place the patient at high risk for treatment-related complications. 10. Inadequate organ function as evidenced by the following tests: - Absolute neutrophil count <1,500 cells / mm 3 . - Platelet count <100,000 cells / mm 3 . - Hemoglobin <9g / dL. - Total bilirubin above the upper limit of normal (ULN) (unless the patient has documented Gilbert syndrome). - AST(SGOT) or ALT(SGPT) > 2.5 × ULN. Serum alkaline phosphatase >2.5 x ULN with AST (SGOT) or ALT (SGPT) >1.5 x ULN; serum alkaline phosphatase may be >2.5 x ULN only if bone metastases are present and AST (SGOT) and ALT (SGPT) <1.5 x ULN. - Serum creatinine >2.0 mg / dL or 177 μmol / L. - International normalized ratio (INR) and activated partial thromboplastin time (aPTT) or partial thromboplastin time (PTT) > 1.5 x ULN (when therapeutic coagulation is not performed). 11. Uncontrolled hypertension (systolic pressure >150 mmHg and / or diastolic pressure >100 mmHg) or clinically significant (i.e., active) cardiovascular disease: cerebrovascular accident (CVA) / stroke or myocardial infarction within 6 months prior to administration of the first study drug, unstable angina, New York Heart Association (NYHA) grade II or greater congestive heart failure (CHF), or serious cardiac arrhythmia requiring medication. 12. Currently known infection with HIV, HBV, or HCV. 13. Dyspnea at rest due to complications of progressive malignant disease or other illness requiring continuous oxygen therapy. 14. Major surgery or significant trauma within 28 days prior to randomization, or anticipated need for major surgery during study treatment. 15. Lack of physical integrity of the upper gastrointestinal tract, clinically significant malabsorption syndrome, or inability to take oral medications. 16. Receipt of intravenous antibiotics for infection within 14 days prior to randomization. 17. Current chronic daily treatment with corticosteroids (dose equivalent to 10 mg / day of methylprednisolone) (excluding inhaled steroids). 18. Known hypersensitivity to any of the study drugs or to any excipients of the recombinant human or humanized antibody. 19. History of any investigational treatment within 28 days prior to randomization. 20. Concurrent participation in any clinical trial.

[0404] Arm A Pertuzumab (IV infusion) A loading dose of 840 mg was administered on day 1 of the first treatment cycle, followed by 420 mg on day 1 of each subsequent 3-week cycle. The first infusion of pertuzumab is administered over 90 (±) minutes, and the patient is observed for infusion-related symptoms, e.g., fever, chills, etc., for at least 30 minutes after the end of the infusion. Interrupting or delaying the infusion may reduce such symptoms. If the infusion is well tolerated, subsequent infusions may be administered over 30 (± 10) minutes, and the patient may be observed for an additional 30 minutes.

[0405] Trastuzumab (administered IV after pertuzumab) as a loading dose of 8 mg / kg on day 1 of the first treatment cycle, followed by 6 mg / kg on day 1 of each 3-week cycle thereafter: Administer according to product labeling.

[0406] AI (oral) Administer according to product label (anastrozole: 1 mg once daily; letrozole: 2.5 mg once daily).

[0407] Induction chemotherapy Patients receiving induction chemotherapy for the first 18 weeks of treatment will receive a taxane (docetaxel every 3 weeks or paclitaxel once a week) according to the respective product label. The chemotherapy will be administered after monoclonal antibody (pertuzumab and / or trastuzumab) infusion.

[0408] In patients receiving induction chemotherapy, treatment with an AI begins after the chemotherapy induction phase.

[0409] Control arm - Arm B Trastuzumab (IV infusion) A loading dose of 8 mg / kg on Day 1 of the first treatment cycle, followed by 6 mg / kg on Day 1 of each 3-week cycle thereafter; administered according to product labeling.

[0410] AI (oral) Administer according to product label (anastrozole: 1 mg once daily; letrozole: 2.5 mg once daily).

[0411] Induction chemotherapy Same as the investigational arm.

[0412] Primary efficacy outcome PFS (defined as the time from randomization to the date of first radiographic progression or death from any cause, whichever occurs first).

[0413] Secondary efficacy outcomes - OS -ORR - CBR - Duration of response - Time to response

[0414] safety - Incidence and severity of adverse events (AEs) and serious adverse events (SAEs) - Incidence of CHF - LVEF during this study - Abnormal laboratory tests We predict that the combination of pertuzumab, trastuzumab, and an AI is safe and effective in this patient population, and that the addition of pertuzumab to trastuzumab and an AI prolongs progression-free survival (PFS) compared with trastuzumab plus an AI without pertuzumab. [Example]

[0415] Pertuzumab for improving overall survival (OS) in cancer patients Background: In the CLEOPATRA study, Example 3 above, 808 patients with HER2-positive first-line (1L) metastatic breast cancer (MBC) were randomized to treatment with placebo, trastuzumab, and docetaxel (Pla+T+D) or pertuzumab, trastuzumab, and docetaxel (P+T+D). The independently reviewed primary endpoint of progression-free survival was significantly improved with P+T+D versus Pla+T+D (hazard ratio (HR) = 0.62; P < 0.0001; median, 18.5 months vs. 12.4 months) (Example 3 above). This example includes a second interim overall survival (OS) analysis after longer follow-up.

[0416] Methods: This interim overall survival (OS) analysis was performed by applying the LanDeMets α-spending function with an O'BrienFleming stopping limit to maintain an overall type I error of 5%. Based on the number of observed OS events, the OBF limit of statistical significance for this analysis was P ≤ 0.0138. OS was compared between arms in the intention-to-treat population using the log-rank test stratified by prior treatment status and geographic region. The Kaplan-Meier approach was used to estimate median OS for both arms; stratified Cox proportional hazards models were used to estimate HRs and 95% CIs. Subgroup analyses of OS were performed for stratification factors and other important baseline characteristics.

[0417] Results: At the time of this analysis, the median follow-up was 30 months, and 267 deaths had occurred (69% of those planned for the final analysis). Results showed a statistically significant improvement in OS in favor of P+T+D (HR = 0.66; 95% confidence interval (CI), 0.52-0.84; P = 0.0008). This HR corresponds to a 34% reduction in the risk of death. The analysis was statistically significant and therefore considered a confirmatory OS analysis. Median OS was 37.6 months in the Pla arm and not yet reached in the P arm. Treatment effects were generally consistent across predefined subgroups based on baseline variables and stratification factors, including prior (neo)adjuvant therapy (HR = 0.66; 95% CI, 0.46-0.94); no prior (neo)adjuvant therapy (HR = 0.66; 95% CI, 0.47-0.93); prior (neo)adjuvant therapy (HR = 0.68; 95% CI, 0.30-1.55); hormone receptor-negative disease (HR = 0.57; 95% CI, 0.41-0.79); and hormone receptor-positive disease (HR = 0.73; 95% CI, 0.50-1.06). Kaplan-Meier estimates of OS rates showed a survival benefit at 1, 2, and 3 years for P+T+D. TIFF2026028254000018.tif44170

[0418] The majority of patients received anticancer therapy after discontinuation of study treatment (Pla arm 64%; P arm 56%). Sequential therapy with HER2-directed agents (T, lapatinib, T, emtansine) was balanced between arms. Causes of death remained unchanged from the first interim OS analysis, with progression being the most common cause of death. Adverse events leading to death were rare and not balanced between arms.

[0419] Conclusions: Treatment of patients with HER2-positive 1L MBC with P+T+D compared with Pla+T+D resulted in improved OS, both of which were statistically significant and clinically meaningful. These results indicate that the combination of HER2 blockade and chemotherapy using a P+T+D regimen can be considered standard of care for patients with HER2-positive MBC in the 1L setting.

[0420] These data regarding OS can be included in the package insert along with the prescribing information for pertuzumab in the product, for example as in Example 4 above. [Example]

[0421] Pertuzumab and trastuzumab in combination with a taxane (PERUSE) as first-line treatment for patients with HER2-positive advanced breast cancer Background: The humanized monoclonal antibody pertuzumab (P) inhibits signaling downstream of HER2 by binding to the receptor's dimerization domain and preventing heterodimerization with other HER family members. Because the epitope recognized by P is distinct from that bound by trastuzumab (H), their complementary mechanisms of action result in more comprehensive HER2 blockade. Data from the phase III trial CLEOPATRA showed significantly improved PFS in patients (pts) receiving P+H+docetaxel as first-line treatment for HER2-positive metastatic breast cancer (BC) compared with H+docetaxel+placebo.

[0422] Study Design: This is a single-arm, Phase IIIb, multicenter, open-label study in patients with HER2-positive metastatic or locally recurrent BC who have not previously received systemic non-hormonal anticancer therapy for metastatic disease. Patients will receive P: loading dose of 840 mg, 420 mg IV every 3 weeks; H: loading dose of 8 mg / kg, 6 mg / kg IV every 3 weeks; taxane: docetaxel, paclitaxel, or nab-paclitaxel according to local guidelines. Treatment will be administered until progression or unacceptable toxicity. A planned protocol modification will allow hormone receptor-positive patients to receive endocrine therapy in parallel with P+H after completion of taxane therapy, in accordance with clinical practice.

[0423] Eligibility criteria: At baseline, patients must have an LVEF of ≥ 50%, an ECOG PS of 0, 1, or 2, a disease-free interval of ≥ 6 months, and must not have received prior anti-HER2 therapy for the treatment of metastatic BC. Prior H and / or lapatinib in the (neo)adjuvant setting is permitted if there has been no progression during treatment. Patients must not have a history of other malignancies within the past 5 years other than cervical intraepithelial neoplasia or basal cell carcinoma. There must be no clinical or radiographic evidence of CNS metastases or clinically significant cardiovascular disease.

[0424] Specific objectives: H was not widely available in the (neo)adjuvant setting prior to CLEOPATRA recruitment, so the proportion of patients with prior H exposure in CLEOPATRA was relatively small. PERUSE will evaluate the safety and tolerability of P+H+taxane option as first-line treatment for patients with HER2-positive metastatic or locally advanced BC in a patient population more likely to have had more extensive prior exposure to H therapy.

[0425] Statistical Methods: The primary endpoints of the PERUSE study are safety and tolerability. Secondary endpoints include PFS, OS, ORR, CBR, duration of response, time to response, and QoL. The final analysis will occur when 1500 patients have been followed for at least 12 months since the last patient received their final study treatment, unless the patient is lost to follow-up, withdraws consent, or dies, or if the study is terminated earlier by the sponsor. Safety analyses are planned after enrollment of approximately 350, 700, and 1000 patients. In addition, a Data Safety Monitoring Board will review safety data after enrollment of approximately 50 patients and every six months thereafter.

[0426] Pertuzumab and trastuzumab in combination with a taxane are predicted to be effective as first-line treatment for patients with HER2-positive advanced breast cancer according to the protocol in this example. [Example]

[0427] Pertuzumab in combination with chemotherapy in HER3-low ovarian cancer Epithelial ovarian cancer, along with primary peritoneal and fallopian tube cancers, is the fifth leading cause of cancer-related deaths among European women (Bray et al., Int. J. Cancer 113:977-90 (2005)). Ovarian cancer is often not diagnosed until it has progressed to an advanced stage, at which point the standard treatment is surgical resection followed by chemotherapy. Although the addition of taxanes to platinum-based chemotherapy has achieved a complete response (CR) in approximately 80% of patients, the disease recurs in most patients, and more than 50% of patients diagnosed with epithelial ovarian cancer ultimately die from the disease (Du Bois et al., Cancer 115:1234-1244 (2009)). After failure of platinum-based chemotherapy, few treatment options remain. Patients with platinum-sensitive disease (disease recurrence more than 6 months after the last cycle of platinum-based chemotherapy) are often retreated with platinum-based therapy and have a progression-free survival (PFS) of approximately 9-10%; however, for patients with primary platinum-resistant disease, the prognosis is significantly worse. For these patients, retreatment with platinum-based therapy or surgery is not warranted; instead, patients with platinum resistance are often treated with single-agent chemotherapy, such as topotecan, pegylated liposomal doxorubicin (PLD), paclitaxel, and gemcitabine.

[0428] In patients with platinum-resistant disease, response rates range from 10 to 20%, and median progression-free survival (PFS) ranges from 3.5 to 4 months. Platinum-resistant disease is not curable; treatment goals for these patients include palliation of symptoms, prolonged survival, and improved quality of life (QoL). Overall, results from major clinical trials conducted over the past 20 years have shown that in patients with progressive disease, median PFS ranges from 16 to 23 months, and median overall survival (OS) ranges from 31 to 65 months.

[0429] The majority of ovarian cancer cell lines and many ovarian cancer biopsies express all members of the HER family of receptors (Campiglio et al., J. Cell Biochem 73:522-32 (1999)). EGFR and HER2 have been the most extensively studied, and several drugs targeting the receptors or associated intracellular tyrosine kinases have been tested.

[0430] Recent studies have demonstrated that quantitative HER2 protein analysis demonstrates that malignant ovarian tumors have significantly higher HER2 levels compared with benign ovarian tumors and normal ovaries. Furthermore, a correlation between HER2 and HER3 protein levels has been observed (Steffensen et al., Int J Oncol. 33:195-204 (2008)). Studies in cell culture systems have shown that heregulin-activated HER3-HER2 heterodimers induce the strongest proliferative and transforming responses of any possible receptor combination (Pinkas-Kramarski et al., EMBO J. 15:2452-67 (1996); Riese et al., Mol Cell Biol 15:5770-6 (1995). Erratum: Mol Cell Biol 16:735 (1996)). The potency of these biological responses is likely the result of efficient dual activation of the MAP kinase and PI3 kinase pathways. Furthermore, HER3 is the most potent activator of the PI3 kinase / AKT pathway (Olayioye et al., EMBO J 19:3159-67 (2000)). Studies in HER2-amplified breast cancer cell lines showed that HER3, but not EGFR, was critical for HER2 signaling, and that HER3 inhibited growth in three-dimensional culture and induced rapid tumor regression in in vivo xenografts (Lee-Hoeflich et al., Cancer Res 68:5878-87 (2008)).

[0431] Furthermore, HER3 expression has been implicated in ovarian cancer as a possible risk factor (Tanner et al. J Clin Oncol 24:4317-23 (2006)).

[0432] In a phase II multicenter trial (TOC2689g) in patients with advanced ovarian cancer that had relapsed after or was refractory to platinum-based chemotherapy, patients enrolled in cohort 1 (n=61) received a loading dose of 840 mg pertuzumab followed by 420 mg pertuzumab on day 1 of each 3-week cycle, and patients enrolled in cohort 2 (n=62) received 1050 mg pertuzumab on day 1 of each 3-week cycle. Similar outcomes were observed in both cohorts with regard to overall response rate and median progression-free survival (PFS). Eight patients (4 in each cohort) had evidence of stable disease (SD) lasting at least 6 months. Median PFS and OS were 6.6 weeks and 52.7 weeks, respectively, in the overall population.

[0433] The results of this study led to two phase II randomized trials in platinum-sensitive and platinum-resistant populations. The TOC3258g study evaluated the efficacy and safety of gemcitabine plus pertuzumab versus gemcitabine plus placebo in patients with advanced ovarian, primary peritoneal, or fallopian tube cancer refractory to platinum-based chemotherapy (Amler et al., J Clin Oncol 26:5552 (2008)). In this study, patients crossed over to receive pertuzumab at the time of progression. The median PFS was 2.6 months in the gemcitabine plus placebo arm and 2.9 months in the gemcitabine plus pertuzumab arm. Median OS was similar between treatment arms. The most common adverse events (AEs) that were increased in the pertuzumab-treated cohort (in at least six patients) included fatigue, nausea, diarrhea, back pain, dyspepsia, stomatitis, headache, epistaxis, rhinorrhea, rash, and grade 3-4 neutropenia.

[0434] In the BO17931 study, 149 patients with ovarian cancer that had recurred after 6 months of platinum-based therapy were randomized to receive paclitaxel and carboplatin or gemcitabine with or without pertuzumab. After six treatment cycles, chemotherapy was discontinued, and patients in the chemotherapy-plus-pertuzumab arm continued to receive pertuzumab alone for up to 11 additional cycles (a total of 17 cycles of pertuzumab). There were no significant differences in progression-free survival (PFS) or overall survival (OS) across groups. Median PFS was 34.1 weeks in the chemotherapy-plus-pertuzumab arm compared with 31.3 weeks in the chemotherapy-alone arm; however, an exploratory subset analysis of HER3 mRNA expression using a 6- to 12-month treatment-free interval showed a trend toward clinical benefit in patients expressing high levels of HER3 mRNA (Kaye et al., J Clin Oncol 26:5520 (2008)).

[0435] Archival tissue samples from patients enrolled in both randomized phase II studies were examined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for mRNA expression levels of the HER receptors EGFR, HER2, HER3, and two HER ligands: amphiregulin and betacellulin.

[0436] A significant difference in PFS was observed only with respect to tumor HER3 mRNA expression. Among patients who achieved a clinical response, PR was observed in nine patients in the gemcitabine plus pertuzumab arm and three patients in the gemcitabine plus placebo arm. Six patients in the gemcitabine plus pertuzumab arm who experienced a PR had tumor HER3 mRNA levels lower than the median level. In contrast, no patients in the gemcitabine plus placebo arm with tumor HER3 mRNA levels lower than the median level in the study population experienced a PR. An additional six patients achieved a PR, all of whom had tumor HER3 mRNA levels equal to or greater than the median level in the study population. Of these patients, three received gemcitabine plus pertuzumab and three received gemcitabine plus placebo, but no benefit of pertuzumab was suggested in this population.

[0437] In patients with low HER3 mRNA expression (below the median level of the study population), the PFS hazard ratio (HR) was 0.32, compared with 1.68 in patients with HER3 mRNA expression above the median level, i.e., the additional effect of pertuzumab was in the opposite direction. No significant benefit in OS was detected in patients with low HER3 mRNA expression, but a trend toward greater OS was observed in patients treated with pertuzumab. For patients with high HER3 mRNA expression, the HR for OS was 1.59.

[0438] To assess prognostic value, HER3 mRNA expression was correlated with PFS and OS in patients in the gemcitabine plus placebo arm. Median PFS was 1.4 months in patients with low HER3 mRNA expression (n=35) compared with 5.5 months in patients with high HER3 mRNA expression. Similarly, OS was 8.4 months in patients with low HER3 mRNA expression compared with 18.2 months in patients with high HER3 mRNA expression.

[0439] In the BO17931 study, no treatment benefit was observed in patients with low HER3 mRNA expression (below the median level in this study population), but in an exploratory analysis of patients with a treatment-free interval of 6 to 12 months, the combination of chemotherapy with pertuzumab showed a trend toward a clinical benefit in terms of PFS.

[0440] Overview of this research This is a two-part, multicenter study, with a non-randomized safety run-in part 1 and a randomized, double-blind part 2.

[0441] Part 1 will evaluate the safety and tolerability of pertuzumab in novel combinations with two chemotherapy agents (topotecan or paclitaxel). Part 2 of this study is a two-arm, prospective, multicenter, randomized, double-blind, placebo-controlled trial of pertuzumab in combination with chemotherapy agents (topotecan, paclitaxel, or gemcitabine). Patients will receive study drug until progression by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, progression by Gynecologic Cancer Intergroup (GCIG) criteria for CA-125-evaluable disease, unacceptable toxicity, withdrawal of consent, or death. PFS will be assessed in Part 1 of this study, but results are descriptive due to the small number of patients and PFS events per cohort. The study design for Part 1 of this study is shown in Figure 30.

[0442] In Part 2 of the study, patients will be randomized in a 1:1 ratio to receive either: - Arm A: Pertuzumab in combination with chemotherapy (topotecan, paclitaxel, or gemcitabine), or - Arm B: Pertuzumab-placebo + chemotherapy (topotecan, paclitaxel or gemcitabine).

[0443] Study drug assignment will be double-blind as to whether patients receive pertuzumab or pertuzumab-placebo. The chemotherapy agent assigned will be at the discretion of the investigator.

[0444] The stratification factors for Part 2 of the study were as follows: - Selective chemotherapy cohort (topotecan vs. paclitaxel vs. gemcitabine). - Prior antiangiogenic therapy (yes vs. no). If a patient has previously participated in a blinded trial of an antiangiogenic drug, they will be enrolled in the same stratum as patients known to have previously received an antiangiogenic drug. - Treatment-free interval (TFI) from platinum therapy (specifically, less than 3 months vs. 3-6 months (inclusive) from first study treatment).

[0445] The study design for Part 2 of this study is shown in Figure 31.

[0446] The main objectives of this study: Part 1: The primary objective of Part 1 of this study is to determine the safety and tolerability of pertuzumab in combination with either topotecan or paclitaxel.

[0447] Part 2: The primary objective of Part 2 of this study is to determine whether pertuzumab plus chemotherapy is superior to placebo plus chemotherapy, as measured by PFS.

[0448] Secondary objectives of this study: Part 1: A secondary objective of Part 1 of this study is to descriptively evaluate the PFS of pertuzumab in combination with either topotecan or paclitaxel.

[0449] Part 2: The secondary objectives of Part 2 of this study are to determine whether pertuzumab plus chemotherapy is superior to placebo plus chemotherapy for: - OS. - Response rate. - Biological progression-free interval (PFI BIO ). - Safety and tolerability. - QoL.

[0450] Efficacy outcome measure

[0451] The following efficacy endpoints will be measured in Part 1 of the study: - PFS, defined as the time from randomization into Part 1 of the study to progression by RECIST version 1.1 or progression by GCIG criteria for CA-125 evaluable disease or death from any cause, whichever occurs first.

[0452] Efficacy endpoints for Part 2 of the study were: - PFS, defined as the time from randomization into Part 2 of the study to progression by RECIST version 1.1 or progression by GCIG criteria for CA-125 evaluable disease or death from any cause, whichever occurs first. - OS, defined as the time from randomization to Part 2 of the study to death from any cause. - Objective response rate (ORR) assessed by best (confirmed) overall response (BOR) according to RECIST version 1.1, defined as the best response recorded from the start of treatment in Part 2 of the study until progression / relapse (the smallest measurement recorded since treatment started in Part 2 of the study is considered the reference for PD). Patients must have two consecutive assessments of partial response (PR) or complete response (CR) to be considered a responder. PR or CR must be confirmed by two consecutive tumor assessments separated by at least 4 weeks. Only patients with measurable disease at baseline will be included in the analysis of objective tumor response. - Responders are defined as patients who have a response according to RECIST version 1.1 and using the CA-125 50% response criteria, RECIST responders are defined as patients who have only a response defined according to RECIST, and CA-125 responders are defined as patients who do not have a response according to RECIST but have a response defined using the CA-125 50% response criteria. PFI, defined as the period from the date of randomization into Part 2 of the study to the first demonstration of an increase in CA-125 level to twice the upper limit of normal (for patients with a normal pretreatment CA-125 or patients with a high pretreatment CA-125 who normalized early in treatment) or to twice the nadir (for patients with a high baseline CA-125 whose levels did not normalize during treatment), based on a gradual sequential rise in serum CA-125 (assessed according to CGIG criteria) BIO .

[0453] Safety outcome measure In Part 1 of the study, safety and tolerability will be assessed after patients have received three cycles of treatment.

[0454] In addition, safety endpoints of this study were assessed in both Part 1 and Part 2 of the study and include: - The incidence, nature, and severity of all AEs, serious adverse events (SAEs), AEs of grade 3 or higher according to NCI-CTCAE version 4.0, and AEs leading to premature discontinuation of study drug. - Early withdrawal from the study and study treatment. - Development of cardiac problems / congestive heart failure. - Abnormal laboratory tests. - Left ventricular ejection fraction.

[0455] Inclusion criteria 1. Female patients aged 18 years or older. 2. Low HER3 mRNA expression levels (concentration ratio ≤ 2.81 as assessed by qRT-PCR on a COBAS z480® instrument). 3. Histologically or cytologically confirmed and documented platinum-resistant or refractory (defined as progression within 6 months of completing at least four platinum therapy cycles or progression during platinum therapy) epithelial ovarian cancer. 4. At least one measurable lesion and / or non-measurable disease by RECIST version 1.1 or cancer antigen-125 (CA-125) evaluable disease by Gynecologic Center Intergroup (GCIG) criteria. The following histologies are eligible: - Adenocarcinoma not otherwise specified. - Clear cell adenocarcinoma. - Endometrioid adenocarcinoma. - Malignant Brenner tumor. - Mixed cell carcinoma, including malignant mixed Müllerian tumor - Mucinous adenocarcinoma. - Serous adenocarcinoma. - Transitional cell carcinoma. - Undifferentiated cancer. 5. Eastern Cooperative Oncology Group (ECOG) performance status 0-2. 6.LVEF 55% or more.

[0456] Pertuzumab Dosage and Administration Pertuzumab and pertuzumab-placebo will be administered intravenously as an 840 mg loading dose on Day 1 of the first treatment cycle, followed by 420 mg on Day 1 of each subsequent 3-week cycle. The first infusion of pertuzumab / pertuzumab-placebo will be administered over 60 minutes; after a 60-minute observation period in the seated position, subsequent infusions may be administered over 30 minutes if the infusion is well tolerated, followed by a 30-minute observation period before chemotherapy administration. Premedication should be administered according to local practice and depending on the chemotherapy selected.

[0457] Topotecan Dosage and Administration Topotecan is administered at 1.25 mg / m daily on days 1 through 5 every 3 weeks as directed in the Summary of Product Characteristics. 2 should be given as a 30-minute IV infusion.

[0458] Paclitaxel Dosage and Administration Paclitaxel was administered at 80 mg / m on days 1, 8, 15, and 22. 2 should be administered as a 1-hour IV infusion. 2 The Summary of Product Characteristics should be followed for dosage preparation and administration guidance.

[0459] Gemcitabine Dosage and Administration Gemcitabine (Part 2 of this study only) was administered at 1000 mg / m on days 1 and 8 every 3 weeks as directed in the Summary of Product Characteristics. 2 should be given as a 30-minute IV infusion.

[0460] HER3 mRNA expression Before agreeing to participate in the study, patients will be asked to specifically consent to the collection and testing of primary tumor tissue samples to assess HER3 mRNA levels, including mRNA and protein levels of other HER family receptors, such as HER2. Only patients whose tumors express low levels of HER3 mRNA will be eligible to participate in the study.

[0461] During initial screening of HER3 mRNA levels, the mRNA and / or protein levels of other receptors in the HER family (e.g., EGFR, HER2, or HER4) are assessed in parallel with the HER3 evaluation to obtain a more complete picture of the status of HER family receptors by mRNA level.

[0462] The cutoff for study eligibility was defined as a concentration ratio of 2.81 or less, as assessed by qRT-PCR on the COBASz480 instrument using the COBAS® HER2 & HER3 (qRT-PCR) mRNA Expression Assay provided by Roche Molecular Diagnostics. The rationale for the cutoff definition was based on cutoff models from previous studies and a conversion function that had to be implemented when the assay was switched to the new instrument, the COBASz480®. It was expected that 40-50% of screened patients would have HER3 mRNA levels below the 2.81 cutoff, and that 30% of patients expressing low levels of HER3 mRNA would be ineligible for enrollment due to other inclusion / exclusion criteria.

[0463] Submission of a formalin-fixed, paraffin-embedded tumor specimen of the primary tumor from the initial surgery is required for all patients prior to screening; cytology specimens are not permitted as a substitute. Patients will be evaluated for HER3 mRNA expression levels, as well as mRNA and protein expression levels of other HER family receptors, using qRT-PCR assays and IHC. Such evaluation of HER receptor mRNA / protein expression will be performed after obtaining patient informed consent at any time after the initial surgery and prior to screening.

[0464] The combination of pertuzumab with topotecan or paclitaxel is predicted to be safe and effective in patients with epithelial ovarian, primary peritoneal, or fallopian tube cancer.

[0465] In addition, pertuzumab plus chemotherapy (topotecan, paclitaxel, or gemcitabine) is predicted to be superior to placebo plus chemotherapy in patients with epithelial ovarian, primary peritoneal, or fallopian tube cancer, when efficacy is measured by PFS.

Claims

1. 1. A method for extending progression-free survival by six months or more in a population of HER2-positive breast cancer patients, comprising administering to patients in said population pertuzumab, trastuzumab and a chemotherapy agent.

2. 10. The method of claim 1, which results in a response rate of 80% or greater in said population of patients.

3. 3. The method of claim 1 or 2, wherein the chemotherapeutic agent comprises a taxane.

4. 4. The method of claim 3, wherein the taxane is docetaxel.

5. 5. The method of any one of claims 1 to 4, wherein the breast cancer is metastatic or locally recurrent unresectable breast cancer or de novo stage IV disease.

6. 6. The method of any one of claims 1 to 5, wherein the patients in the population are previously untreated or have relapsed after adjuvant therapy.

7. 7. The method of any one of claims 1 to 6, wherein HER2-positive breast cancer is defined as immunohistochemistry (IHC) 3+ and / or fluorescence in situ hybridization (FISH) amplification ratio ≧2.

0.

8. 8. The method of any one of claims 1 to 7, wherein the patients in the population have a left ventricular ejection fraction (LVEF) of 50% or greater at baseline.

9. 9. The method of any one of claims 1 to 8, wherein the patients in the population have an Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 or 1.

10. 1. A method of combining two HER2 antibodies to treat HER2-positive cancer without increasing cardiotoxicity in a population of HER2-positive cancer patients, comprising administering pertuzumab, trastuzumab and a chemotherapeutic agent to patients in said population.

11. 11. The method of claim 10, wherein the patient population is monitored for cardiotoxicity in terms of incidence of symptomatic left ventricular systolic dysfunction (LVSD) or congestive heart failure (CHF) or reduced left ventricular ejection fraction (LVEF).

12. The method of claim 10 or 11, wherein the HER2-positive cancer is breast cancer.

13. 13. The method of any one of claims 10 to 12, wherein the breast cancer is metastatic or locally recurrent unresectable breast cancer or de novo stage IV disease.

14. 1. An article of manufacture comprising a vial containing pertuzumab and a package insert, wherein the package insert provides the safety data in Table 3 or Table 4.

15. 15. The article of manufacture of claim 14, wherein the package insert provides the efficacy data of Table 2, Table 5, Figure 8 or Figure 10.

16. 16. The article of manufacture of claim 14 or claim 15, wherein the vial is a single dose vial containing about 420 mg of pertuzumab.

17. 10. A method of manufacturing an article of manufacture comprising packaging a vial containing pertuzumab together with a package insert, wherein the package insert provides the safety data of Table 3 or Table 4.

18. 10. A method for ensuring the safe and effective use of pertuzumab, comprising packaging a vial containing pertuzumab together with a package insert, wherein the package insert provides the safety data in Table 3 or Table 4 and the efficacy data in Table 2, Table 5, Figure 8 or Figure 10.

19. A method for treating early stage HER2-positive breast cancer, comprising administering to a breast cancer patient pertuzumab, trastuzumab and a chemotherapeutic agent, wherein the chemotherapeutic agent comprises an anthracycline chemotherapeutic agent or a carboplatin chemotherapeutic agent.

20. 20. The method of claim 19, wherein the chemotherapeutic agent comprises an anthracycline chemotherapeutic agent, including 5-FU, epirubicin, and cyclophosphamide (FEC).

21. 21. The method of claim 20, wherein pertuzumab is administered simultaneously with the anthracycline chemotherapy agent.

22. 20. The method of claim 19, wherein the chemotherapeutic agent comprises a carboplatin-based chemotherapeutic agent, including docetaxel and carboplatin.

23. 23. The method of claim 22, wherein pertuzumab is administered simultaneously with a carboplatin-based chemotherapy agent.

24. 24. The method of any one of claims 19 to 23, wherein administration of pertuzumab does not increase cardiotoxicity compared to treatment without pertuzumab.

25. 25. The method of any one of claims 19 to 24, comprising neoadjuvant or adjuvant therapy.

26. 1. A method of treating HER2-positive cancer in a patient, comprising co-administering to the patient a mixture of pertuzumab and trastuzumab from the same intravenous infusion bag.

27. 27. The method of claim 26, further comprising administering a chemotherapeutic agent to the patient.

28. An intravenous (IV) bag containing a stable mixture of pertuzumab and trastuzumab suitable for administration to cancer patients.

29. 30. The IV bag of claim 28, wherein the mixture is a saline solution.

30. 30. The IV bag of claim 29, wherein the saline solution comprises about 0.9% NaCl or about 0.45% NaCl.

31. 31. The IV bag according to any one of claims 28 to 30, which is an IV bag made of polyolefin or polyvinyl chloride and contains 250 mL of 0.9% saline.

32. 32. The IV bag of any one of claims 28 to 31, comprising a mixture of about 420 mg or about 840 mg of pertuzumab and about 200 mg to about 1000 mg of trastuzumab.

33. 33. The IV bag of any one of claims 28 to 32, wherein the mixture is stable at 5°C or 30°C for up to 24 hours.

34. 34. The IV bag of any one of claims 28 to 33, wherein stability has been assessed by an assay selected from the group consisting of color, appearance and clarity (CAC), concentration and turbidity analysis, particle analysis, size exclusion chromatography (SEC), ion exchange chromatography (IEC), capillary zone electrophoresis (CZE), image capillary isoelectric focusing (iCIEF), and potency assay.

35. 1. A method of treating HER2-positive gastric cancer in a human subject, comprising administering pertuzumab, trastuzumab and a chemotherapeutic agent to a subject with HER2-positive gastric cancer.

36. 36. The method of claim 35, wherein the gastric cancer comprises unresectable locally advanced or metastatic gastric cancer or progressive post-operative recurrent gastric cancer that may not be amenable to curative therapy by known methods, or adenocarcinoma of the stomach or gastroesophageal junction.

37. 37. The method of claim 35 or claim 36, wherein the patient has not received prior anti-cancer therapy for metastatic gastric cancer and has an Eastern Cooperative Oncology Group Performance Status Scale (ECOG PS) of 0-1 or a HER2-positive status of IHC3+ or IHC2+ / ISH+.

38. 38. The method of any one of claims 35 to 37, wherein the chemotherapeutic agent comprises a platin (cisplatin) and / or a fluoropyrimidine (capecitabine or 5-fluorouracil (5-FU)).

39. 39. The method of claim 38, wherein pertuzumab, trastuzumab, cisplatin and capecitabine or 5-FU is administered.

40. 40. The method of any one of claims 35 to 39, wherein pertuzumab is administered at a dose of 840 mg in all treatment cycles.

41. 41. The method of any one of claims 35 to 40, which improves overall survival (OS) compared to patients treated with trastuzumab and a chemotherapy agent alone, or improves progression-free survival (PFS) or response rate (RR) compared to treatment with trastuzumab and a chemotherapy agent alone.

42. 1. A method of treating gastric cancer in a human subject, comprising administering pertuzumab to a subject having gastric cancer, wherein pertuzumab is administered at a dose of 840 mg in all treatment cycles.

43. 43. The method of claim 42, wherein pertuzumab is administered at a dose of 840 mg for six treatment cycles.

44. 44. The method of claim 42 or claim 43, wherein the subject maintains a trough level of pertuzumab above about 20 μg / mL.

45. 45. The method of any one of claims 42 to 44, further comprising administering trastuzumab and a chemotherapeutic agent to the subject.

46. 1. A method of treating HER2-positive unresectable or metastatic adenocarcinoma of the stomach or gastroesophageal junction in a human patient who has not received prior chemotherapy for metastatic disease except for previous adjuvant or neoadjuvant therapy completed more than 6 months prior to the current treatment, the method comprising administering to the patient pertuzumab, trastuzumab, cisplatin and capecitabine or fluorouracil (5-FU) in amounts that improve progression-free survival (PFS) and / or overall survival (OS), wherein the patient has an Eastern Cooperative Oncology Group Performance Status Scale (ECOG PS) of 0 to 1.

47. A method for improving progression-free survival (PFS) in a human patient with HER2-positive unresectable or metastatic adenocarcinoma of the stomach or gastroesophageal junction, comprising administering pertuzumab in combination with trastuzumab and a chemotherapeutic agent to the patient.

48. 1. A method of treating HER2-positive breast cancer in a patient, comprising administering to the patient pertuzumab, trastuzumab and vinorelbine.

49. 49. The method of claim 48, wherein pertuzumab and trastuzumab are administered simultaneously to the patient from a single intravenous infusion bag.

50. 50. The method of claim 48 or 49, wherein the breast cancer is metastatic or locally advanced.

51. 51. The method of any one of claims 48 to 50, wherein the patient has not been previously treated with systemic non-hormonal anti-cancer therapy in the metastatic setting.

52. 1. A method of treating HER2-positive breast cancer in a patient, comprising administering to the patient pertuzumab, trastuzumab and an aromatase inhibitor.

53. 53. The method of claim 52, wherein the aromatase inhibitor is anastrozole or letrozole.

54. 54. The method of claim 52 or 53, wherein the breast cancer is hormone receptor-positive advanced breast cancer.

55. 55. The method of claim 54, wherein the hormone receptor is an estrogen receptor (ER) and / or a progesterone receptor (PgR).

56. 56. The method of any one of claims 52 to 55, wherein the patient has not been previously treated with systemic non-hormonal anti-cancer therapy in the metastatic setting.

57. 57. The method of any one of claims 52 to 56, wherein the patient undergoes induction chemotherapy.

58. 58. The method of claim 57, wherein the induction chemotherapy comprises a taxane.

59. 19. The method of claim 17 or 18, wherein the package insert further comprises the warning box of Example 4.

60. 1. A method of treating a cancer patient, comprising administering to the patient an initial dose of 840 mg of pertuzumab, followed by doses of 420 mg of pertuzumab every three weeks thereafter, and further comprising readministering to the patient an 840 mg dose of pertuzumab when the interval between two successive 420 mg doses is 6 weeks or more.

61. 61. The method of claim 60, further comprising administering 420 mg of pertuzumab every three weeks after the re-administration of the 840 mg dose.

62. 62. The method of claim 60 or claim 61, wherein the cancer patient has HER2-positive breast cancer.

63. 14. The method of any of claims 1 to 13, which reduces the risk of death by about 34% or more compared to patients treated with trastuzumab and a chemotherapy agent.

64. 19. The method of claim 17 or 18, wherein the package insert further provides the overall survival (OS) efficacy data of Example 9 or Table 14.

65. A method for treating HER2-positive metastatic or locally recurrent breast cancer in a patient, comprising administering to the patient pertuzumab, trastuzumab and a taxoid, wherein the patient has been previously treated with trastuzumab and / or lapatinib as adjuvant or neoadjuvant therapy.

66. 66. The method of claim 65, wherein the taxoid is docetaxel, paclitaxel, or nab-paclitaxel.

67. 66. The method of claim 65, wherein the taxoid is paclitaxel or nab-paclitaxel.

68. 68. The method of any one of claims 65 to 67, wherein the patient has been previously treated with trastuzumab as neoadjuvant therapy.

69. A method for treating HER3-low ovarian, primary peritoneal or fallopian tube cancer in a patient, comprising administering pertuzumab and a chemotherapeutic agent to the patient, wherein the HER3-low cancer expresses HER3 mRNA at a concentration ratio of about 2.81 or less as assessed by polymerase chain reaction (PCR).

70. 70. The method of claim 69, wherein the chemotherapeutic agent comprises gemcitabine, carboplatin, paclitaxel, docetaxel, topotecan, or pegylated liposomal doxorubicin (PLD).

71. 71. The method of claim 70, wherein the chemotherapeutic agent comprises paclitaxel or topotecan.

72. 72. The method of any one of claims 69 to 71, wherein the cancer is platinum-resistant or platinum-refractory epithelial ovarian cancer.

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