HK40091923A - Use of her2 dimerization inhibitor pertuzumab and articles comprising her2 dimerization inhibitor pertuzumab - Google Patents

Description

Uses of the HER2 dimerization inhibitor pertuzumab and products containing the HER2 dimerization inhibitor pertuzumab

This application is a second divisional application of the invention application filed on October 11, 2012, with Chinese application number 201811518146.X and invention title "Use of HER2 dimer inhibitor pertuzumab and articles comprising HER2 dimer inhibitor pertuzumab" (original application number 201280061896.3).

This non-provisional application was filed pursuant to 37 CFR §1.53(b) and claims, pursuant to 35 USC §119(e), the rights 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, which are incorporated in their entirety by reference.

Preservation of biological materials

The following biological materials have been deposited at the American Type Culture Collection (12301 Parklawn Drive, Rockville, MD 20852, USA) (ATCC):

The following biological materials have been deposited at the American Type Culture Collection (10801 University Blvd., Manassas, VA 20110-2209, USA) (ATCC):

Invention Field

This invention relates to the use of pertuzumab, a first type of HER2 dimerization inhibitor, and to articles comprising pertuzumab, a first type of HER2 dimerization inhibitor.

Specifically, this invention relates to prolonging progression-free survival in a population of patients with HER2-positive breast cancer; 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 pocket; 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 low-HER3 ovarian cancer, primary peritoneal cancer, or fallopian tube cancer.

It also relates to an article comprising a tubular vial containing pertuzumab and a packaging insert thereon providing safety and/or efficacy data; a method for preparing said article; and a method for ensuring the safe and effective use of pertuzumab in connection therewith.

In addition, the present invention relates to an intravenous (IV) bag containing a stable mixture of pertuzumab and trastuzumab suitable for administration to cancer patients.

Background of the Invention

Members of the HER receptor tyrosine kinase family are important mediators of cell growth, differentiation, and survival. This receptor family includes four distinct members: epidermal growth factor receptor (EGFR, ErbB1, or HER1), HER2 (ErbB2 or ^p185neu ), HER3 (ErbB3), and HER4 (ErbB4 or tyro2). Members of this receptor family have been involved in various types of human malignancies.

Recombinant humanized forms of mouse anti-HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, trastuzumab or U.S. Patent No. 5,821,337) are clinically active in patients with metastatic breast cancer that overexpresses HER2 and have received extensive prior anticancer therapy (Baselga et al., J. Clin. Oncol. 14: 737-744 (1996)).

Trastuzumab received marketing authorization 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 monotherapy or in combination with chemotherapy or hormone therapy in a metastatic context, and as a monotherapy or in combination with chemotherapy as adjuvant therapy for patients with early-stage HER2-positive breast cancer. Trastuzumab-based therapies are now the recommended treatment for patients with early-stage HER2-positive breast cancer who do not have contraindications to its use (prescription information; NCCN Guidelines, version 2.2011). Trastuzumab plus docetaxel (or palitaxacin) is a registered standard of care in the context of first-line treatment for metastatic breast cancer (MBC) (Slamon et al., N Engl J Med. 2001; 344(11): 783-792; Marty et al., J Clin Oncol. 2005; 23(19): 4265-4274).

Although trastuzumab has yielded good results in the treatment of breast cancer, recent data from the lapatinib clinical trial appear to suggest that HER2 plays an active role in tumor biology even with trastuzumab administration (Geyer et al., N Engl J Med 2006; 355: 2733-2743).

Patients are selected for treatment with the HER2 antibody trastuzumab based on HER2 expression. See, for example, 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 2003/0152987, Cohen et al., which involve immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for detecting HER2 overexpression and amplification. Thus, optimal management of metastatic breast cancer now considers not only the patient's general condition, medical history, and receptor status, but also HER2 status.

Pertuzumab (also known as recombinant humanized monoclonal antibody 2C4 (rhuMAb 2C4); Genentech, Inc., South San Francisco) represents the first in a new class of agents called HER dimerization inhibitors (HDIs) and functions to inhibit the formation of active heterodimers or homodimers of HER2 with other HER receptors such as EGFR/HER1, HER2, HER3, and HER4. See, for example, 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).

Pertuzumab has been shown to inhibit key cellular signaling by blocking the formation of HER2-HER3 heterodimers in tumor cells, which leads to reduced tumor proliferation and survival (Agus et al. Cancer Cell 2: 127-37 (2002)).

Pertuzumab has been clinically tested as a single agent in Phase Ia trials in patients with advanced cancer and Phase II trials in patients with ovarian and breast cancer, as well as lung and prostate cancer. In Phase I studies, 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 is generally well tolerated. Tumor regression was achieved in 3 of 20 evaluable patients. Partial responses were confirmed in 2 patients. Stable disease lasting longer 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 2.0–15 mg/kg, the pharmacokinetics of pertuzumab are linear, with mean clearance ranging from 2.69 to 3.74 mL/d/kg and mean terminal elimination half-life ranging from 15.3 to 27.6 days. No antibody against pertuzumab was detected (Allison et al. Pro Am Soc Clin Oncol 22:197 (2003)).

US 2006/0034842 describes a method of treating ErbB-expressing cancers using a combination of anti-ErbB2 antibodies. US 2008/0102069 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 reported the treatment of patients with pre-treatment HER2-positive breast cancer who had progressed during trastuzumab treatment using a combination of trastuzumab and pertuzumab. 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 trastuzumab plus pertuzumab combination therapy in HER2-positive breast cancer patients whose disease had progressed on trastuzumab-based therapy. The authors concluded that further evaluation of the efficacy of this combination therapy is needed to define the overall risks and benefits of this treatment regimen.

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

Patent publications involving HER2 antibodies include: US 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; and 6,054. 297; 6,407,213; 6,639,055; 6,719,971; 6,800,738; 8,075,890; 5,648,237; 7,018,809; 6,267,958; 6,685,940; 6,821,515; 7,060,268; 7,682,609; 7,371,376; 6,127,526; 6,333,398; 6,797,8 14; 6,339,142; 6,417,335; 6,489,447; 7,074,404; 7,531,645; 7,846,441; 7,892,549; 8,075,892; 6,573,043; 6,905,830; 7,129,051; 7,344,840; 7,468,252; 7,674,589; 7,919,254; 6,949,24 5; 7,485,302; 7,498,030; 7,501,122; 7,537,931; 7,618,631; 7,862,817; 7,041,292; 6,627,196; 7,371,379; 6,632,979; 7,097,840; 7,575,748; 6,984,494; 7,279,287; 7,811,773; 7,993,834; 8,076,066; 8,044,017; 7,435,797; 7,850,966; 7,485,704; 7,807,799; 8,142,784; 7,560,111; 7,879,325; 8,241,630; 7,449,184; 8,163,287; 7,700,299; 7,981,418; 8,247,397; and US2010/001 6556; US 2005/0244929; US 2001/0014326; US 2003/0202972; US 2006/0099201; US 2010/015 8899; US 2011/0236383; US 2011/0033460; US 2008/0286280; US 2005/0063972; US 2006/0182 739; US 2009/0220492; US 2003/0147884; US 2004/0037823; US2005/0002928; US 2007/02924 19; US 2008/0187533; US 2011/0250194; US 2012/0034213; US 2003/0152987; US 2005/010094 4; US 2006/0183150; US 2008/0050748; US 2009/0155803; US 2010/0120053; US 2005/0244417 ;US 2007/0026001;US 2008/0160026;US2008/0241146;US 2005/0208043;US 2005/0238640;U S 2006/0034842; US 2006/0073143; US 2006/0193854; US 2006/0198843; US 2011/0129464; U S 2007/0184055; US 2007/0269429; US 2008/0050373; US 2006/0083739; US 2009/0087432; US 2006/0210561; US2002/0035736; US 2002/0001587; US 2008/0226659; US 2002/0090662; US 2006/0046270; US 2008/0108096; US 2007/0166753; US 2008/0112958; US 2009/0239236; US 2 012/0034609; US 2012/0093838; US 2004/0082047; US 2012/0065381; US 2009/0187007; US20 11/0159014; US 2004/0106161; US 2011/0117096; US 2004/0258685; US 2009/0148402; US 200 9/0099344; US 2006/0034840; US 2011/0064737; US 2005/0276812; US 2008/0171040; US 2009 /0202536; US 2006/0013819; US 2012/0107391; US 2006/0018899; US2009/0285837; US 2011/0 117097; US 2006/0088523; US 2010/0015157; US 2006/0121044; US 2008/0317753; US 2006/0 165702; US 2009/0081223; US 2006/0188509; US 2009/0155259; US 2011/0165157; US 2006/02 04505; US 2006/0212956; US 2006/0275305; US2012/0003217; US 2007/0009976; US 2007/002 0261; US 2007/0037228; US 2010/0112603; US 2006/0067930; US 2007/0224203; US 2011/0064 736; US 2008/0038271; US 2008/0050385; US 2010/0285010; US 2011/0223159; US 2008/0102 069; US 2010/0008975; US 2011/0245103; US 2011/0246399; US 2011/0027190; US 2010/029815 6; US 2011/0151454; US 2011/0223619; US 2012/0107302; US 2009/0098135; US 2009/0148435 ; US 2009/0202546; US 2009/0226455; US 2009/0317387; US 2011/0044977; US 2012/0121586.

Invention Overview

In a first aspect, the present invention relates to a method for prolonging progression-free survival for 6 months or longer in a population of patients with HER2-positive breast cancer, comprising administering pertuzumab, trastuzumab, and chemotherapy (e.g., taxanes, such as docetaxel) to patients in the population. Optionally, the method results in an objective response rate of 80% or higher 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, patients in the population are: untreated or recurrent after adjuvant therapy, have a left ventricular ejection fraction (LVEF) ≥50% 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 immunohistochemical (IHC) 3+ and/or fluorescence in situ hybridization (FISH) amplification ratio ≥2.0. Optionally, the method reduces the risk of death by approximately 34% or more compared to patients treated with trastuzumab and chemotherapy.

In another aspect, the present invention relates to a method for treating HER2-positive cancer in a patient population by combining two HER2 antibodies without increasing cardiotoxicity, comprising administering pertuzumab, trastuzumab, and chemotherapy 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). Optionally, the HER2-positive cancer is breast cancer, such as metastatic or locally recurrent, unresectable breast cancer, or de novo stage IV disease.

In another aspect, the present invention relates to an article comprising a tubular vial containing pertuzumab and a packaging insert, wherein the packaging insert provides safety data in Table 3 or Table 4 and/or efficacy data in Table 2, Table 5, Figure 8 or Figure 10.

The present invention also relates to a method for preparing an article comprising packaging a tubular vial containing pertuzumab together with a packaging insert, wherein the packaging insert provides safety data in Table 3 or Table 4 and/or efficacy data in Table 2, Table 5, Figure 8 or Figure 10.

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

Optionally, the product comprises a single-dose tubular vial containing approximately 420 mg of pertuzumab.

Optionally, the packaging insert further includes the warning box from Embodiment 4.

Optionally, the packaging insert further provides overall survival (OS) efficacy data from Example 9 or Table 14.

In another aspect, the present invention relates to a method for treating early HER2-positive breast cancer, comprising administering pertuzumab, trastuzumab, and chemotherapy to a patient with breast cancer, wherein the chemotherapy comprises anthracycline-based chemotherapy (e.g., 5-FU, epirubicin, and cyclophosphamide (FEC)) or carboplatin-based chemotherapy (e.g., docetaxel and carboplatin). Optionally, pertuzumab is administered concurrently with anthracycline-based chemotherapy or carboplatin-based chemotherapy. In one embodiment of the method, pertuzumab administration does not increase cardiotoxicity relative to treatment without pertuzumab. Optionally, this type of treatment for early HER2-positive breast cancer comprises neoadjuvant or adjuvant therapy.

The present invention also relates to a method for treating HER2-positive cancer in a patient, comprising co-administering to the patient a mixture of pertuzumab and trastuzumab from the same intravenous pocket. Such methods optionally further include administration of chemotherapy to the patient.

In one related aspect, the present invention provides an intravenous (IV) bag containing a stable mixture of pertuzumab and trastuzumab suitable for administration to cancer patients. Optionally, the mixture is in a saline solution; for example, containing about 0.9% NaCl or about 0.45% NaCl. Optionally, the IV bag is a 250 mL 0.9% saline polyolefin or polyvinyl chloride infusion bag. 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 can be assessed by one or more assays selected from: color, appearance and clarity (CAC), concentration and turbidity analysis, particulate analysis, size exclusion chromatography (SEC), ion exchange chromatography (IEC), capillary zone electrophoresis (CZE), imaging capillary isopoint focusing (iCIEF), or potency assays.

This invention provides a novel treatment regimen for gastric cancer. Specifically, this invention relates to the treatment of HER2-positive gastric cancer in human subjects using a combination of trastuzumab, pertuzumab, and at least one chemotherapy drug.

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 chemotherapy to the subject.

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

In another aspect, the present invention relates to a method for improving survival in human subjects with HER2-positive gastric cancer, comprising administering pertuzumab, trastuzumab, and chemotherapy to said subjects.

In another aspect, the present invention relates to pertuzumab for the treatment of HER2-positive gastric cancer in human subjects in combination with trastuzumab and chemotherapy.

In another 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 said treatment comprises administration of pertuzumab in combination with trastuzumab and chemotherapy.

In another 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 said treatment comprises administration of trastuzumab in combination with pertuzumab and chemotherapy.

In another aspect, the present invention relates to a kit comprising a container including pertuzumab and instructions for use relating to the administration of pertuzumab in combination with trastuzumab and chemotherapy for the treatment of HER2-positive gastric cancer in a subject.

In another aspect, the present invention relates to a kit comprising a container including trastuzumab and instructions for use relating to the administration of trastuzumab in combination with pertuzumab and chemotherapy for the treatment of HER2-positive gastric cancer in a subject.

In all respects, gastric cancer can be, for example, unresectable locally advanced gastric cancer, or metastatic gastric cancer, or advanced, recurrent gastric cancer after surgery, which may not be suitable for treatment by curative methods known. In all respects, gastric cancer includes adenocarcinoma of the stomach or gastroesophageal junction. In all respects, in one specific embodiment, the patient has not received prior anticancer therapy for metastatic gastric cancer. In all respects, in one specific embodiment, chemotherapy comprises the administration of platinum and/or fluoropyrimidine. In some embodiments, the platinum is cisplatin. In other embodiments, the fluoropyrimidine comprises capecitabine and/or 5-fluorouracil (5-FU). In all respects, the patient's HER2-positive status can be, for example, IHC 3+ or IHC2+/ISH+. In all respects, in one specific embodiment, the treatment improves survival, including overall survival (OS) and/or progression-free survival (PFS) and/or response rate (RR). In all respects, in one specific embodiment, the patient has an ECOG PS of 0-1. In all respects, treatment cycles are generally spaced 4 weeks or less, or 3 weeks or less, or 2 weeks or less, or 1 week or less.

In one specific aspect, the present invention relates to a method for treating HER2-positive unresectable or metastatic gastric or gastroesophageal junction adenocarcinoma in patients who have not received prior chemotherapy for metastatic disease (other than prior adjuvant or neoadjuvant therapy completed more than 6 months prior to current treatment), comprising administering pertuzumab, trastuzumab, cisplatin, and capecitabine and/or fluorouracil (5-FU) to said patient in an amount that improves progression-free survival (PFS) and/or overall survival (OS), wherein said patient has an ECOG PS of 0-1. In one specific embodiment, said patient has not received prior treatment with platinum.

In another aspect, the present invention relates to a method for improving progression-free survival in patients with HER2-positive unresectable or metastatic gastric or gastroesophageal junction adenocarcinoma, comprising administering pertuzumab to said patients in combination with trastuzumab and chemotherapy.

In another aspect, the present invention relates to a method of treating HER2-positive breast cancer in a patient, comprising administering pertuzumab, trastuzumab, and vinorelbine to the patient. Optionally, pertuzumab and trastuzumab are co-administered to the patient via a single intravenous pouch. Optionally, the breast cancer is metastatic or locally advanced. In one embodiment, the patient has not previously received systemic non-hormonal anticancer therapy in a metastatic background.

In another aspect, the present invention relates to a method of treating HER2-positive breast cancer in a patient, comprising administering pertuzumab, trastuzumab, and an aromatase inhibitor (e.g., anastrozole or letrozole) to the patient. Optionally, the breast cancer is hormone receptor-positive advanced breast cancer, wherein the hormone receptor is, for example, estrogen receptor (ER) and/or progesterone receptor (PgR). According to this embodiment of the invention, the patient has not previously received systemic non-hormonal anticancer therapy in a metastatic background. Moreover, the patient optionally receives induction chemotherapy (e.g., containing taxanes).

In another embodiment, the present 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 420 mg doses of pertuzumab every 3 weeks, and further comprising administering a follow-up 840 mg dose of pertuzumab to the patient if the interval between two consecutive 420 mg doses is 6 weeks or longer. Optionally, the method further comprises administering 420 mg of pertuzumab every 3 weeks after the follow-up 840 mg dose. In one embodiment, the cancer patient has HER2-positive breast cancer.

In another aspect, the present invention relates to a method of treating HER2-positive metastatic or locally recurrent breast cancer in a patient, comprising administering pertuzumab, trastuzumab, and a taxane (e.g., docetaxel, palitaxel, or nab-palitaxel) to said patient, said patient having previously been treated with trastuzumab and/or lapatinib as adjuvant or neoadjuvant therapy.

In another aspect, the present invention relates to a method for treating low HER3 ovarian cancer, primary peritoneal cancer, or fallopian tube cancer in a patient, comprising administering pertuzumab and chemotherapy to said patient, wherein said chemotherapy comprises taxanes (e.g., palitaxetine) or topotecan.

In another aspect, the present invention relates to a method for treating low-HER3 ovarian cancer, primary peritoneal cancer, or fallopian tube cancer in a patient, comprising administering pertuzumab and chemotherapy to said patient, wherein said low-HER3 cancer expresses HER3 mRNA at a concentration ratio equal to or less than about 2.81, as assessed by polymerase chain reaction (PCR). In one embodiment, said chemotherapy comprises gemcitabine, carboplatin, palitaxil, docetaxel, topotecan, or PEGylated liposomal doxorubicin (PLD). Optionally, said chemotherapy comprises palitaxil or topotecan. In one embodiment, said cancer is platinum-resistant or platinum-insensitive epithelial ovarian cancer.

This application relates to the following implementation scheme.

1. A method for prolonging progression-free survival for 6 months or longer in a population of patients with HER2-positive breast cancer, comprising administering pertuzumab, trastuzumab, and chemotherapy to patients in said population.

2. The method of implementation scheme 1, which results in an objective response rate of 80% or higher among patients in the said population.

3. The method of implementation scheme 1 or implementation scheme 2, wherein the chemotherapy comprises taxane.

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

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

6. The method of any one of implementation schemes 1 to 5, wherein the patients in said population have not received prior treatment or have relapsed after adjuvant therapy.

7. The method of any one of embodiments 1 to 6, wherein the HER2-positive breast cancer is defined as immunohistochemical (IHC) 3+ and/or fluorescence in situ hybridization (FISH) amplification ratio ≥2.0.

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

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

10. A method for treating HER2-positive cancer in a population of patients with HER2-positive cancer by combining two HER2 antibodies without increasing cardiotoxicity, comprising administering pertuzumab, trastuzumab, and chemotherapy to patients in said population.

11. The method of implementation plan 10, wherein cardiotoxicity in the 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).

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

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

14. An article comprising a tubular vial containing pertuzumab and a packaging insert, wherein the packaging insert provides safety data as shown in Table 3 or Table 4.

15. The article of embodiment 14, wherein the packaging insert provides efficacy data as shown in Table 2, Table 5, Figure 8 or Figure 10.

16. The product of embodiment 14 or embodiment 15, wherein the tubular vial is a single-dose tubular vial containing approximately 420 mg of pertuzumab.

17. A method of preparing an article of articles comprising packaging a tubular vial containing pertuzumab with a packaging insert, wherein the packaging insert provides safety data in Table 3 or Table 4.

18. A method for ensuring the safe and effective use of pertuzumab, comprising packaging a tubular vial containing pertuzumab with a packaging insert, wherein the packaging insert provides safety data in Table 3 or Table 4 and 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 pertuzumab, trastuzumab, and chemotherapy to a patient with breast cancer, wherein the chemotherapy comprises anthracycline-based chemotherapy or carboplatin-based chemotherapy.

20. The method of embodiment 19, wherein the chemotherapy comprises anthracycline-based chemotherapy comprising 5-FU, epirubicin, and cyclophosphamide (FEC).

21. The method of implementation scheme 20, wherein pertuzumab is administered in parallel with anthracycline-based chemotherapy.

22. The method of embodiment 19, wherein the chemotherapy comprises carboplatin-based chemotherapy comprising docetaxel and carboplatin.

23. The method of implementation scheme 22, wherein pertuzumab is administered in parallel with carboplatin-based chemotherapy.

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

25. The method of any one of implementation schemes 19 to 24, which includes neoadjuvant or adjuvant therapy.

26. 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 bag.

27. The method of implementation scheme 26, further comprising administering chemotherapy to the patient.

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

29. The IV bag of embodiment 28, wherein the mixture is in a brine solution.

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

31. The IV bag of any one of embodiments 28 to 30 is a 250 mL 0.9% saline polyolefin or polyvinyl chloride infusion bag.

32. An IV bag according to any one of embodiments 28 to 31, containing a mixture of about 420 mg or about 840 mg of pertuzumab and about 200 mg to about 1000 mg of trastuzumab.

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

34. An IV bag according to any one of embodiments 28 to 33, wherein stability has been evaluated by a method selected from the group consisting of: color, appearance and clarity (CAC), concentration and turbidity analysis, particulate analysis, size exclusion chromatography (SEC), ion exchange chromatography (IEC), capillary zone electrophoresis (CZE), imaging capillary isofocusing (iCIEF), and potency assay.

35. A method for treating HER2-positive gastric cancer in human subjects, comprising administering pertuzumab, trastuzumab, and chemotherapy to a subject with HER2-positive gastric cancer.

36. The method of implementation scheme 35, wherein the gastric cancer includes unresectable, locally advanced gastric cancer, or metastatic gastric cancer, or advanced postoperative recurrent gastric cancer that may not be suitable for curative treatment by known methods, or adenocarcinoma of the stomach or gastroesophageal junction.

37. The method of implementation scheme 35 or implementation scheme 36, wherein the patient has not received prior anticancer therapy for metastatic gastric cancer, has an Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0-1, or has an IHC 3+ or IHC 2+/ISH+ HER2-positive status.

38. The method of any one of embodiments 35 to 37, wherein the chemotherapy comprises platinum (cisplatin) and/or fluoropyrimidine (capecitabine or 5-fluorouracil (5-FU)).

39. The method of implementation scheme 38, wherein pertuzumab, trastuzumab, cisplatin, and capecitabine or 5-FU are administered.

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

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

42. 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 throughout all treatment cycles.

43. The method of implementation plan 42, wherein pertuzumab is administered at a dose of 840 mg for 6 treatment cycles.

44. The method of implementation scheme 42 or implementation scheme 43, wherein the pertuzumab trough level in the subject is maintained at or above about 20 μg/mL.

45. The method of any one of embodiments 42 to 44, further comprising administering trastuzumab and chemotherapy to the subject.

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

47. A method for improving progression-free survival (PFS) in human patients with HER2-positive unresectable or metastatic gastric or gastroesophageal junction adenocarcinoma, comprising administering pertuzumab to said patient in combination with trastuzumab and chemotherapy.

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

49. The method of implementation scheme 48, wherein the patient is co-administered pertuzumab and trastuzumab from a single intravenous bag.

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

51. The method of any one of embodiments 48 to 50, wherein the patient has not previously received systemic non-hormonal anticancer therapy in a metastatic background.

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

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

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

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

56. The method of any one of embodiments 52 to 55, wherein the patient has not previously received systemic non-hormonal anticancer therapy in a metastatic background.

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

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

59. The method of embodiment 17 or embodiment 18, wherein the packaging insert further includes the warning box of embodiment 4.

60. A method of treating a cancer patient, comprising administering to the patient an initial dose of 840 mg of pertuzumab, followed by 420 mg of pertuzumab every 3 weeks thereafter, and further comprising administering an 840 mg dose of pertuzumab if the interval between two consecutive 420 mg doses is 6 weeks or longer.

61. The method of embodiment 60, further comprising administering 420 mg of pertuzumab every 3 weeks after re-administering the 840 mg dose.

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

63. The method of any one of implementation schemes 1 to 13, which reduces the risk of death by approximately 34% or more relative to patients treated with trastuzumab and chemotherapy.

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

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

66. The method of embodiment 65, wherein the taxane is docetaxel, pallitaxel, or nab-pallitaxel.

67. The method of embodiment 65, wherein the taxane is palitaxetine or nab-palitaxetine.

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

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

70. The method of embodiment 69, wherein the chemotherapy comprises gemcitabine, carboplatin, palitaxetine, docetaxel, topotecan, or PEGylated liposomal doxorubicin (PLD).

71. The method of embodiment 70, wherein the chemotherapy comprises palitaxetine or topotecan.

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

Brief description of the attached diagram

Figure 1 provides a schematic diagram of the HER2 protein structure and the amino acid sequences of domains I-IV of its extracellular domain (SEQ ID No. 1-4, respectively).

Figures 2A and 2B illustrate the following amino acid sequence alignments: the variable light ( _VL ) (Figure 2A) and variable heavy ( _VH ) (Figure 2B) domains of mouse monoclonal antibody 2C4 (SEQ ID Nos. 5 and 6, respectively); the _VL and _VH domains of variant 574/pertuzumab (SEQ ID Nos. 7 and 8, respectively); and the human _VL and _VH common framework (humκ1, light subgroup I; humIII, heavy subgroup III) (SEQ ID Nos. 9 and 10, respectively). An asterisk indicates differences between the variable domains of pertuzumab and mouse monoclonal antibody 2C4, or between the variable domains of pertuzumab and the human framework. Complementarity-determining regions (CDRs) are enclosed in parentheses.

Figures 3A and 3B show the amino acid sequences of the pertuzumab light chain (Figure 3A; SEQ ID NO. 11) and heavy chain (Figure 3B; SEQ ID NO. 12). CDRs are shown in bold. The calculated molecular weights of the light and heavy chains are 23,526.22 Da and 49,216.56 Da (cysteine in reduced form), respectively. The carbohydrate module is attached to Asn 299 of the heavy chain.

Figures 4A and 4B show the amino acid sequences of the trastuzumab light chain (Figure 4A; SEQ ID NO. 13) and heavy chain (Figure 4B; SEQ ID NO. 14), respectively. The boundaries between the variable light and variable heavy domains are indicated by arrows.

Figures 5A and 5B show the light chain sequence of the variant pertuzumab (Figure 5A; SEQ ID NO.15) and the heavy chain sequence of the variant pertuzumab (Figure 5B; SEQ ID NO.16), respectively.

Figure 6 shows the study protocol in Example 1. ECOG = Eastern Cooperative Oncology Group; PD = Progressive Disease. Note: Trastuzumab, pertuzumab, and cisplatin were administered via IV infusion on day 1 of each 3-week cycle. Capecitabine was 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 were defined as IHC 3+ or IHC 2+ combined with ISH+ (i.e., IHC 3+/ISH+ or IHC 2+/ISH+); (b) Trastuzumab was administered at a loading dose of 8 mg/kg for cycle 1 and at a dose of 6 mg/kg for subsequent cycles; (c) Pertuzumab was administered at a loading dose of 840 mg for cycle 1 and at a dose of 420 mg for cycles 2-6 on day 1 of each cycle.

Figure 7 illustrates the recruitment, intention to treat, and safety groups and patient exit in the study of Example 3.

Figure 8 shows the Kaplan-Meier curve for progression-free survival (PFS), as evaluated by the Independent Review Board (IRF) for the study in Example 3.

Figure 9 illustrates the PFS according to patient subgroups in the study of Example 3.

Figure 10 illustrates the overall survival in the study of Example 3.

Figure 11 is an overview of the dosing schedule in HER2-positive neoadjuvant breast cancer patients with low cardiac risk factors in Example 5. Additional radiotherapy, hormone therapy, and chemotherapy are permitted after surgery and during adjuvant trastuzumab treatment, if deemed necessary by the investigators.

Figure 12 illustrates the mean change in LVEF (central reading) for the study in Example 5.

Figure 13 shows the pathological complete response (pCR) for the study in Example 5.

Figure 14 illustrates the complete pathological response according to hormone receptor status for the study in Example 5.

Figure 15 shows the pertuzumab/trastuzumab mixture (840 mg) in a 0.9% saline PO IV infusion bag at 30°C. (1) Time = 0; (2) Time = 24 hours. Developed view; full view (inset).

Figure 16 shows the trastuzumab SEC profile of the pertuzumab/trastuzumab mixture (840 mg) in a 0.9% saline PO IV infusion bag at 30°C (1) time = 0; (2) time = 24 hours. Unfolded diagram; full view (inset).

Figure 17 shows the pertuzumab IEC profile of the pertuzumab/trastuzumab mixture in a 0.9% saline POIV infusion bag at 30°C (1) time = 0; (2) time = 24 hours. Full view.

Figure 18 shows the trastuzumab IEC profile of the pertuzumab/trastuzumab mixture in a 0.9% saline PO IV infusion bag at 30°C. (1) Time = 0; (2) Time = 24 hours. Developed view; full view (inset).

Figure 19 illustrates the non-reducing profile of the pertuzumab/trastuzumab mixture in a 0.9% saline PO IV infusion bag at 30°C for (1) time = 0; (2) time = 24 hours. (Expanded diagram)

Figure 20 shows the CE-SDS LIF reduction profile of the pertuzumab/trastuzumab mixture in a 0.9% saline PO IV infusion bag at 30°C (1) time = 0; (2) time = 24 hours. (Expanded diagram)

Figure 21 shows the CZE of the pertuzumab/trastuzumab mixture in a 0.9% saline PO IV infusion bag at 30°C for (1) time = 0; (2) time = 24 hours. Full view.

Figure 22 shows the iCIEF (1) time = 0; (2) time = 24 hours for the pertuzumab/trastuzumab mixture in a 0.9% saline PO IV infusion bag at 30°C. Full view.

Figure 23 shows the potency dose-response curves (μg/mL vs RFU) of pertuzumab/trastuzumab mixture, pertuzumab alone, and trastuzumab alone in a 0.9% saline PO IV infusion bag. (1) Time = 0; (2) Time = 24 hours.

Figure 24 shows the pertuzumab/trastuzumab mixture (1560 mg) in a 0.9% saline PO IV infusion bag as follows: (1) PO 5℃ T0; (2) PO 5℃ T24 hours; (3) PO 30℃ T0; (4) PO 30℃ T24 hours; (5) PVC 5℃ T0; (6) PVC 5℃ T24 hours; (7) PVC 30℃ T0; (8) PVC 30℃ T24 hours. Expanded view; full view (inset).

Figure 25 shows the trastuzumab SEC profile of the pertuzumab/trastuzumab mixture (1560 mg) in a 0.9% saline PO IV infusion bag: (1) PO 5℃ T0; (2) PO 5℃ T24 hours; (3) PO 30℃ T0; (4) PO 30℃ T24 hours; (5) PVC 5℃ T0; (6) PVC 5℃ T24 hours; (7) PVC 30℃ T0; (8) PVC 30℃ T24 hours. Expanded view; full view (inset).

Figure 26 shows the pertuzumab IEC (pertuzumab-rapid) profile of the pertuzumab/trastuzumab mixture (1560 mg) in a 0.9% saline PO IV infusion bag: (1) PO 5℃ T0; (2) PO 5℃ T24 hours; (3) PO 30℃ T0; (4) PO 30℃ T24 hours; (5) PVC 5℃ T0; (6) PVC 5℃ T24 hours; (7) PVC 30℃ T0; (8) PVC 30℃ T24 hours. Full view.

Figure 27 shows the trastuzumab IEC profile of the pertuzumab/trastuzumab mixture (1560 mg) in a 0.9% saline PO IV infusion bag: (1) PO 5℃ T0; (2) PO 5℃ T24 hours; (3) PO 30℃ T0; (4) PO 30℃ T24 hours; (5) PVC 5℃ T0; (6) PVC 5℃ T24 hours; (7) PVC 30℃ T0; (8) PVC 30℃ T24 hours. Full view.

Figure 28 illustrates the research plan for Example 7.

Figure 29 shows the study design of Example 8.

Figure 30 shows the research design of Part 1 of Example 11.

Figure 31 shows the research design of Part 2 of Example 11.

Figure 32 shows the samples and time points taken in the stage IIa gastric cancer (GC) study in Example 1.

Figure 33 shows the demographics of the patient populations in both arms of the GC study (treated with 420 mg (arm A) or 840 mg (arm B) of pertuzumab).

Figure 34 shows the GC history of the patients in arms A and B, respectively.

Figure 35 shows the deployment of GC patients in arms A and B, respectively.

Figure 36 shows the overall response rate in arms A and B of the GC study, respectively.

Figure 37 shows the results of pertuzumab concentration assessment on day 42 in gastric cancer (GC) versus metastatic breast cancer (MBC). The day 42 C-valence was ~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 resulted in a day 42 C-valence ≥20 μg/mL in 90% of patients. The JOSHUA 840/840 mg regimen resulted in a day 42 C-valence comparable to that observed in MBC (CLEO 840/420 mg).

Invention Details

Glossary of abbreviations used in this article: Adverse drug reaction (ADR), Adverse event (AE), Alkaline phosphatase (ALP), Absolute neutrophil count (ANC), Area under 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 ( _Cmax ). Complete Response (CR), Case Report Form (CRF), Computed Tomography (CT), Common Terminology Standard for Adverse Events (CTCAE), Docetaxel (D), Dose-Limiting Toxicity (DLT), Ethics Committee (EC), Epirubicin, Cisplatin and 5-Fluorouracil (ECF), Echocardiography (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 (GCP), Human Epidermal Growth Factor Receptor 2 (HER2), Ion Exchange Chromatography (IEC), Immunohistochemistry (IHC), Independent Reviewing Authority (IRF), Institutional Review Interventional Biometrics (IRB), In situ Hybridization (ISH), Intravenous (IV), Imaging Capillary Isoelectric Focusing (iCIEF), Left Ventricular Ejection Fraction (LVEF), Mitomycin C, Cisplatin and 5-Fluorouracil (MCF), Magnetic Resonance Imaging (MRI), Metastatic Breast Cancer (MBC), Multigate Acquisition (MUGA), Not Significant (NS), Overall Survival (OS), Pathological Complete Response (pCR), Polyolefin (PO), Polyvinyl Chloride (PVC), Progressive Disease (PD), Progression-Free Survival (PFS), Pharmacokinetics (PK), Partial Response (PR), Progesterone Receptor (PgR), Response Evaluation Criteria in Solid Tumors (RECIST), Serious Adverse Events (SAE), Size Exclusion Chromatography (SEC), Stable Disease (SD), Study Management Group (SMT), Sterile Water for Injection (SWFI), Time to Maximum Plasma Concentration ( _tmax ), Upper Limit of Normal (ULN).

I. Definition

As used herein, the term “chemotherapy” refers to a treatment that includes the administration of chemotherapy as defined below.

"Survival" refers to a patient remaining alive, and includes overall survival as well as progression-free survival.

"Overall survival" or "OS" refers to a patient's survival for a defined period of time, such as 1 year or 5 years, from the date of diagnosis or treatment. For the purposes of the clinical trials described in this example, overall survival (OS) is defined as the time from the date the patient population was randomized to the date of death from any cause.

"Progression-free survival" or "PFS" refers to a patient remaining alive without cancer progression or worsening. For the purposes of the clinical trials described in this example, progression-free survival (PFS) is defined as the time from randomization of the study population to the first documented progressive disease or refractory toxicity or death from any cause (whichever occurs first). Disease progression can be documented by any clinically accepted method, such as, for example, radiographically progressive disease as determined by the 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 monitoring for recurrence of subcutaneous lesions in the chest wall.

"Prolonged survival" means extending overall or progression-free survival in patients treated according to the present invention relative to untreated patients and/or patients treated with one or more approved antitumor agents but not treated according to the present invention. In one specific example, "prolonged survival" means extending the progression-free survival (PFS) and/or overall survival (OS) of cancer patients receiving the combination therapy of the present invention (e.g., a combination of pertuzumab, trastuzumab, and chemotherapy) relative to patients treated with trastuzumab and chemotherapy alone. In another specific example, "prolonged survival" means extending the progression-free survival (PFS) and/or overall survival (OS) of cancer patients receiving the combination therapy of the present invention (e.g., a combination of pertuzumab, trastuzumab, and chemotherapy) relative to patients treated with pertuzumab and chemotherapy alone.

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

"Complete response" or "CR" means that all symptoms of cancer disappear in response to treatment. This does not always mean that the cancer is cured.

"Partial response" or "PR" refers to the reduction in size or extent of a tumor or lesion or cancer in the body in response to treatment.

"HER receptors" are receptor protein tyrosine kinases belonging to the HER receptor family, including EGFR, HER2, HER3, and HER4 receptors. HER receptors typically contain an extracellular domain that binds to HER ligands and/or dimers with another HER receptor molecule; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain containing several phosphorylated tyrosine residues. HER receptors can be the "natural sequence" HER receptor or its "amino acid sequence variants." Preferably, the HER receptor is the natural sequence human HER receptor.

The terms “ErbB2” and “HER2” are used interchangeably in this document, referring, for example, to the human HER2 protein described in Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al., Nature 319:230-234 (1986) (Genebank accession number X03363). The term “erbB2” refers to the gene encoding human ErbB2, while “neu” refers to the gene encoding rat p185 ^neu . The preferred HER2 is the naturally occurring human HER2 sequence.

In this document, "HER2 extracellular domain" or "HER2ECD" refers to the extracellular HER2 domain anchored to the cell membrane or circulating in the cell, including fragments thereof. The amino acid sequence of HER2 is shown in Figure 1. In one embodiment, the extracellular domain of HER2 may 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" (approximately 320-488 amino acid residues; SEQ ID NO: 3), and "domain IV" (approximately 489-630 amino acid residues; SEQ ID NO: 4) (without residue numbering for 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 in this paper.

"HER3" or "ErbB3" in this document refers to, for example, the receptor disclosed in U.S. Patent Nos. 5,183,884 and 5,480,968 and Kraus et al., PNAS (USA) 86:9193-9197 (1989).

"Low HER3" cancer is cancer that expresses HER3 at a level below the median level of HER3 expression in that cancer type. In one embodiment, the low HER3 cancer is epithelial ovarian cancer, peritoneal cancer, or fallopian tube cancer. The levels of HER3 DNA, protein, and/or mRNA in the cancer can be assessed to determine whether the cancer is a low HER3 cancer. More information on low HER3 cancer can be found, for example, in U.S. Patent No. 7,981,418. Optionally, a HER3 mRNA expression assay is performed to determine whether the cancer is a low HER3 cancer. In one embodiment, the HER3 mRNA level in the cancer is assessed, for example using polymerase chain reaction (PCR), such as quantitative reverse transcription PCR (qRT-PCR). Optionally, the cancer expresses HER3 at a concentration ratio equal to or less than about 2.81, as assessed by qRT-PCR, for example using a COBAS instrument.

"HER dimer" in this document refers to a non-covalently bound dimer containing at least two HER receptors. Such complexes may form when cells expressing two or more HER receptors are exposed to HER ligands and can be isolated by immunoprecipitation and analyzed by SDS-PAGE, as described, for example, by Sliwkowski et al., J. Biol. Chem. 269(20): 14661-14665 (1994). Other proteins, such as cytokine receptor subunits (e.g., gp130), may bind to the dimer. Preferably, the HER dimer contains HER2.

In this article, "HER heterodimer" refers to a non-covalently linked heterodimer containing at least two different HER receptors, such as EGFR-HER2, HER2-HER3, or HER2-HER4 heterodimer.

"HER antibody" is an antibody that binds to the HER receptor. Optionally, the HER antibody further intervenes in HER activation or function. Preferably, the HER antibody binds to the HER2 receptor. The HER2 antibodies of interest in this paper are pertuzumab and trastuzumab.

"HER activation" refers to the activation or phosphorylation of any one or more HER receptors. Generally, HER activation leads to signal transduction (e.g., phosphorylation of tyrosine residues in the HER receptor or substrate peptide, induced by the intracellular kinase domain of the HER receptor). HER activation can be mediated by HER ligands binding to HER dimers containing the HER receptor of interest. HER ligands binding to HER dimers can activate the kinase domains of one or more HER receptors in the dimer, thereby leading to phosphorylation of tyrosine residues in one or more HER receptors and/or phosphorylation of tyrosine residues in other substrate peptides such as Akt or MAPK intracellular kinases.

"Phosphorylation" refers to the addition of one or more phosphate groups to a protein, such as the HER receptor or its substrate.

Antibodies that “inhibit HER dimerization” are antibodies that inhibit or interfere with the formation of HER dimers. Preferably, such antibodies bind to HER2 at its heterodimer binding site. The most preferred dimerization inhibitory antibodies described herein are 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., J. 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.

"HER2 dimerization inhibitors" are drugs that inhibit the formation of dimers or heterodimers containing HER2.

The "heterodimer binding site" on HER2 refers to a region in the extracellular domain of HER2 that, when forming a dimer with EGFR, HER3, or HER4, contacts or forms an interface with a region in the extracellular domain of EGFR, HER3, or HER4. This region has been found in domain II of HER2 (SEQ ID NO: 15). Franklin et al., Cancer Cell 5:317-328 (2004).

"A HER2 antibody that binds residues in the HER2 antibody-binding domain II (SEQ ID NO: 2) to the heterodimer binding site of HER2, and optionally also binds residues in other domains of the HER2 extracellular domain, such as domains I and III (SEQ ID NO: 1 and 3), and at least partially spatially inhibits the formation of HER2-EGFR, HER2-HER3, or HER2-HER4 heterodimers. Franklin et al., Cancer Cell 5:317-328 (2004) characterized the crystal structure of HER2-pertuzumab stored in the RCSB Protein Database (ID Code IS78), illustrating an exemplary antibody that binds to the heterodimer binding site of HER2."

The antibody binding domain II (SEQ ID NO: 2) binds residues of the antibody binding domain II of HER2 and optionally residues of other domains of HER2, such as domains I and III (SEQ ID NO: 1 and 3, respectively). Preferably, the antibody binding domain II binds to the junction between HER2 domains I, II and III.

For the purposes of this document, the interchangeable terms “pertuzumab” and “rhuMAb 2C4” refer to antibodies comprising the variable light chain and variable heavy chain amino acid sequences of SEQ ID NO: 7 and 8, respectively. Where pertuzumab is a complete 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. Optionally, the antibody is produced from recombinant Chinese hamster ovary (CHO) cells. The terms “pertuzumab” and “rhuMAb 2C4” herein cover biosimilars of drugs having a US Adopted Name (USAN) or International Nonproprietary Name (INN): pertuzumab.

For the purposes of this document, the interchangeable terms “trastuzumab” and “rhuMAb4D5” refer to antibodies comprising the variable light chain and variable heavy chain amino acid sequences from SEQ ID NO: 13 and 14, respectively. Where the trastuzumab is a complete antibody, it preferably comprises an IgG1 antibody; in one embodiment, it comprises the light chain amino acid sequence of SEQ ID NO: 13 and the heavy chain amino acid sequence of SEQ ID NO: 14. Optionally, the antibody is produced by Chinese hamster ovary (CHO) cells. The terms “trastuzumab” and “rhuMAb4D5” herein cover biosimilars of drugs having a United States Adopted Name (USAN) or International Nonproprietary Name (INN): trastuzumab.

The term “antibody” is used in the broadest sense in this article, specifically covering monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity.

The “humanized” form of non-human (e.g., rodent) antibodies refers to chimeric antibodies that contain at least a minimum sequence derived from non-human immunoglobulins. For most, humanized antibodies are human immunoglobulins (receptor antibodies), where residues from the receptor hypervariable region are replaced with residues from the hypervariable region of a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, rabbit, or non-human primate. In some cases, the frame region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the receptor or donor antibody. These modifications are made to further improve antibody performance. Typically, humanized antibodies contain at least one, usually two, variable domains substantially entirely of the following: all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Optionally, humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of the 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 as described in Table 3 of U.S. Patent 5,821,337 (expressly incorporated herein by reference) and as defined herein; and humanized 2C4 antibodies, such as pertuzumab as described and defined herein.

"Intact antibody" in this document refers to an antibody containing two antigen-binding regions and an Fc region. Preferably, an intact antibody has a functional Fc region.

An "antibody fragment" comprises a portion of a complete antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab′, F(ab′) ₂ , and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

"Natural antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, the number of which varies among heavy chains of different immunoglobulin isoforms. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V_{_H} ) at one end, followed by multiple constant domains. Each light chain has a variable domain (V_{_L}) at one end and a constant domain at the other. The constant domains of the light chains align with the first constant domain of the heavy chains, while the variable domains of the light chains align with the variable domains of the heavy chains. Specific amino acid residues are thought to form interfaces between the variable domains of the light and heavy chains.

The term "hypervariate region," as used in this article, refers to the amino acid residues in the antibody responsible for antigen binding. Hypervariate regions typically contain amino acid residues from the complementarity-determining region (CDR) or CDR (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service). ce, National Institutes of Health, Bethesda, MD. (1991)) and/or residues from the “hypervariate ring” (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). “Frame region” or “FR” residues are those variable domain residues other than the hypervariate residues as defined herein.

The term "Fc region" is used herein to define the C-terminal region of the immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. 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 recombinant engineering of the nucleic acid encoding the antibody heavy chain. Therefore, compositions of complete antibodies can comprise antibody populations with all K447 residues removed, antibody populations without any K447 residues removed, and antibody populations containing a mixture of antibodies with and without K447 residues.

Unless otherwise indicated, the residues in the immunoglobulin heavy chain are numbered in the manner of the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), which is explicitly incorporated herein by reference. "EU index as described in Kabat" refers to the residue numbering method used for human IgG1 EU antibodies.

The “functional Fc region” possesses the “effector function” of the native Fc region. Exemplary “effector functions” include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptor; BCR). These effector functions generally require the Fc region to be coupled with a binding domain (e.g., antibody variable domain) and can be assessed using various assays, such as those disclosed herein.

The “natural sequence Fc region” contains the same amino acid sequence as the Fc region found in nature. The natural sequence human Fc region includes the natural sequence human IgG1 Fc region (non-A and A allotypes); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, as well as its naturally occurring variants.

The “mutated/variant Fc region” comprises an amino acid sequence that differs from the amino acid sequence of the native Fc region due to 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 compared to the native Fc region or the Fc region of the parent polypeptide, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably shares at least about 80% homology with the native Fc region and/or the Fc region of the parent polypeptide, most preferably at least about 90% homology, and more preferably at least about 95% homology.

Based on the amino acid sequence of the heavy chain constant domain of intact antibodies, they can be classified into different "classes." There are five main classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different antibody classes are respectively named α, δ, ε, γ, and μ. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

"Naked antibody" refers to an antibody that is not conjugated to a heterologous molecule, such as a cytotoxic module or a radiolabeled substance.

"Affinity-matured" antibodies are those with one or more alterations in one or more of their hypervariable regions, which result in improved affinity for the antigen compared to parental antibodies without such alterations. Preferred affinity-matured antibodies exhibit nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced using procedures known in the art. Marks et al. (Bio/Technology 10:779-783, 1992) described affinity maturation via VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described in the following: 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).

"Deamidated" antibodies are antibodies in which one or more asparagine residues have been derivatized into, for example, aspartic acid, succinimide, or isoaspartic acid.

The terms “cancer” and “cancerous” refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth.

"Gastric cancer" specifically includes metastatic or locally advanced unresectable gastric cancer, including but not limited to histologically confirmed gastric or gastroesophageal junction adenocarcinoma with inoperable (unresectable) locally advanced or metastatic disease that is not suitable for curative treatment, and postoperative recurrence of advanced gastric cancer, such as gastric or gastroesophageal junction adenocarcinoma, when the surgical intent is to cure the disease.

"Late-stage" cancer refers to cancer that has spread beyond its original site or organ through local invasion or metastasis. Therefore, the term "late-stage" cancer includes both locally advanced and metastatic disease.

"Refractory" cancer refers to cancer that continues to progress despite the administration of antitumor agents such as chemotherapy to the cancer patient. An example of refractory cancer is platinum-resistant cancer.

"Recurrent" cancer refers to cancer that grows again at the initial site or a distant site after a response to initial treatment such as surgery.

"Locally recurrent" cancer is cancer that returns to the same location as the previously treated cancer after treatment.

"Unresectable" cancer is cancer that cannot be removed (surgery).

In this article, "early-stage breast cancer" refers to breast cancer that has not yet spread beyond the breast or axillary lymph nodes. This type of cancer is generally treated with neoadjuvant or adjuvant therapy.

"Neoadjuvant therapy" refers to systemic therapy given before surgery.

"Adjunctive therapy" refers to systemic therapies given after surgery.

Metastatic cancer refers to cancer that has spread from one part of the body (such as the breast) to another part of the body.

In this article, "patient" or "subject" refers to a human patient. The patient may be a "cancer patient," that is, a patient who has or is at risk of developing one or more symptoms of cancer (particularly stomach or breast cancer).

"Patient population" refers to a group of cancer patients. This population can be used to demonstrate the statistically significant efficacy and/or safety of drugs such as pertuzumab.

A "relapsed" patient is one who has developed symptoms or signs of cancer after regression. Optionally, the patient relapses after adjuvant or neoadjuvant therapy.

Cancer or biological samples that “display HER expression, amplification, or activation” are cancer or biological samples that express (including overexpress) the HER receptor in diagnostic tests, have an amplified HER gene, and/or otherwise exhibit HER receptor activation or phosphorylation.

Cancer or biological samples that “display HER activation” are those that show HER receptor activation or phosphorylation in diagnostic tests. This type of activation can be determined directly (e.g., by measuring HER phosphorylation via ELISA) or indirectly (e.g., by gene expression profiling or by detecting HER heterodimers, as described herein).

"HER receptor overexpression or amplification" refers to cancer cells that have significantly higher levels of HER receptor protein or genes compared to non-cancerous cells of the same tissue type. This overexpression can be caused by gene amplification or increased transcription or translation. HER receptor overexpression or amplification can be detected in diagnostic or prognostic assays by assessing elevated levels of HER protein on the cell surface (e.g., via immunohistochemistry; IHC). Alternatively/otherwise, the level of nucleic acid encoding HER in cells can be measured, for example by in situ hybridization (ISH), including fluorescence in situ hybridization (FISH; see WO 98/45479, October 1998) and chromogenic in situ hybridization (CISH; see, for example, 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) techniques, such as quantitative real-time PCR (qRT-PCR). HER receptor overexpression or amplification can also be studied by measuring exfoliated antigens (e.g., the HER extracellular domain) in biological fluids such as serum (see, for example, U.S. Patent No. 4,933,294, published June 12, 1990; WO 91/05264, published April 18, 1991; U.S. Patent No. 5,401,638, published March 28, 1995; and Sias et al. J. Immunol. Methods 132:73-80 (1990)). In addition to the above assays, skilled practitioners can utilize a variety of in vivo assays. For example, cells in a patient can be exposed to antibodies optionally labeled with detectable markers such as radioisotopes, and the binding of the antibody to the patient's cells can be assessed, for example, by external scanning for radioactivity or by analyzing biopsy samples taken from patients who have been previously exposed to the antibody.

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. Optionally, HER2-positive cancers have an immunohistochemical (IHC) score of 2+ or 3+ and/or an in situ hybridization (ISH) amplification ratio of ≥2.0.

In this article, "antitumor agent" refers to a drug used to treat cancer. Non-limiting examples of antitumor agents in this article include chemotherapeutic agents, HER dimerization inhibitors, HER antibodies, antibodies against tumor-associated antigens, anti-hormonal compounds, cytokines, EGFR-targeting drugs, anti-angiogenic agents, tyrosine kinase inhibitors, growth inhibitors and growth-inhibiting antibodies, cytotoxic agents, apoptosis-inducing antibodies, COX inhibitors, farnesyltransferase inhibitors, antibodies binding to carcinoembryonic protein CA125, HER2 vaccines, Raf or ras inhibitors, doxorubicin liposomes, topotecan, taxane, dual tyrosine kinase inhibitors, TLK286, EMD-7200, pertuzumab, trastuzumab, erlotinib, and bevacizumab.

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

“Epitope 4D5” refers to the region in the extracellular domain of HER2 where antibody 4D5 (ATCC CRL 10463) binds to trastuzumab. This epitope is close to the transmembrane domain of HER2 and is within domain IV (SEQ ID NO: 4) of HER2. To screen for antibodies that substantially bind to the 4D5 epitope, conventional cross-blocking assays can be performed, as described in Antibodies, A Laboratory Manual, ColdSpring Harbor Laboratory, Ed Harlow, and David Lane (1988). Alternatively, epitope mapping can be performed to assess whether the antibody substantially binds to the 4D5 epitope of HER2 (e.g., any one or more residues in the region from approximately residue 529 to approximately residue 625, containing HER2ECD; residue numbers include the signal peptide).

"Treatment" refers to both therapeutic procedures and protective or preventative measures. Those who require treatment include those who already have cancer and those who want to prevent cancer. Therefore, the patients in this article who require treatment may have already been diagnosed with cancer or may have a predisposition or susceptibility to developing cancer.

The term "effective dose" refers to the amount of a drug that is effective in treating cancer in a patient. An effective dose of a drug can reduce the number of cancer cells; shrink the size of the tumor; inhibit (i.e., slow down to a certain extent and preferably prevent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down to a certain extent and preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more cancer-related symptoms to some extent. In terms of the extent to which a drug can stop growth and/or kill existing cancer cells, it can be cellular inhibition and/or cytotoxicity. An effective dose can prolong progression-free survival (e.g., as measured by RECIST or CA-125 change criteria for evaluating response in solid tumors), result in an objective response (including 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).

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cellular damage. This term is intended to include radioactive isotopes (e.g., radioactive isotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , p ³² , and 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.

"Chemotherapy agents" refer to chemical compounds that can be used to treat cancer. Examples of chemotherapeutic agents used in chemotherapy include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; TLK 286 (TELCYTA ^™ ) ); acetogenins (especially bullatacin and bullatacinone); δ-9-tetrahydrocannabinol (dronabinol); β-lapachone; lapachol; colchicines; betulinic acid Podophyllin; camptothecin (including synthetic analogs topotecan CPT-11 (irinotecan), acetylcamptothecin, scopoletin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid acid); teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, and mechlorethamine oxide. Hydrochlorides, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; bisphosphonates, such as clodronate; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see, for example, Agnew, Chem Intl.Ed.Engl., 33:183-186(1994)) and anthracycline antibiotics such as annamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubicin, KRN5500, menogaril, and anthracycline antibiotics (dynemicin, including dynemicin) A. Esperamicin, neocarzinostatin chromophore and related chromopeptides of enediyne antibiotics, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin C, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin D n), detorubicin, 6-diaza-5-oxo-L-leucine, doxorubicin (including morpholine doxorubicin, cyanomorpholine doxorubicin, 2-pyrrole doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, and 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 and 6-mercaptopurine. ercaptopurine), thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azouridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; and androgens, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenergics, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as folinic acid (leucovorin); aceglatone; anti-folate and anti-vesicle agents such as LY231514 pemetrexed; dihydrofolate reductase inhibitors such as methotrexate; antimetabolites such as 5-fluorouracil (5-FU) and its prodrugs such as UFT, S-1, and capecitabine; and thymidylate synthase inhibitors and glycine ribonucleotide formyltransferase inhibitors such as raltitrexed (TOMUDEX ^RM ). TDX); inhibitors of dihydropyrimidine dehydrogenase such as eniluracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine (defosfamide); demecolcine; diaziquone; elfornithine; elliptinium acetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural) Products, Eugene, OR; Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid acid); triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin, verrucarin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes; chlorambucil; gemcitabine 6 -Thioguanine; mercaptopurine; platinum; platinum analogs or platinum-based analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vincaalkaloid; vinorelbine; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; and any pharmaceutically acceptable salts, acids, or derivatives of the foregoing; and combinations of two or more of the foregoing, such as CHOP (an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone) and FOLFOX (an abbreviation for a treatment regimen of oxaliplatin (ELOXATIN ^™ ) in combination with 5-FU and leucovorin).

Also included within this definition are anti-hormonal agents that act to regulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; aromatase inhibitors; and anti-androgens such as flutamide and nilumethamide. tamide), bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane cytosine analog); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as gene therapy vaccines, e.g., vaccines, vaccines, and vaccines; rIL-2; topoisomerase 1 inhibitors; rmRH; and pharmaceutically acceptable salts, acids, or derivatives of any of the above substances.

Taxanes are a type of chemotherapy that inhibits mitosis and interferes with microtubules. Examples of taxanes include palitaxal (Bristol-Myers Squibb Oncology, Princeton, NJ); nab-palitaxal, an albumin-engineered nanoparticle formulation that does not contain cremophor, or nab-palitaxal (ABRAXANE ^™ ; American Pharmaceutical Partners, Schaumberg, Illinois); and docetaxel (Rorer, Antony, France).

"Anthracycline antibiotics" are a type of antibiotic derived from the fungus Streptococcus peucetius. Examples include daunorubicin, doxorubicin, and epirubicin.

"Anthracycline-based chemotherapy" refers to chemotherapy regimens that consist of or contain one or more anthracycline antibiotics. Examples include 5-FU, epirubicin, and cyclophosphamide (FEC); 5-FU, doxorubicin, and cyclophosphamide (FAC); doxorubicin and cyclophosphamide (AC); epirubicin and cyclophosphamide (EC), etc.

For the purposes of this article, "carboplatin-based chemotherapy" refers to a chemotherapy regimen consisting of or containing one or more carboplatin. An example is TCH (docetaxel/carboplatin and trastuzumab/).

"Aromatase inhibitors" inhibit aromatase, which regulates estrogen production in the adrenal glands. Examples of aromatase inhibitors include: 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formate, fadrozole, vorozole, letrozole, and anastrozole. In one embodiment, the aromatase inhibitor described herein is letrozole or anastrozole.

Antimetabolite chemotherapy uses drugs that are structurally similar to metabolites but cannot be utilized by the body in a productive manner. Many antimetabolite chemotherapy agents interfere with the production of nucleic acids, RNA, and DNA. Examples of antimetabolite chemotherapy agents include gemcitabine, 5-fluorouracil (5-FU), capecitabine (XELODA ^™ ), 6-mercaptopurine, methotrexate, 6-thioguanine, pemetrexed, raltitrexed, arabinosylcytosine (ARA-C cytarabine), dacarbazine, azocytosine, deoxycytosine, pyridmidene, fludarabine, cladrabine, and 2-deoxy-D-glucose.

"Chemotherapy-resistant" cancer refers to cancer that progresses while the patient is receiving chemotherapy (i.e., the patient is "chemotherapy-resistant"), or cancer that progresses within 12 months (e.g., within 6 months) after the patient completes chemotherapy.

The term “platinum” is used in this article to refer to platinum-based chemotherapy, including but not limited to cisplatin, carboplatin, and oxaliplatin.

The term "fluoropyrimidine" as used in this article refers to an antimetabolite chemotherapy, including but not limited to capecitabine, fluorouridine, and fluorouracil (5-FU).

In this article, a “fixed” or “flat” dose of a chemotherapy agent refers to the dose administered to a human patient regardless of the patient’s weight (WT) and body surface area (BSA). Therefore, a fixed or flat dose is not provided as a mg/kg dose or mg/ ^m² dose, but as an absolute amount of the therapeutic agent.

The term "loading" generally refers to the initial dose of the therapeutic agent administered to the patient, followed by one or more maintenance doses. Typically, a single loading dose is administered, but multiple loading doses are also covered herein. Often, the amount of loading dose administered exceeds the amount of maintenance dose administered, and/or loading doses are administered more frequently than maintenance doses, thereby achieving the desired steady-state concentration of the chemotherapeutic agent earlier than with maintenance doses.

The term "maintenance dose" in this document refers to one or more doses of the therapeutic agent administered to a patient during treatment. Typically, maintenance doses are administered at certain treatment intervals, such as approximately once a week, approximately once every two weeks, approximately once every three weeks, or approximately once every four weeks, preferably once every three weeks.

"Infusion" refers to the introduction of a drug-containing solution into the body through a blood vessel for therapeutic purposes. Generally, this is done via an intravenous (IV) bag.

An "intravenous bag" or "IV bag" is a bag capable of holding a solution that can be administered intravenously to a patient. In one embodiment, the solution is an aqueous saline solution (e.g., about 0.9% or about 0.45% NaCl). Optionally, the IV bag is formed of polyolefin or polyvinyl chloride.

"Co-administration" means administering two (or more) drugs intravenously during the same administration period, rather than infusing the two or more drugs consecutively. Generally, this involves combining two (or more) drugs into the same IV bag before their co-administration.

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

The phrase “no increase in cardiotoxicity” for a combination of drugs containing pertuzumab means an incidence of cardiotoxicity equal to or lower than that observed in patients treated with drugs other than pertuzumab in the combination of drugs (e.g., equal to or lower than that from administration of trastuzumab and chemotherapy such as docetaxel).

A "vial" is a container suitable for storing liquids or lyophilized preparations. In one embodiment, the vial is a disposable vial, such as a 20-cc disposable vial with a stopper.

"Packaging inserts" are loose printed materials that the Food and Drug Administration (FDA) or other regulatory agencies mandate to be placed inside the packaging of every prescription drug. These inserts typically contain the drug's trademark, generic name, and mechanism of action; information on indications, contraindications, warnings, precautions, adverse effects, and dosage forms; and instructions for use regarding recommended dosage, timing, and route of administration.

The term "safety data" refers to data obtained in controlled clinical trials that show the prevalence and severity of adverse events to guide users on drug safety, including guidance on how to monitor and prevent adverse reactions to the drug. Tables 3 and 4 in this document provide safety data for pertuzumab. The safety data include any one or more (e.g., two, three, four, or more) of the most common adverse events (AEs) or adverse reactions (ADRs) listed in Tables 3 and 4. For example, the safety data may include information on neutropenia, febrile neutropenia, diarrhea, and/or cardiotoxicity as disclosed herein.

"Efficacy data" refers to data obtained in controlled clinical trials that demonstrate the efficacy of a drug in treating a disease, such as cancer. Efficacy data for pertuzumab are provided in the embodiments described herein. For HER2-positive metastatic or locally recurrent, unresectable breast cancer, efficacy data for pertuzumab are presented in Tables 2, 5, Figures 8 and 10 herein. The safety data include any one or more (e.g., two, three, four or more) of the primary endpoint (progression-free survival, PFS, by IRF) and/or secondary endpoints (overall survival (OS); progression-free survival (PFS, by investigator); objective response rate (ORR), including complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD), and/or duration of response) as disclosed herein. For example, efficacy data may include information regarding progression-free survival (PFS) and/or overall survival (OS) as disclosed herein.

When referring to a mixture of two or more drugs, such as pertuzumab and trastuzumab, the term "stable mixture" means that each drug in the mixture substantially retains its physical and chemical stability within 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, particulate analysis, size exclusion chromatography (SEC), ion exchange chromatography (IEC), capillary zone electrophoresis (CZE), imaging capillary isoelectric focusing (iCIEF), and potency assays. In one embodiment, the mixture has been shown to be stable at 5°C or 30°C for up to 24 hours.

A drug administered "concurrently" with one or more other drugs is administered on the same day as treatment with the one or more other drugs during the same treatment cycle, and optionally, at the same time as the one or more other drugs. For example, in a cancer therapy administered every 3 weeks, the concurrently administered drugs are each administered on day 1 of the 3-week cycle.

II. Antibody and Chemotherapy Combinations

The HER2 antigen to be used for antibody production can be, for example, a soluble form of the extracellular domain of the HER2 receptor or a portion thereof, containing the desired epitope. Alternatively, cells expressing HER2 at their 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 generate antibodies. Other forms of the HER2 receptor that can be used to generate antibodies will be apparent to those skilled in the art.

Various methods exist in the art for preparing the monoclonal antibodies described herein. For example, the hybridoma method first described in Kohler et al., Nature, 256:495 (1975), using a recombinant DNA method (US Patent No. 4,816,567), can be used to prepare monoclonal antibodies.

The anti-HER2 antibodies trastuzumab and pertuzumab used according to this invention are commercially available.

(i) Humanized antibodies

Methods for humanizing nonhuman antibodies have been described in the art. Preferably, the humanized antibody has one or more amino acid residues introduced from a nonhuman source. These nonhuman amino acid residues are often referred to as “input” residues, and they are typically derived from the “input” variable domain. Humanization can be performed in a manner that largely follows the approach of Winter et al. (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)) by replacing the corresponding sequence of the human antibody with a hypervariable region sequence. Thus, such “humanized” antibodies are chimeric antibodies (US Patent No. 4,816,567), in which substantially less than the entire human variable domain is replaced with a corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced with residues from similar sites in rodent antibodies.

The selection of human variable domains for constructing humanized antibodies, including both light and heavy chains, is crucial for reducing antigenicity. Following the so-called "best-fit" approach, an entire library of known human variable domain sequences is screened using the variable domain sequences of rodent antibodies. The human sequence closest to the rodent sequence is then selected as the human frame region (FR) of the humanized antibody (Sims et al., J. Immunol. 151: 2296 (1993); Chothia et al., J. Mol. Biol. 196: 901 (1987)). Another approach uses specific frame regions derived from common sequences of all human antibodies from specific light or heavy chain subgroups. The same frame can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993)).

More importantly, antibodies retain high affinity for antigens and other favorable biological properties after humanization. To achieve this, humanized antibodies are prepared according to a preferred method by analyzing parental sequences and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are widely available and well-known to those skilled in the art. Computer programs are available to diagram and display the possible three-dimensional conformations of selected candidate immunoglobulin sequences. Examining these displayed images allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., analyzing residues that affect the ability of the candidate immunoglobulin to bind its antigen. Thus, FR residues can be selected from the receptor and input sequences and combined to obtain desired antibody characteristics, such as increased affinity for the target antigen. Generally, hypervariable residues are directly and most substantially involved in the effect on antigen binding.

U.S. Patent No. 6,949,245 describes the generation of an exemplary humanized HER2 antibody that binds to HER2 and blocks ligand activation of the HER receptor.

Humanized HER2 antibodies specifically include trastuzumab as described in Table 3 of U.S. Patent 5,821,337 (expressly incorporated herein by reference) and as defined herein, as well as humanized 2C4 antibodies such as pertuzumab as described and defined herein.

The humanized antibodies described herein may, for example, comprise nonhuman hypervariable region residues incorporated into human variable heavy domains, and may also comprise frame region (FR) substitutions at positions selected from the group consisting of 69H, 71H, and 73H, utilizing the variable domain numbering system listed 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 the positions of 69H, 71H, and 73H.

The exemplary humanized antibodies of interest herein comprise the variable heavy domain complementarity-determining residue GFTFTDYTMX (SEQ ID NO: 17), wherein X is preferably D or S; DVNPNSGGSIYNQRFKG (SEQ ID NO: 18); and/or NLGPSFYFDY (SEQ ID NO: 19), optionally including amino acid modifications of those CDR residues, for example wherein the modifications substantially maintain or improve the antibody affinity. For example, antibody variants for use in the methods of the present invention may have about 1 to about 7 or about 5 amino acid substitutions in the above-described variable heavy CDR sequence. Such antibody variants can be prepared by affinity maturation, for example as described below.

For example, in addition to the variable heavy domain CDR residues mentioned above, humanized antibodies may also contain variable light domain complementarity-determining residues KASQDVSIGVA (SEQ ID NO: 20); SASYX1 ^{X2 X3} , wherein ^X1 is preferably R or L, ^X2 is preferably Y or E, and ^X3 is preferably T or S ( ^SEQ ID NO: 21); and/or QQYYIYPYT (SEQ ID NO: 22). Such humanized antibodies may optionally include amino acid modifications to the aforementioned CDR residues, for example, where the modifications substantially maintain or improve the antibody's affinity. For example, the target antibody variant may have about one to about seven or about five amino acid substitutions in the aforementioned variable light CDR sequence. Such antibody variants can be prepared by affinity maturation, for example, as described below.

This application also covers affinity-matured antibodies that bind to HER2. The parent antibody may be a human antibody or a humanized antibody, such as an antibody containing light chain and/or heavy chain variable region sequences of SEQ ID NO: 7 and 8, respectively (i.e., VL and/or VH containing pertuzumab). Affinity-matured variants of pertuzumab preferably bind to the HER2 receptor with a superior affinity to mouse 2C4 or pertuzumab (e.g., an affinity increase of about 2-fold or about 4-fold to about 100-fold or about 1000-fold, based on assessment using a HER2 extracellular domain (ECD) ELISA). Exemplary heavy chain variable region CDR residues for substitution include H28, H30, H34, H35, H64, H96, H99, or combinations of two or more (e.g., 2, 3, 4, 5, 6, or 7 of these residues). Examples of light chain variable region CDR residues used for modification include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97, or combinations of two or more (e.g., 2 to 3, 4, 5, or up to about 10 of these residues).

Humanization of mouse 4D5 antibodies to generate their 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 mouse 4D5 antibodies and possesses secondary immune function (ADCC) that allows the humanized antibody to exhibit targeted cytotoxic activity in the presence of human effector cells. HuMAb4D5-8 contains variable light ( _VL ) CDR residues incorporated into the _VLκ subgroup I common framework and variable heavy ( _VH ) CDR residues incorporated into the _VH subgroup III common framework. The antibody also contains frame region (FR) substitutions at the following locations: 71, 73, 78, and 93 of _VH (Kabat numbers of FR residues); and FR substitution at position 66 of _VL (Kabat number of FR residues). Trastuzumab contains a non-A allogeneic human γ1Fc region.

This encompasses various forms of humanized antibodies or affinity-matured antibodies. For example, humanized antibodies or affinity-matured antibodies can be antibody fragments. Alternatively, humanized antibodies or affinity-matured antibodies can be complete antibodies, such as complete IgG1 antibodies.

(ii) Pertuzumab composition

In one embodiment of the HER2 antibody composition, the composition comprises a mixture of a major class pertuzumab antibody and one or more variants thereof. A preferred embodiment of the major class pertuzumab antibody herein is an antibody comprising the light chain and heavy chain variable domain amino acid sequences of SEQ ID No. 7 and 8, most preferably comprising 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 those sequences). In one embodiment, the composition comprises a mixture of a major class pertuzumab antibody and an amino acid sequence variant comprising an N-terminal leader extension. Preferably, the N-terminal leader extension is located on the light chain of the antibody variant (e.g., on one or both light chains of the antibody variant). The major class HER2 antibody or antibody variant may be a full-length antibody or an antibody fragment (e.g., a Fab or F(ab') ₂ fragment), but preferably both are full-length antibodies. The antibody variant herein may comprise an N-terminal leader extension on any one or more of its heavy or light chains. Preferably, the N-terminal leader extension is located on one or both light chains of the antibody. The N-terminal leader extension preferably contains or is composed of VHS-. The presence of the N-terminal leader extension in the composition can be detected by a variety of analytical techniques, including but not limited to N-terminal sequence analysis, assays of charge heterogeneity (e.g., cation exchange chromatography or capillary zone electrophoresis), mass spectrometry, etc. The amount of antibody variant in the composition is generally within the range that is at least greater than the amount of the major class antibody, from the detection limit constituting any assay (preferably N-terminal sequence analysis) for detecting the variant. Typically, about 20% or less of the antibody molecules in the composition (e.g., from about 1% to about 15%, e.g., from 5% to about 15%) contains the N-terminal leader extension. Such a percentage is preferably determined using quantitative N-terminal sequence analysis or cation exchange analysis (preferably using a high-resolution, weak cation exchange column, such as a PROPACWCX-10 ^™ cation exchange column). In addition to N-terminal leader extension variants, amino acid sequence changes of the major class antibody and/or variants are also included, including but not limited to antibodies containing a C-terminal lysine residue on one or all two heavy chains, deamidated antibody variants, etc.

In addition, major types of antibodies or variants may also contain glycosylation variations. Non-limiting examples include antibodies containing G1 or G2 oligosaccharide structures attached to their Fc regions, antibodies containing carbohydrate modules attached to their light chains (e.g., one or two carbohydrate modules, such as glucose or galactose, attached to one or two light chains of the antibody, for example, attached to one or more lysine residues), antibodies containing one or two non-glycosylated heavy chains, or antibodies containing sialylated oligosaccharides attached to one or two heavy chains.

The composition can be recovered from genetically engineered cell lines expressing HER2 antibodies, such as the Chinese hamster ovary (CHO) cell line, or it can be prepared by peptide synthesis.

For more information on exemplary pertuzumab compositions, see U.S. Patent Nos. 7,560,111 and 7,879,325 and US 2009/0202546A1.

(iii) Trastuzumab composition

Trastuzumab compositions typically comprise a mixture of major antibody species (containing the light and heavy chain sequences of SEQ ID NO: 13 and 14, respectively) and their 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, B 752:233-245 (2001), which relates to trastuzumab forms resolvable by cation exchange chromatography, including peak A (Asn30 deamidated to Asp in two 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 antibody class); peak 4 (Asp102 isomerized to isoAsp in one heavy chain); and peak C (Asp102 is succinimide (Asu) in one heavy chain). Such variant forms and compositions are included in the invention herein.

(iv) 5-FU and cisplatin

There is no single, standard, globally recognized chemotherapy regimen for advanced gastric cancer, but 5-fluorouracil (5-FU) plus cisplatin is widely used for this indication. In phase II studies in patients who had not received prior chemotherapy, 5-FU plus cisplatin produced a response rate of approximately 40% and a median overall survival of 7–10.6 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).

(v) capecitabine

Capecitabine has been extensively tested in patients with advanced gastric cancer. Phase II efficacy results for capecitabine monotherapy showed response rates of 19% and 26%, and overall survival of 8.1 and 10.0 months, respectively (in the study by Koizumi et al. 2003, 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). For capecitabine in combination with platinum, numerous studies have shown response rates ranging from 28% to 65%, time to progression from 5.8 to 9 months, and overall survival 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; 13:1893-8; Park YH, Kim BS, Ryoo BY, et al. Br J Cancer 2006; 94:959-63).

III. Selection of patients for the therapy

HER2 testing can be used to select patients for treatment according to this invention. Several FDA-approved commercial assays are available for identifying HER2-positive cancer patients. These methods include (Dako) and HER2 (immunohistochemical (IHC) assay) and HER2 FISH pharmDx ^™ (FISH assay). Users should refer to the packaging insert of the specific assay kit for information on the validation and performance of each assay.

For example, HER2 overexpression can be detected by IHC analysis, such as using (Dako). Paraffin-embedded tissue sections from tumor biopsies can be subjected to IHC analysis, comparing the results against the following HER2 protein staining intensity criteria:

Score 0: No staining was observed or membrane staining was observed in less than 10% of tumor cells.

Score 1+: Weak/just barely perceptible membrane staining was detected in more than 10% of tumor cells. The cells were stained only in a portion of their membranes.

Score 2+: Weak to moderate complete membrane staining was observed in more than 10% of tumor cells.

Score 3+: Moderate to intense complete membrane staining was observed in more than 10% of tumor cells.

Tumors with a HER2 overexpression score of 0 or 1+ can be characterized as HER2 negative, while those with a score of 2+ or 3+ can be characterized as HER2 positive.

Tumors overexpressing HER2 can be graded based on immunohistochemical scores corresponding to the number of HER2 molecules expressed per cell, and can also be determined by biochemical methods.

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.

HER2 overexpression at levels 3+ leading to ligand-independent activation of tyrosine kinases (Hudziak et al., Proc. Natl. Acad. Sci. USA 84: 7159-7163 (1987)) occurs in approximately 30% of breast cancers, and in these patients, recurrence-free survival and overall survival are reduced (Slamon et al., Science 244: 707-712 (1989); Slamon et al., Science 235: 177-182 (1987)).

The presence of HER2 protein overexpression is highly correlated with gene amplification; therefore, or/otherwise, in situ hybridization (ISH), such as fluorescence in situ hybridization (FISH), can be used to detect gene amplification assays for selecting patients suitable for treatment according to the present invention. FISH assays, such as INFORM ^™ (sold by Ventana, Arizona) or (Vysis, Illinois), can be performed on formalin-fixed, paraffin-embedded tumor tissue to determine the extent (if any) of HER2 amplification in the tumor.

Most commonly, archival paraffin-embedded tumor tissue is used to confirm HER2 positivity using any of the aforementioned methods.

Preferably, HER2-positive patients with an IHC score of 2+ or 3+ or FISH or ISH positivity are selected for treatment according to the present invention.

See also U.S. Patent No. 7,981,418 and Example 11 for other assays for screening patients for pertuzumab treatment.

IV. Pharmaceutical Preparations

Therapeutic formulations of HER2 antibodies prepared according to this invention are stored by mixing an antibody of desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), generally in lyophilized or aqueous form. Antibody crystals are also covered (see U.S. Patent Application 2002/0136719). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used, including buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride). Chloride); phenol, butanol, or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; anti-charge ions that form salts such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN ^™ , PLURONICS ^™ , or polyethylene glycol (PEG). Lyophilized antibody formulations are described in WO 97/04801, which is explicitly incorporated herein by reference.

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 (trastuzumab) formulation is a sterile, white to pale yellow, preservative-free lyophilized powder for intravenous (IV) administration, comprising 440 mg trastuzumab, 400 mg α,α-trehalose dehydrate, 9.9 mg L-histidine-HCl, 6.4 mg L-histidine, and 1.8 mg polysorbate 20, USP. Reconstitution with 20 mL of bacteriostatic water for injection (BWFI) containing 1.1% benzyl alcohol as a preservative yields a multidose solution containing 21 mg/mL trastuzumab at a pH of approximately 6.0. See the trastuzumab formulation information for further details.

Preferred pertuzumab formulations for therapeutic use comprise 30 mg/mL pertuzumab in 20 mM histidine acetate, 120 mM sucrose, 0.02% polysorbate 20, pH 6.0. Alternative pertuzumab formulations comprise 25 mg/mL pertuzumab, 10 mM histidine-HCl buffer, 240 mM sucrose, 0.02% polysorbate 20, pH 6.0.

The placebo formulation used in the clinical trials described in the examples is equivalent to pertuzumab without an active agent.

The formulations described herein may also contain more than one active compound necessary for the specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. Various drugs that can be combined with HER dimerization inhibitors are described in the Methods section below. These molecules are suitably combined in amounts effective for the intended purpose.

Preparations intended for internal administration must be sterile. This can be easily achieved by filtration through a sterile filter membrane.

V. Treatment Methods

In a first aspect of the treatment method described herein, a method is provided for prolonging progression-free survival (PFS) by 6 months or longer in a population of patients with HER2-positive breast cancer, comprising administering pertuzumab, trastuzumab, and chemotherapy (e.g., taxanes such as docetaxel) to patients in the population. Optionally, the patient population comprises an appropriate number of patients (e.g., more than 200, 300, or 400 patients) to allow for the assessment of statistically significant PFS prolongation.

The Phase III CLEOPATRA clinical data in Example 3 below showed that the investigator-assessed median PFS was 12.4 months for placebo plus trastuzumab plus docetaxel and 18.5 months for pertuzumab plus trastuzumab plus docetaxel. Thus, compared with patients who did not receive pertuzumab (i.e., patients who received only trastuzumab and docetaxel), the median PFS was improved by 6 months or longer (e.g., 6.1 months).

In another or alternative implementation, a method for achieving an objective response rate of 80% or higher in a population of patients with HER2-positive breast cancer is provided, comprising administering pertuzumab, trastuzumab, and chemotherapy (e.g., taxanes, such as docetaxel) to patients in the population.

In a related aspect, a method is provided for treating HER2-positive cancer in a patient population by combining two HER2 antibodies without increasing cardiotoxicity, comprising administering pertuzumab, trastuzumab, and chemotherapy to patients in the population. Optionally, the patient population comprises an appropriate number of patients (e.g., more than 200, 300, or 400 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 show that combining pertuzumab and trastuzumab does not exacerbate cardiotoxicity. Cardiotoxicity can be monitored in terms of the incidence of symptomatic left ventricular systolic dysfunction (LVSD) or congestive heart failure (CHF), or a decrease in left ventricular ejection fraction (LVEF), as disclosed, for example, in Example 3 below.

Optionally, the breast cancer is a metastatic or locally recurrent, unresectable breast cancer or de novo stage IV disease, defined as immunohistochemical (IHC) 3+ and/or fluorescence in situ hybridization (FISH) amplification ratio ≥2.0.

Optionally, patients in the population who have not received prior treatment or have relapsed after adjuvant therapy, have a left ventricular ejection fraction (LVEF) of ≥50% at baseline, and/or have an Eastern Cooperative Oncology Performance Status (ECOG PS) of 0 or 1.

In one alternative embodiment, the present invention relates to a method of treating early HER2-positive breast cancer, comprising administering pertuzumab, trastuzumab, and chemotherapy to a patient with breast cancer, wherein the chemotherapy comprises anthracycline-based chemotherapy or carboplatin-based chemotherapy. This aspect of the invention is supported by clinical data in Example 5. In one embodiment, the chemotherapy comprises anthracycline-based chemotherapy, such as 5-FU, epirubicin, and cyclophosphamide (FEC). In an alternative embodiment, the chemotherapy comprises carboplatin-based chemotherapy, such as taxanes (e.g., docetaxel) or carboplatin (e.g., the TCH regimen) in addition to trastuzumab. In one embodiment, pertuzumab is administered concurrently with anthracycline-based chemotherapy or carboplatin-based chemotherapy, for example, wherein pertuzumab, trastuzumab, and chemotherapy are administered in a 3-week cycle, with pertuzumab, trastuzumab, and chemotherapy administered on day 1 of each cycle. Data from the examples presented in this article demonstrate that pertuzumab administration does not increase cardiotoxicity relative to no pertuzumab treatment (i.e., relative to trastuzumab with anthracycline-based chemotherapy (e.g., FEC) and no pertuzumab; or relative to trastuzumab with carboplatin-based chemotherapy and no pertuzumab (i.e., TCH)). Early HER2-positive breast cancer therapies covered in this article include neoadjuvant and adjuvant therapies.

The invention herein also relates to a method for treating HER2-positive cancer in a patient, comprising co-administering to the patient a mixture of pertuzumab and trastuzumab from the same intravenous pocket. 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 HER2-positive breast cancer, etc. Optionally, the method further includes administering chemotherapy to the patient.

In another embodiment, the treatment method of the present invention includes, is essentially composed of, or is composed of: administration of pertuzumab, trastuzumab and chemotherapy such as platinum (e.g., cisplatin) and/or fluoropyrimidine (e.g., capecitabine and/or 5-fluorouracil (5-FU)) to treat HER2-positive gastric cancer.

Specifically, the treatment method of the present invention includes, is essentially composed of, or comprises the following: administration of pertuzumab, trastuzumab, and chemotherapy, such as platinum and/or fluoropyrimidine, such as cisplatin and/or capecitabine and/or 5-fluorouracil (5-FU), to a patient with metastatic gastric cancer, unresectable locally advanced gastric cancer, or postoperative recurrent gastric cancer. In some embodiments, the gastric cancer is not treatable by curative therapy.

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 received systemic non-hormonal anticancer therapy in a metastatic background.

In another aspect, the present invention provides a method for treating HER2-positive breast cancer in a patient, comprising administering pertuzumab, trastuzumab, and an aromatase inhibitor (e.g., anastrozole or letrozole) to the patient. According to this embodiment, the breast cancer is advanced breast cancer, including hormone receptor-positive breast cancer such as estrogen receptor (ER)-positive and/or progesterone receptor (PgR)-positive breast cancer. Optionally, the patient has not previously received systemic non-hormonal anticancer therapy in a metastatic background. The treatment method optionally further includes administering induction chemotherapy (e.g., containing taxanes) to the patient.

The therapy according to the present invention prolongs progression-free survival (PFS) and/or overall survival (OS) of the treated patients.

The antibodies and chemotherapeutic agents were administered to human patients according to known methods. Specific administration regimens and formulations are described in the examples herein.

According to one implementation scheme, pertuzumab is administered _{at a minimum} steady-state C dose of ≥20 μg/mL in 90% of patients receiving pertuzumab and trastuzumab.

According to one specific embodiment of the invention, approximately 840 mg (loading dose) of pertuzumab is administered, followed by one or more doses of approximately 420 mg (maintenance dose) of the antibody. The maintenance dose is preferably administered approximately every 3 weeks for a total of at least 2 doses until clinical progression of disease or refractory toxicity, preferably up to approximately 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 doses or more. Longer treatment periods, including more treatment cycles, are also covered.

According to another specific implementation plan, pertuzumab was administered at a dose of 840 mg for all treatment cycles.

Trastuzumab is typically administered intravenously at a loading dose of approximately 8 mg/kg, followed by doses of 6 mg/kg in subsequent cycles. Trastuzumab is usually administered every 3 weeks until clinical progression of disease or refractory toxicity, preferably up to approximately 17 doses or more.

In one specific implementation, trastuzumab is administered intravenously (IV) on day 1 of each treatment cycle until disease progression or refractory toxicity is assessed by the investigator, at a loading dose of 8 mg/kg in cycle 1 and at a dose of 6 mg/kg for subsequent cycles.

In another specific implementation, pertuzumab is administered via IV infusion on day 1 of each treatment cycle for a total of 6 cycles or until investigator-assessed disease progression or refractory toxicity (whichever occurs first), with a loading dose of 840 mg in cycle 1 and a dose of 420 mg for subsequent cycles, or with a loading dose of 840 mg in cycle 1 and a dose of 840 mg for subsequent cycles.

For the treatment of gastric cancer, cisplatin 80 mg/ ^m² is usually administered via IV infusion on day 1 of each treatment cycle, for a total of at least 6 cycles.

For the treatment of gastric cancer, capecitabine 1000 mg/ ^m² is usually administered orally twice daily, from the evening of day 1 of each cycle to the morning of day 15, for a total of at least 6 cycles. Capecitabine administration may be extended at the discretion of the attending physician after a careful risk-benefit assessment of the individual patient.

Dosage and arrangements of chemotherapy for the treatment of HER2-positive breast cancer are disclosed in the examples below, but other dosages and arrangements are also known and covered in the inventions pursuant to this document.

VI. Products

One embodiment of the product described herein includes an intravenous (IV) bag containing a stable mixture of pertuzumab and trastuzumab suitable for administration to cancer patients. Optionally, the mixture is a saline solution; for example, containing about 0.9% NaCl or about 0.45% NaCl. An exemplary IV bag is a polyolefin or polyvinyl chloride infusion bag, such as a 250 mL IV bag. According to one embodiment of the 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).

Optionally, the mixture in the IV bag is stable at 5°C or 30°C for up to 24 hours. 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, particulate analysis, size exclusion chromatography (SEC), ion-exchange chromatography (IEC), capillary zone electrophoresis (CZE), imaging capillary isoelectric focusing (iCIEF), and potency assays.

In an alternative embodiment, the present invention provides an article of manufacture comprising a tubular vial containing pertuzumab and a packaging insert, wherein the packaging insert provides safety data from Table 3 or Table 4 and/or efficacy data from Table 2, Table 5, Figure 8 or Figure 10. Optionally, the vial is a single-dose vial containing approximately 420 mg of pertuzumab. In one embodiment, the vial is provided in a cardboard carton.

In one related aspect, the present invention relates to a method of preparing an article comprising packaging a reagent vial containing pertuzumab together with a packaging insert, wherein the packaging insert provides safety data in Table 3 or Table 4 and/or efficacy data in Table 2, Table 5, Figure 8 or Figure 10.

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

VII. Preservation of biological materials

The following hybridoma cell lines are deposited at the American Center for Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, USA:

Further details of the invention are illustrated by the following non-limiting embodiments. All disclosures referenced in this specification are expressly incorporated herein by reference.

Example

Example 1: A Phase IIa study evaluating pertuzumab in combination with trastuzumab and chemotherapy in patients with HER2-positive advanced gastric cancer.

Although the incidence and mortality rates of gastric cancer declined sharply worldwide in the second half of the 20th century, it remains the second leading cause of cancer death globally after lung cancer (Parkin, D. Oncogene 23:6329-40 (2004)). The incidence of gastric cancer varies widely across geographic regions (Kelley et al. J Clin Epidemiol 56:1-9 (2003); Plummer et al. Epidemiology of gastric cancer. In: Butlet et al. eds. Mechanisms of carcinogenesis: contribution of molecular epidemiology. Lyon: IARC Scientific Publications No. 157, IARC (2004)). In Japan, South Korea, China, and some Central and South American countries, the incidence rate is 20 to 95 cases per 100,000 people. In contrast, in the United States, India, and Thailand, the incidence rate is 4 to 8 cases per 100,000 people. In Western Europe, the incidence rate ranges from 37 cases per 100,000 people (in some parts of Italy) to 12 cases per 100,000 people (in France). The incidence rate in women follows a similar geographical pattern but is about 50% lower than in men. There is a clear epidemiological difference between cancers located at the cardia (the junction of the stomach and esophagus) and those located in the rest of the stomach. Cardia cancer accounts for 39% of stomach cancer cases in white Americans, but only 4% in Japanese. For reasons that are not yet fully understood, cancers of the cardia and lower esophagus have increased rapidly in developed countries since the 1970s.

To date, surgery remains the only potentially curative treatment for gastric cancer. Survival rates for gastric cancer have improved significantly in Japan in recent years, 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 overall 5-year 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)).

Regardless of geographic location, patients with unresectable disease have a poor prognosis due to locally advanced growth or metastatic spread, with an overall 5-year survival rate ranging from 5% to 15% (Cunningham et al., Annals of Oncology 16 Suppl 1: 122-3 (2005)). For patients with unresectable disease at diagnosis and those with postoperative recurrence, the primary treatment option is chemotherapy (National Comprehensive Cancer Network. NCC Clinical Practice Guidelines in Oncology. Gastric Cancer. Version 1. National Comprehensive Cancer Network, (2006)). Chemotherapy intended for palliative care 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)).

The BO18255 study (ToGA) was a randomized, open-label, multicenter, international, comparative phase III trial designed to evaluate the efficacy and safety of trastuzumab in combination with chemotherapy compared to chemotherapy alone as first-line treatment in patients with unresectable, locally advanced, recurrent, and/or metastatic HER2-positive adenocarcinoma of the stomach or gastroesophageal junction. The primary objective of the study was to compare overall survival in patients treated with trastuzumab in combination with fluoropyrimidine (5-FU or capecitabine) plus cisplatin. Results from the BO18255 study demonstrated a significant clinical benefit when trastuzumab was used in combination with chemotherapy in patients with gastric cancer. Compared to the chemotherapy alone arm, the primary endpoint of overall survival was significantly improved in the trastuzumab plus chemotherapy 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, with a 26% reduction in the risk of death in patients in the trastuzumab plus chemotherapy arm. All other secondary endpoints showed clinical significance with similar hazard ratios and odds ratios. (See, for example, Bang et al., Lancet 28;376(9742):687-97(2010)).

The study results indicate that trastuzumab in combination with cisplatin plus capecitabine or 5-FU is now indicated (including in the EU and the US) for the treatment of patients with HER2-positive metastatic gastric or gastroesophageal junction adenocarcinoma who have not received prior therapy for metastatic disease.

Currently, there is no single, standard, globally accepted chemotherapy regimen for advanced gastric cancer. Despite the success of the ToGA trial, there remains a great need to provide new and effective treatment options for this serious disease. Specifically, new treatments are needed that aim to avoid treatment-related morbidity and/or increase survival in patients with gastric cancer. Therefore, this embodiment is a randomized, multicenter, open-label study evaluating two different doses of pertuzumab in patients with HER2-positive adenocarcinoma of the stomach or gastroesophageal junction. Patients were randomized 1:1 to two treatment arms. Patients in arm A received a loading dose of 840 mg pertuzumab for cycle 1 and 420 mg for cycles 2-6, while patients in arm B received 840 mg pertuzumab for all 6 cycles. Patients in both treatment arms received trastuzumab, cisplatin, and capecitabine. The study protocol is shown in Figure 6. The study length was approximately 24 months (4 months for recruitment and 20 months of follow-up after the last patient was recruited). The study will end when progressive disease occurs in all patients, or when all patients withdraw from or stop the study, whichever comes first.

target group

The trial involved approximately 30 patients.

Patients must meet the following criteria to be eligible for the study :

● Histologically confirmed adenocarcinoma of the stomach or gastroesophageal junction, with inoperable locally advanced or metastatic disease, which cannot be treated with curative therapies.

● Patients with advanced disease who present with postoperative recurrence (when the purpose of the surgery is to cure) are also eligible for admission.

●Measurable diseases, assessed using imaging techniques (computed tomography (CT) or magnetic resonance imaging (MRI)) according to the Response Evaluation Criteria in Solid Tumors (RECIST) v1.1, or non-measurable diseases that can be followed up.

●Limited to HER2-positive tumors with IHC 3+ or IHC 2+ in combination with ISH+, such as those assessed by a central laboratory for primary or metastatic tumors. ISH positivity is defined as a ratio of HER2 gene copy number to CEP17 signal number ≥2.0.

●Formalin-fixed, paraffin-embedded (FFPE) tissue with an invasive tumor of at least 5 mm must be available for center confirmation of HER2 eligibility.

● The Eastern Cooperative Oncology Group (ECOG) status is 0 or 1.

● Baseline left ventricular ejection fraction (LVEF) ≥ 55% (measured by echocardiography (ECHO) or multigate acquisition (MUGA) scan).

●Life expectancy is at least 3 months.

● Male or female.

●Age ≥ 18 years old.

●Sign an informed consent form.

● For women of childbearing potential and male participants with partners of childbearing potential: The patient and/or partner agree to use one highly effective form of non-hormonal contraception or two effective forms of non-hormonal contraception.

● Contraceptive use must continue for at least 6 months after the last dose of the study drug, while the study treatment duration is ongoing.

Patients meeting any of the following criteria were excluded from the study:

● There has been prior chemotherapy for advanced or metastatic disease, with the exception that prior adjuvant or neoadjuvant therapy is permitted if at least 6 months have passed between the completion of adjuvant or neoadjuvant therapy and enrollment in the study.

● Platinum-based adjuvant or neoadjuvant therapies are not permitted.

● Lack of solid integrity of the upper gastrointestinal tract or malabsorption syndrome (e.g., patients with partial or total gastrectomy can be included in the study, but those with jejunostomy probes cannot be included).

● Active (major or uncontrolled) gastrointestinal bleeding.

● Residual toxicities from previous therapies (e.g., neurotoxicity ≥ grade 2 (NCI CTCAE)), except for alopecia.

●Other malignancies within the past 5 years, excluding cervical carcinoma in situ or basal cell carcinoma.

● One of the following anomalous laboratory tests immediately preceding randomization:

Serum total bilirubin > 1.5 times the upper limit of normal (ULN), or for patients known to have Gilbert's syndrome, serum total bilirubin > 2 x ULN.

For patients without liver or bone metastases:

AST or ALT > 2.5 × ULN and alkaline phosphatase (ALP) > 2.5 × ULN

In patients with liver metastases but no bone metastases: AST or ALT > 5 x ULN and ALP > 2.5 x ULN

In patients with liver and bone metastases: AST or ALT > 5 x ULN and ALP > 10 x ULN

In patients with bone metastases but no liver metastases: AST or ALT > 2.5 x ULN and ALP > 10 x ULN

albumin <25g/L

Creatinine clearance rate <60 mL/min

Total white blood cell (WBC) count <2500/μL (<2.5 x ^10⁹ /L)

Absolute neutrophil count (ANC) <1500/μL (<1.5 x ^10⁹ /L)

Platelet count <100,000/μL (<100 x ^10⁹ /L)

●Serious heart disease or medical condition, including but not limited to:

A documented history of heart failure or systolic dysfunction (LVEF < 50%);

High-risk uncontrolled arrhythmias, such as atrial tachycardia with a resting heart rate ≥100/min;

Major ventricular arrhythmias (ventricular tachycardia) or higher-grade AV block (second-degree AV block type 2 (Mobitz II) or third-degree AV block);

Angina pectoris that requires antianginal medication;

Clinically serious valvular heart disease;

Evidence of transmural infarction on ECG; poorly controlled hypertension (e.g., systolic blood pressure >180 mmHg or diastolic blood pressure >100 mmHg);

Difficulty breathing at rest or the need for supplemental oxygen therapy due to late-stage malignant complications or other diseases;

Treatment using long-term or high-dose corticosteroid therapy;

Inhaled steroids and short-acting oral steroids are permitted for use as antiemetics or as gastrointestinal stimulants;

Clinically significant hearing abnormalities; known dihydropyrimidine dehydrogenase deficiency;

A history of brain metastases or clinical evidence; severe, uncontrolled systemic intercurrent illness (e.g., infection or poorly controlled diabetes).

●Pregnancy 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 administered within 4 weeks prior to the start of the study treatment, or within 2 weeks prior to the start of the study treatment (if palliative radiation therapy was administered around the bone metastasis site and the patient recovered from any acute toxicity).

●The patient underwent a major surgery within four weeks prior to the start of the study and had not fully recovered.

● Known active infection with HIV, hepatitis B virus, or hepatitis C virus.

●A known allergy to any of the investigational drugs.

● Failure to comply with follow-up tests or procedures, as determined by the researcher.

Investigational medicine products: dosage, pathway, and regimen

The treatment period lasts up to 3 weeks.

● Trastuzumab is administered intravenously (IV) on day 1 of each cycle until disease progression or refractory toxicity is assessed by the investigator, with a loading dose of 8 mg/kg for cycle 1 and a dose of 6 mg/kg for subsequent cycles.

● Pertuzumab is administered intravenously (IV) on day 1 of each cycle for a total of 6 cycles or until investigator-assessed disease progression or refractory toxicity (whichever occurs first), for each arm as follows:

Arm A: Patients received pertuzumab at a loading dose of 840 mg for cycle 1 and at a dose of 420 mg for cycles 2-6.

Arm B: The patient received 840 mg of pertuzumab for cycles 1-6.

Non-research medical products

The treatment period lasts up to 3 weeks.

● Cisplatin 80 mg/ ^m² was administered via IV infusion on day 1 of each cycle for a total of 6 cycles.

●Capecitabine 1000 mg/ ^m² orally twice daily, from the evening of day 1 of each cycle to the morning of day 15, for a total of 6 cycles. (Capecitabine administration may be extended at the investigator's discretion after careful risk-benefit assessment of individual patients).

preparation

pertuzumab formulation

Each batch of recombinant antibodies produced for clinical purposes meets viral safety requirements as well as sterility requirements of the United States Pharmacopeia and the European Pharmacopoeia. Each batch meets the specifications required for identity, purity, and potency.

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

trastuzumab formulation

The investigational trastuzumab is supplied as a lyophilized preparation, in most countries at a nominal content of 150 mg per vial (via size varies by country).

Trastuzumab is formulated with histidine, trehalose, and polysorbate 20. Once reconstituted, each solution contains 21 mg/mL of the active drug at pH approximately 6.0.

Evaluate

effect

The tumor response assessed by the researchers will be used to summarize the best overall response at the end of cycles 3 and 6 for each treatment arm, defined as patients with a complete or partial response as determined by RECIST.

Security

Safety will be assessed by summarizing adverse events, changes in laboratory test results, and changes in vital signs.

Pharmacokinetics/Pharmacodynamics

The minimum (cup) serum concentration ( _Cmin ) of pertuzumab on day 43 will be evaluated. Additionally, PK parameters such as CL, Vss, AUC, and half-life will be estimated. PK parameter evaluation from data collected up to day 43 will enable modeling and simulation of the estimated dose predicted to be ≥20 μg/mL in 90% of patients with a steady-state cup concentration.

Statistical analysis

Pharmacokinetic Analysis

Individual and mean serum pertuzumab concentration-time data will be listed and plotted according to dose levels. Serum pharmacokinetics of pertuzumab will be summarized by estimating total exposure (area under the curve (AUC)), maximum serum concentration ( _Cmax ), minimum serum concentration ( _Cmin ), time before steady-state _Cmax and _Cmin , total serum clearance, volume of distribution, and elimination half-life (t1/2). Estimates of these parameters will be listed and summarized based on descriptive statistics (mean, standard deviation, minimum, and maximum). Based on the observed pertuzumab serum concentration-time data, population PK methods can be used to estimate the dose required to achieve the target PK concentration.

The observed C- _maxima and C- _min for trastuzumab will be listed and summarized for each specified PK sampling time point using descriptive statistics. For all PK analyses, the actual time of sample collection (not the scheduled time) will be used.

The PK parameters (AUC, _Cmax , t1/2) of pertuzumab will be calculated using a non-compartmental method, and the systemic clearance rate will be obtained from plasma concentration using standard methods.

Analysis of groups

Intended treatment group

All patients who received at least one dose of the investigational drug will be included in the intended treatment population (patients will be assigned to a treatment group that is randomized for analysis purposes).

safe groups

All randomized patients who received at least one dose of an investigational drug will be included in a safety-evaluable population (patients will be assigned to the treatment group).

Sample size

The aim of this study was to evaluate the C- _minimum of pertuzumab on day 43 in patients receiving two different pertuzumab dosing regimens. These data were then analyzed using a population PK model to identify a pertuzumab dose that would achieve a PK target steady-state trough concentration ≥20 μg/mL 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, with a sample size of 15 patients per arm (30 patients in total in this study), the dose required to achieve the desired target concentration can be estimated with acceptable accuracy (coefficient of variation <15%).

Clinical results

The clinical results of this Ha-stage gastric cancer (GC) study are shown in Figures 32-37.

Figure 32 shows the samples taken and the time points.

Figure 33 shows the demographics of the patient populations in both arms of the GC study (treated with 420 mg (arm A) or 840 mg (arm B) of pertuzumab).

Figure 34 shows the GC history of the patients in arms A and B, respectively.

Figure 35 shows the patient distribution in arms A and B, respectively.

Figure 36 shows the overall response rates in arms A and B, respectively.

Security data

Diarrhea was the most common event in 90% of the subjects, and was usually grade 1 or 2, starting in cycle 1; no patients discontinued treatment due to diarrhea.

Grade ≥3 adverse events (AEs) (>13%) included diarrhea, stomatitis, fatigue/asthenia, decreased appetite, hyponatremia, anemia, and neutropenia. Except for neutropenia and hyponatremia (more pronounced in arm A) and decreased appetite (more pronounced in arm B), the incidence of these events was similar in both the standard and high-dose pertuzumab arms.

Asymptomatic changes in ejection fraction (EF), neutropenia, fever, rash, and drug allergic reactions were not associated with higher pertuzumab doses.

Serious adverse events (SAEs) occurred in 60% of patients, and the incidence was not related to high-dose pertuzumab.

Although more patients discontinued treatment in arm B, it is unclear whether this was due to a higher pertuzumab dose, as the events leading to treatment discontinuation were different.

Pharmacokinetic (PK) Results

Figure 37 shows the results of pertuzumab concentration assessment on day 42 in gastric cancer (GC) (JOSHUA) versus metastatic breast cancer (MBC) (CLEOPATRA).

Summary of Results

- The concentration of pertuzumab in the GC was lower than that in the MBC.

● The concentrations during the cycle (i.e., days 7 and 14) were consistent with the expected MBC concentrations because the clearance rate was linear at these higher concentrations.

● The tank level using an 840/420 mg dose was approximately 37% lower than that in the CLEOPATRA trial (Example 3), most likely due to nonlinear clearance at lower concentrations (incomplete receptor saturation).

The 840/840 mg dose in the GC provides a cell concentration similar to the 840/420 mg dose in the MBC.

- Covariates have no effect on PK.

in conclusion

Based on the pertuzumab PK in GC, an 840/840 mg dose will be used to treat gastric cancer. This dose is expected to maintain gastric levels above the target >20 μg/mL in 90% of patients and provide similar gastric levels as those observed in MBC.

Example 2: 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, randomized, open-label, multicenter clinical study designed to evaluate the efficacy of pertuzumab in combination with trastuzumab and chemotherapy in patients with HER2-positive, locally advanced, or metastatic gastric cancer.

Patients in the treatment arm received trastuzumab, cisplatin, and capecitabine and/or 5-fluorouracil. In the other arm, patients received either placebo or pertuzumab.

Treatment plan :

Pertuzumab:

The dosage for cycles 1-6 is 840mg.

Trastuzumab

A loading dose of 8 mg/kg was followed by 6 mg/kg every 3 weeks.

Capecitabine

1000mg/ ^m² bid d1-14 q3w x 6

5-Fluorouracil

800 mg/ ^m² /day, continuous IV infusion, days 1-5, every 3 weeks, 6 times a week.

Cisplatin

800mg/ ^m² q3w x 6

Primary endpoint:

Overall Survival (OS)

Secondary endpoint :

Progression-free survival (PFS), time to disease progression (TTP), objective response rate (ORR), clinical benefit rate, duration of response, Qol, safety, pain intensity, analgesic consumption, weight change, and pharmacokinetics.

Primary patient selection criteria

Inclusion criteria :

●Adenocarcinoma of the stomach or GEJ

● Inoperable, locally advanced and/or metastatic disease

●Measurable (RECIST), or non-measurable assessable diseases

●HER2-positive tumors: IHC 2+ or 3+ and/or ISH+

●Adequate organ function and ECOG performance ≤2

● Written informed consent form

Exclusion criteria

●Adjuvant chemotherapy within 6 months

●Chemotherapy for advanced diseases

● Congestive heart failure or baseline LVEF <50%

● Creatinine clearance rate < 60 mL/min

The treatments described in this article (including administration of pertuzumab, trastuzumab, and chemotherapy such as cisplatin and capecitabine) are expected to meet the primary endpoint (OS). Specifically, the treatments described in this article are expected to be effective in the treated gastric cancer patients, for example, by prolonging survival relative to treatment with trastuzumab and chemotherapy alone, including overall survival (OS) and/or progression-free survival (PFS) and/or time to progression (TTP) and/or objective response rate (ORR).

Example 3: Results of a Phase III, randomized, double-blind, placebo-controlled registry trial (CLEOPATRA) evaluating the efficacy and safety of placebo plus trastuzumab plus docetaxel versus pertuzumab plus trastuzumab plus docetaxel in patients with previously untreated HER2-positive metastatic breast cancer.

Regimens for evaluating pertuzumab in HER2- positive metastatic breast cancer can be found at http://clinicaltrials.gov/ct2/show/NCT00567190 and US 2009/0137387 and WO2009/154651.

This embodiment relates to clinical data obtained from a randomized, double-blind, placebo-controlled phase III trial in patients with HER2-positive MBC who had not received chemotherapy or biologic therapy for their metastatic disease. Patients were randomized 1:1 to receive either placebo plus trastuzumab plus docetaxel or pertuzumab plus trastuzumab plus docetaxel. The primary endpoint was progression-free survival (PFS) based on tumor assessment. PFS was defined as the time from randomization to the first recorded radiographically progressive disease (PD) (Therasse et al. J Natl Cancer Inst 92:205-16 (2000)) or death from any cause (if within 18 weeks of the patient's last tumor assessment) according to the RECIST version 1.0 (Therasse et al. J Natl Cancer Inst 92:205-16 (2000)). Secondary endpoints included overall survival (OS), investigator-assessed PFS, objective response rate (ORR), and safety.

Patients: Eligible patients were centrally confirmed to be HER2 positive (defined as immunohistochemical (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)), with locally recurrent, unresectable, or metastatic breast cancer, or de novo stage IV disease. Patients were ≥18 years old, had a left ventricular ejection fraction (LVEF) at baseline (measured by echocardiography or multigate acquisition), and an Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 or 1. Patients may have received one hormone therapy for MBC, or neoadjuvant or adjuvant systemic breast cancer therapy (including trastuzumab and/or taxanes) prior to randomization, provided they had a disease-free interval of ≥12 months between completion of neoadjuvant or adjuvant therapy and diagnosis of metastatic disease. Exclusion criteria included treatment for MBC (other than those listed above); central nervous system metastases; experience of cumulative exposure to >360 mg/ ^m² of doxorubicin or its equivalents; experience of LVEF decreasing to <50% during or after trastuzumab treatment; currently uncontrolled hypertension; history of impaired cardiac function; impaired bone marrow, kidney, or liver function; currently known HIV, HBV, or HCV infection; pregnancy; breastfeeding; and refusal to use non-hormonal contraceptives.

Protocol: Patients receive a loading dose of 8 mg/kg trastuzumab, followed by a maintenance dose of 6 mg/kg every 3 weeks until investigator-assessed radiographic or clinical PD, or refractory toxicities. Docetaxel is administered at an initial dose of 75 mg/ ^m² every 3 weeks, increasing to 100 mg/ ^m² if tolerated. Investigators may reduce the dose by 25% to 55 mg/ ^m² or 75 mg/ ^m² (if the patient has undergone dose escalation) to manage tolerability, as per protocol. At least 6 cycles of docetaxel are recommended. Pertuzumab or placebo is administered at a fixed loading dose of 840 mg followed by 420 mg every 3 weeks until investigator-assessed radiographic or clinical PD, or refractory toxicities. In cases where chemotherapy is discontinued due to cumulative toxicities, antibody therapy continues until PD, unacceptable toxicities, or consent to withdrawal. All medications are administered intravenously.

Assessment: PFS was assessed every 9 weeks by each center and IRF using standard RECIST-accepted methodologies until PD was assessed by IRF. LVEF was assessed at baseline, every 9 weeks during treatment, at treatment discontinuation, every 6 months in the first year after treatment discontinuation, and then annually for up to 3 years during the follow-up period. Laboratory parameters and ECOG status were assessed at each cycle. Adverse events (AEs) were continuously monitored and classified according to NCI-CTCAE version 3.0. All cardiac events and serious adverse events (SAEs) occurring at treatment discontinuation were followed up until resolution or stabilization was achieved up to 1 year after the final dose. Cardiac events and treatment-related SAEs occurring after treatment were also monitored.

result

Study population: A total of 808 patients were recruited and randomized to receive either placebo plus trastuzumab plus docetaxel (n=406) or pertuzumab plus trastuzumab plus docetaxel (n=402) (Figure 7). Baseline characteristics were similar across treatment arms (Table 1).

Table 1: Baseline characteristics of the intended treatment group

*Including Native Americans and Alaskan Natives

No prior chemotherapy or biological therapy

In the context of new support/support or transfer

Progression-free survival: Compared with placebo plus trastuzumab plus docetaxel, treatment with pertuzumab plus trastuzumab plus docetaxel significantly improved PFS-IRF (stratified by pretreatment status and region) (HR = 0.62; 95% CI 0.51 to 0.75; p < 0.0001) (Figure 8). Median PFS-IVRF was prolonged by 6.1 months from 12.4 months with placebo plus trastuzumab plus docetaxel to 18.5 months with pertuzumab plus trastuzumab plus docetaxel. PFS benefit was observed in all predefined subgroups (Figure 9).

The investigators' assessment of PFS closely matched the PFS-IRF. The median PFS assessed by the investigators 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 to 0.78; p < 0.0001).

Key secondary efficacy endpoint: Interim analysis of OS was performed when 43% of the events planned for the final OS analysis (n = 165) 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 to 0.88; p = 0.0053) did not meet the O’Brien-Fleming stopping boundary of the Lan-DeMets α-exhaustion function used for this survival period analysis (HR ≤ 0.603, p ≤ 0.0012) and was therefore not statistically significant. However, the data showed a strong trend indicating a bias towards the survival benefit of pertuzumab plus trastuzumab plus docetaxel. At the data cutoff, the median overall survival (OS) for patients followed up in both treatment arms was 19.3 months (Kaplan-Meier estimate). The objective response rate (ORR) was 69.3% in the placebo plus trastuzumab plus docetaxel arm and 80.2% in the pertuzumab plus trastuzumab plus docetaxel arm. The difference in response rate between treatment arms was 10.8% (95% CI 4.2 to 17.5; p = 0.0011) (Table 2).

Table 2: Overall Response Rate

IRF, Independent Review Board

Treatment exposure: For placebo plus trastuzumab plus docetaxel and pertuzumab plus trastuzumab plus docetaxel, the median number of treatment cycles per patient was 15 and 18, respectively, with median treatment duration estimated at 11.8 and 18.1 months, respectively. Dose reductions for placebo, pertuzumab, or trastuzumab were not permitted. Patients received a median of 8 cycles of docetaxel in each arm. Based on the safety cohort, 61 (15.4%) patients in the placebo plus trastuzumab plus docetaxel arm received an increased docetaxel dose to 100 mg/ ^m² in any cycle, compared to 48 (11.8%) patients in the pertuzumab plus trastuzumab plus docetaxel arm. The median docetaxel dose intensity was 24.8 mg/ ^m² /week in the placebo plus trastuzumab plus docetaxel arm and 24.6 mg/ ^m² /week in the pertuzumab plus trastuzumab plus docetaxel arm. The reasons for permanent discontinuation of treatment in all studies are presented in Figure 7.

Tolerability and Cardiac Safety: The profile of adverse events (AEs) during the treatment period generally balanced between treatment arms (Table 3). With pertuzumab plus trastuzumab plus docetaxel, the following AEs (all grades) had a high incidence of >5%: diarrhea, rash, mucosal inflammation, fever, neutropenia, and dry skin.

Table 3: Adverse events (all grades) with an incidence of ≥25% in any arm or a difference of ≥5% between arms in the safety population, and grade ≥3 adverse events with an incidence of ≥2%.

AE, adverse events

The incidence of the following ≥ grade 3 adverse events was >2% when pertuzumab plus trastuzumab plus docetaxel was used: neutropenia, fever, and diarrhea (Table 3). The incidence of ≥ grade 3 fever and neutropenia was 12% in the placebo plus trastuzumab plus docetaxel arm and 26% in the pertuzumab plus trastuzumab plus docetaxel arm in patients from Asia; in all other geographic regions, the incidence was ≤10% in both arms.

LVSD (all grades) was reported more frequently in the placebo plus trastuzumab plus docetaxel arm than in the pertuzumab plus trastuzumab plus docetaxel arm (8.3% and 4.4%, respectively). Grade ≥3 LVSD was reported in 2.8% of patients receiving placebo plus trastuzumab plus docetaxel, compared to 1.2% of patients receiving pertuzumab plus trastuzumab plus docetaxel. In patients with post-baseline LVEF assessment, a decrease in LVEF from ≥10 percentage points at baseline to <50% was reported at any stage during treatment in 6.6% of patients in the placebo plus trastuzumab plus docetaxel arm and in 3.8% of patients in the pertuzumab plus trastuzumab plus docetaxel arm.

In the safety cohort, the primary cause of death in both treatment arms was attributed to disease progression (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 a similar number of patients died from adverse events (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 AEs.

discuss

These data show that the combination of the anti-HER2 monoclonal antibodies pertuzumab and trastuzumab with docetaxel prolongs PFS in patients with HER2-positive MBC in the first-line setting. Treatment with pertuzumab plus trastuzumab plus docetaxel exceeded expectations, resulting in a statistically significant reduction in the risk of PFS (HR = 0.62) and a median PFS improvement of 6.1 months.

The combination was well tolerated, and pertuzumab did not increase the rate of symptomatic or asymptomatic cardiac dysfunction. Prior to the data presented in this paper, treatment with two HER2 antibodies was expected to exacerbate cardiotoxicity. However, these data indicate that this was not the case, based on the following tests used in this paper to assess cardiotoxicity: symptomatic left ventricular systolic dysfunction (LVSD), including the incidence of congestive heart failure (CHF) and a decrease in left ventricular ejection fraction (LVEF).

Pertuzumab-related adverse events (AEs), including skin rash, mucosal inflammation, and dry skin, were mostly mild. Treatment with pertuzumab plus trastuzumab plus docetaxel was associated with an increased rate of ≥ grade 3 diarrhea, fever, and neutropenia. The control arm in CLEOPATRA showed PFS similar to that of a previous randomized study that demonstrated a median PFS of 11.7 months in the combination of trastuzumab and docetaxel in HER2-positive MBC (Marty et al. J Clin Oncol 23:4265-74 (2005)).

Unbound by any single theory, these data indicate that targeting HER2-positive tumors with two anti-HER2 monoclonal antibodies with complementary mechanisms of action produces more comprehensive HER2 blockade and highlight the clinical importance of preventing ligand-dependent HER2 dimer formation for optimal HER2 signaling silencing. This study shows that combination HER2 blockade using trastuzumab and pertuzumab improves outcomes in patients with advanced HER2-positive disease in a first-line background. These data are significant because they support the use of the first-approved HER2 dimerization inhibitor for the treatment of patients with HER2-positive cancer.

Example 4: Products containing pertuzumab

The Phase III clinical data from Example 3 were used to develop an article comprising a tubular vial (e.g., a single-dose vial) containing pertuzumab and a packaging insert providing information about its safety and/or efficacy, and a method of preparing the article comprising packaging the pertuzumab in the tubular vial (e.g., a single-dose vial) together with the packaging insert having prescribing information about pertuzumab (as described below).

Pertuzumab is a sterile, clear to slightly milky white, colorless to pale yellow IV infusion solution. Each disposable 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.

Pertuzumab is supplied in single-dose vials containing a preservative-free liquid concentrate at a concentration of 30 mg/mL, ready for immediate infusion. Each vial contains a total of 420 mg of pertuzumab. Store the vials in a refrigerator at 2°C to 8°C (36°F to 46°F) until use. Keep the vials in their outer cardboard box to protect them from light.

Complete prescription information

1. Indications and Usage

Pertuzumab is indicated for use in combination with trastuzumab and docetaxel to treat patients with HER2-positive metastatic breast cancer who have not received prior anti-HER2 therapy or chemotherapy for metastatic disease.

2. Dosage and administration

2.1 Recommended dosage and timing

The starting dose of pertuzumab is 840 mg administered intravenously over 60 minutes, followed by 420 mg every 3 weeks via intravenous infusion over 30 to 60 minutes. When used in combination with pertuzumab, the recommended starting dose of trastuzumab is 8 mg/kg administered intravenously over 90 minutes, followed by 6 mg/kg every 3 weeks via intravenous infusion over 30 to 90 minutes. When used in combination with pertuzumab, the recommended starting dose of docetaxel is 75 mg/ ^m² administered intravenously. This dose may be increased to 100 mg/ ^m² every 3 weeks if the starting dose is well tolerated.

2.2 Dosage Modification

For delayed or missed doses, if the interval between two consecutive infusions is less than 6 weeks, a 420 mg dose of pertuzumab should be administered. Do not wait until the next scheduled dose. If the interval between two consecutive infusions is 6 weeks or longer, an initial dose of 840 mg pertuzumab should be administered via intravenous infusion over 60 minutes, followed by a 420 mg dose administered via intravenous infusion over 30 to 60 minutes every 3 weeks thereafter. The infusion rate of pertuzumab may be slowed or interrupted if the patient experiences an infusion-related reaction. If the patient experiences a severe anaphylactic reaction, the infusion should be stopped immediately [see Warnings and Precautions (5.2)].

Left ventricular ejection fraction (LVEF):

For any of the following conditions, pertuzumab and trastuzumab administration should be suspended for at least 3 weeks:

●LVEF reduced to below 40% or

●LVEF is 40% to 45%, with an absolute reduction of 10% or more below the pre-treatment value [see Warnings and Prevention (5.2)]

If LVEF has recovered to above 45% or is associated with an absolute reduction of 40% to 45% below the pre-treatment value, then pertuzumab can be continued.

If LVEF does not improve or further decreases after a repeat assessment within approximately 3 weeks, discontinuation of pertuzumab and trastuzumab should be strongly considered unless the individual patient's benefit is deemed to outweigh the risk [see Warnings and Precautions (5.2)]. If trastuzumab treatment is paused or discontinued, pertuzumab should also be paused or discontinued. If docetaxel is discontinued, treatment with pertuzumab and trastuzumab can continue. Dose reduction is not recommended for pertuzumab. For docetaxel dose modifications, see the docetaxel prescribing information.

2.3 Preparations for application

Administer by intravenous infusion only. Do not administer by intravenous push or bolus. Do not mix pertuzumab with other medications.

Preparation : The solution for infusion is prepared as follows, using aseptic techniques:

● Before application, parenteral drug products should be visually inspected for microparticles and discoloration.

● Take out an appropriate volume of pertuzumab solution from the tubular bottle.

● Dilute to 250 mL of 0.9% sodium chloride in a PVC or non-PVC polyolefin infusion bag.

● Mix the diluted solution by gently inverting it. Do not shake.

● Apply immediately once prepared.

● If the diluted infusion solution is not used immediately, it can 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.

3. Dosage form and intensity

Pertuzumab 420mg/14mL (30mg/mL) in a single-use tubular vial

4 contraindications

none

5 Warnings and Prevention

5.1 Embryo-fetal toxicity

Pertuzumab can cause fetal harm when administered to pregnant women. Treatment of pregnant rhesus monkeys with pertuzumab resulted in oligohydramnios, delayed fetal kidney development, and embryo-fetal death. If pertuzumab is administered during pregnancy, or if a patient becomes pregnant while receiving the drug, the patient should be informed of the potential risks to the fetus [see Use in Specific Populations (8.1)]. Verify pregnancy status before initiating pertuzumab. Warn patients about the risks of embryo-fetal death and birth defects, and the need for contraception during and after treatment. Advise patients to contact their healthcare provider immediately if they suspect they may be pregnant. If pertuzumab is administered during pregnancy, or if a patient becomes pregnant while receiving pertuzumab, report the exposure immediately to Genentech Adverse Events at 1-888-835-2555. Women who may have been exposed during pregnancy are encouraged to participate in the MotHER Pregnancy Registry by contacting 1-800-690-6720 [see Patient Advice Information (17)]. Monitoring for oligohydramnios in patients who become pregnant during pertuzumab treatment. If oligohydramnios occurs, perform fetal testing appropriate for gestational age and consistent with community standards of care. The efficacy of intravenous hydration in managing oligohydramnios due to pertuzumab exposure is unknown.

5.2 Left ventricular dysfunction

Decreased LVEF has been reported with drugs that block HER2 activity, including pertuzumab. In randomized trials, pertuzumab in combination with trastuzumab and docetaxel was not associated with an increased incidence of symptomatic left ventricular systolic dysfunction (LVSD) or decreased LVEF compared to placebo in combination with trastuzumab and docetaxel [see Clinical Studies (14.1)]. Left ventricular dysfunction occurred in 4.4% of patients in the pertuzumab treatment group and 8.3% of patients in the placebo group. Symptomatic left ventricular systolic dysfunction (congestive heart failure) occurred in 1.0% of patients in the pertuzumab treatment group and 1.8% of patients in the placebo group [see Adverse Reactions (6.7)]. Patients who have received prior anthracycline antibiotics or prior radiotherapy to the chest region may have a higher risk of decreased LVEF. Pertuzumab has not been studied in patients with ≤50% of their pre-treatment LVEF, a history of CHF, a decrease in LVEF to <50% during prior trastuzumab therapy, or conditions that may impair left ventricular function such as uncontrolled hypertension, recent myocardial infarction, serious arrhythmias requiring treatment, or prior cumulative exposure to anthracyclines >360 mg/ ^m² of doxorubicin or its equivalents. LVEF should be assessed regularly (e.g., every 3 months) before initiating pertuzumab and during treatment to ensure it remains within the institution’s normal limits. If LVEF is <40%, or an absolute decrease of 40% to 45% along with 10% or more below the pre-treatment value, pertuzumab and trastuzumab should be discontinued and LVEF reassessed within approximately 3 weeks. If LVEF does not improve or decreases further, pertuzumab and trastuzumab should be discontinued unless the individual patient’s benefit outweighs the risk [see Dosage and Administration (2.2)].

5.3 Infusion-related reactions, allergic reactions/allergies

Pertuzumab is associated with infusion and allergic reactions [see Adverse Reactions (6.1)]. Infusion reactions are defined in randomized trials as any event described as an allergic reaction, anaphylactic reaction, acute infusion reaction, or cytokine release syndrome that occurs during or on the day of infusion. The starting dose of pertuzumab is administered one day prior to trastuzumab and docetaxel to allow for examination of pertuzumab-related reactions. On day 1, when pertuzumab alone was administered, the overall frequency of infusion reactions was 13.0% in the pertuzumab treatment group and 9.8% in the placebo treatment group. Less than 1% were grade 3 or 4. The most common infusion reactions (≥1.0%) were fever, chills, fatigue, headache, asthenia, anaphylaxis, and vomiting. During the second cycle, when all drugs were administered on the same day, the most common infusion reactions (≥1.0%) in the pertuzumab treatment group were fatigue, taste disturbance, anaphylaxis, myalgia, and vomiting. In randomized trials, the overall frequency of allergic/anaphylactic reactions was 10.8% in the pertuzumab treatment group and 9.1% in the placebo group. The incidence of grade 3-4 allergic/anaphylactic reactions was 2% in the pertuzumab treatment group and 2.5% in the placebo group, according to the National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE) (Version 3). Overall, 4 patients in the pertuzumab treatment group and 2 patients in the placebo group experienced allergic reactions. Patients were closely monitored for 60 minutes after the first infusion and 30 minutes after subsequent pertuzumab infusions. If a major infusion-related reaction occurred, the infusion was delayed or interrupted and appropriate medical treatment was administered. Patients were carefully monitored until the symptoms and signs completely resolved. Permanent discontinuation was considered in patients with severe infusion reactions [see Dosage and Administration (2.2)].

5.4 HER2 Test

Detection of HER2 protein overexpression is essential for selecting patients eligible for pertuzumab therapy, as these are the only studied patients who have demonstrated benefit [see Indications and Use (1) and Clinical Studies (14)]. In randomized trials, patients with breast cancer need to have evidence of HER2 overexpression, defined as 3+ IHC (by Dako) or a FISH amplification ratio ≥2.0 (by the Dako HER2 FISH PHARMDX ^™ assay kit). Limited data are available for patients who are positive for breast cancer by FISH but do not demonstrate protein overexpression by IHC. HER2 status assessment should be performed by a laboratory proficient in the specific techniques used. Inappropriate assay performance, including the use of suboptimal fixative tissue, failure to utilize specialized reagents, deviation from specific assay instructions, and failure to include appropriate controls for assay validation, can lead to unreliable results.

6 Adverse Reactions

The following adverse reactions are discussed in more detail in other sections of the chart:

● Embryo-fetal toxicity [see Warnings and Precautions (5.1)]

●Left ventricular dysfunction [see Warnings and Prevention (5.2)]

●Infusion-related reactions, allergic reactions/allergies [see Warnings and Prevention (5.3)]

6.1 Clinical Trial Experience

Because clinical trials are conducted under widely varying conditions, the adverse reaction rates observed in a drug's clinical trials cannot be directly compared to rates in another drug's clinical trials and may not reflect rates observed in clinical practice. Pertuzumab has been evaluated in over 1400 patients with various malignancies in clinical trials, and treatment with pertuzumab is primarily in combination with other anti-tumor agents.

Adverse events described in Table 4 were identified in 804 patients with HER2-positive metastatic breast cancer treated in a randomized trial. Patients were randomized to receive 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 in the pertuzumab group and 11.8 months in the placebo group. Dose adjustments to pertuzumab or trastuzumab were not permitted. The rate of adverse events leading to permanent discontinuation of all study treatments was 6.1% in the pertuzumab group and 5.3% in the placebo group. Adverse events led to discontinuation of docetaxel alone in 23.6% of patients in the pertuzumab group and 23.2% in the placebo group. Table 4 reports adverse events occurring in at least 10% of patients in the pertuzumab group. The most common adverse reactions (>30%) seen with pertuzumab in combination with trastuzumab and docetaxel were diarrhea, alopecia, neutropenia, nausea, fatigue, rash, and peripheral neuropathy.

The most common NCI-CTCAE (version 3) grade 3-4 adverse events (>2%) were neutropenia, febrile neutropenia, leukopenia, diarrhea, peripheral neuropathy, anemia, asthenia, and fatigue. In Asian patients, an increased incidence of febrile neutropenia was observed in both treatment arms compared to patients of other races and geographic regions. In Asian patients, the incidence of febrile neutropenia was higher in the pertuzumab treatment group (26%) than in the placebo treatment group (12%).

Table 4: Summary of adverse events occurring in ≥10% of patients in the pertuzumab treatment arm during randomized trials

*In this table, it indicates adverse reactions that have been reported and are associated with fatal outcomes.

The following clinically relevant adverse reactions were reported in <10% of patients in the pertuzumab treatment group:

Skin and subcutaneous tissue disorders: paronychia (7.1% in the pertuzumab group vs. 3.5% in the placebo group); respiratory, chest, and septal 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: allergies (10.1% in the pertuzumab group vs. 8.6% in the placebo group).

Adverse reactions reported in patients receiving pertuzumab and trastuzumab after discontinuation of docetaxel.

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

6.2 Immunogenicity

Like all therapeutic proteins, there is a possibility of an immune response to pertuzumab. Antibodies against pertuzumab were tested in patients in randomized trials 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 group tested positive for anti-pertuzumab antibodies. None of these 34 patients experienced any allergic reactions or anaphylactic reactions clearly associated with anti-therapeutic antibodies (ATAs). The presence of pertuzumab at the expected level in patient serum at the time of ATA sampling may interfere with the assay's ability to detect anti-pertuzumab antibodies. Additionally, the assay can detect antibodies against trastuzumab. As a result, the data may not accurately reflect the true occurrence of anti-pertuzumab antibody formation. Immunogenicity data are highly dependent on the sensitivity and specificity of the assay used. Furthermore, the occurrence of positive results observed in the assay can be influenced by several factors, including sample handling, timing of sample collection, drug interference, concomitant drug therapy, and underlying disease. For these reasons, comparing the occurrence of antibodies against pertuzumab with the occurrence of antibodies against other products may be misleading.

7 Drug Interactions

No drug-drug interactions were observed between pertuzumab and trastuzumab, or between pertuzumab and docetaxel.

8. Use in specific groups

8.1 Pregnancy

Pregnancy Category D

Risk Summary

There are no best-case or appropriate studies of pertuzumab in pregnant women. Based on findings in animal studies, pertuzumab administration to pregnant women may cause fetal harm. The effects of pertuzumab are likely to be present throughout all three months of pregnancy. Administration of pertuzumab to pregnant macaques resulted in oligohydramnios, delayed fetal kidney development, and embryo-fetal death at clinically relevant exposures at doses 2.5 to 20 times higher than _{the maximum} recommended human dose based on C. If pertuzumab is administered during pregnancy, or if a patient becomes pregnant while receiving pertuzumab, the patient should be informed of the potential risks 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 Genentech Adverse Events at 1-888-835-2555. Women who may have been exposed during pregnancy are encouraged to participate in the MotHER Pregnancy Registry by contacting 1-800-690-6720 [see Patient Advice Information (17)].

Animal data

Repeatable toxicity studies have been conducted in rhesus monkeys. Pregnant monkeys were treated with loading doses of 30 to 150 mg/kg pertuzumab at day of gestation (GD) 19, followed by doses of 10 to 100 mg/kg every 2 weeks. These dose levels resulted in clinically relevant exposures 2.5 to 20 times higher than _{the maximum} recommended human dose based on C. Intravenous administration of pertuzumab from GD19 to GD50 (organogenesis period) was embryotoxic, with a dose-dependent increase in embryo-fetal death at GD25 to GD70. The incidence of embryo-fetal loss was 33%, 50%, and 85% for maternal monkeys treated with pertuzumab doses of 10, 30, and 100 mg/kg every two weeks (2.5 to 20 times higher than _{the maximum} recommended human dose based on C), respectively. At GD100 cesarean section, oligohydramnios, reduced relative lung and kidney weight, and microscopic evidence of renal dysplasia consistent with delayed kidney development were identified in all pertuzumab dose groups. Pertuzumab exposure was reported in offspring from all treatment groups at levels ranging from 29% to 40% of maternal serum levels in GD100.

8.3 Breastfeeding mothers

It is unknown whether pertuzumab is excreted in human milk, but human IgG is excreted in human milk. Given that many drugs are excreted in human milk and the possibility of serious adverse reactions from pertuzumab in breastfeeding infants, a decision should be made regarding whether to discontinue breastfeeding or discontinue the drug, taking into account the elimination half-life of pertuzumab and the importance of the drug to the mother [see Warnings and Precautions (5.1), Clinical Pharmacology (12.3)].

8.4 Pediatric use

The safety and efficacy of pertuzumab have not yet been established in pediatric patients.

8.5 Used by the elderly

Of the 402 patients who received pertuzumab in the randomized trial, 60 (15%) were ≥65 years old and 5 (1%) were ≥75 years old. No overall differences in the efficacy and safety of pertuzumab were observed between these patients and younger patients. Based on population pharmacokinetic analysis, no significant differences in the pharmacokinetics of pertuzumab were observed between patients <65 years old (n=306) and patients ≥65 years old (n=175).

8.6 Women with fertility potential

Pertuzumab administration during pregnancy can cause embryo-fetal harm. Advice is provided to patients regarding pregnancy prevention and planning. Women of childbearing potential are advised to use effective contraception during and for 6 months after receiving pertuzumab. If pertuzumab is administered during pregnancy, or if a patient becomes pregnant while receiving pertuzumab, report the exposure immediately to Genentech Adverse Events at 1-888-835-2555. Women who may have been exposed during pregnancy are encouraged to participate in the MotHER Pregnancy Registry by contacting 1-800-690-6720 [see Patient Advice Information (17)].

8.7 Kidney injury

No dose adjustment of pertuzumab is required in patients with mild (creatinine clearance [CLcr] 60 to 90 mL/min) or moderate (CLcr 30 to 60 mL/min) renal impairment. Due to the limited availability of pharmacokinetic data, dose adjustment cannot be recommended for patients with severe renal impairment (CLcr less than 30 mL/min) [see Clinical Pharmacology (12.3)].

8.8 Liver damage

No clinical studies have been conducted to evaluate the impact of liver injury on the pharmacokinetics of pertuzumab.

10 Overdose

To date, there have been no reports of pertuzumab overdose.

11 Description

Pertuzumab is a recombinant humanized monoclonal antibody that targets the extracellular dimer domain (subdomain II) of the human epidermal growth factor receptor 2 (HER2) protein. Pertuzumab is generated using recombinant DNA technology in mammalian cell (Chinese hamster ovary) cultures containing the antibiotic gentamicin. Gentamycin was not detected in the final product. Pertuzumab has an approximate molecular weight of 148 kDa. Pertuzumab is a sterile, clear to slightly milky white, colorless to pale yellow intravenous infusion solution. 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.

12 Clinical Pharmacology

12.1 Mechanism of Action

Pertuzumab targets the extracellular dimerization domain (subdomain II) of human epidermal growth factor receptor 2 (HER2), thereby blocking ligand-dependent heterodimerization of HER2 with other HER family members, including EGFR, HER3, and HER4. As a result, pertuzumab inhibits intracellular signaling initiated via ligands of two major signaling pathways: mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3K). Inhibition of these signaling pathways leads to cell growth arrest and apoptosis, respectively. Additionally, pertuzumab mediates antibody-dependent cell-mediated cytotoxicity (ADCC). Although pertuzumab alone inhibits the proliferation of human tumor cells, the combination of pertuzumab and trastuzumab significantly increases antitumor activity in a HER2-overexpressing xenograft model.

12.2 Pharmacokinetics

Pertuzumab exhibits linear pharmacokinetics across a dose range of 2–25 mg/kg. Based on a population PK analysis of 481 patients, the median clearance (CL) of pertuzumab was 0.24 L/day and the median half-life was 18 days. Steady-state concentrations of pertuzumab were reached after the first maintenance dose, using an initial dose of 840 mg followed by a maintenance dose of 420 mg every 3 weeks. Population PK analysis showed no PK differences based on age, sex, or ethnicity (Japanese vs. non-Japanese). Baseline serum albumin levels and lean body weight, as covariates, had only minor effects on PK parameters. Therefore, dose adjustments based on weight or baseline albumin levels were not required. No drug-drug interactions were observed between pertuzumab and trastuzumab or between pertuzumab and docetaxel in a sub-study of 37 patients in a randomized trial. No specific renal injury studies for pertuzumab have been conducted. Based on population pharmacokinetic analysis, pertuzumab exposure in patients with mild (CLcr 60–90 mL/min, n = 200) and moderate renal impairment (CLcr 30–60 mL/min, n = 71) was similar to exposure in patients with normal renal function (CLcr > 90 mL/min, n = 200). No relationship between CLcr and pertuzumab exposure was observed within the observed CLcr range (27–244 mL/min).

12.3 Cardiac Electrophysiology

In a subgroup of 20 patients with HER2-positive breast cancer in a randomized trial, the effect of a starting dose of 840 mg pertuzumab followed by a maintenance dose of 420 mg every 3 weeks on the QT interval was evaluated. No large changes (i.e., greater than 20 ms) in the mean QT interval from placebo were detected in the trial based on the Fridricia correction method. Due to limitations in the trial design, small increases in the mean QT interval (i.e., less than 10 ms) could not be ruled out.

13 Non-clinical toxicities

13.1 Carcinogenic effects, mutagenicity, and reproductive damage

Long-term animal studies to assess the carcinogenic potential of pertuzumab have not been conducted. Studies to assess the mutagenic potential of pertuzumab have not been conducted. Animal-specific fertility studies to assess the efficacy of pertuzumab have not been conducted. No adverse effects on male and female reproductive organs were observed in repeated-dose toxicity studies lasting up to 6 months in rhesus monkeys.

14 Clinical Studies

14.1 Metastatic breast cancer

This randomized trial was a multicenter, double-blind, placebo-controlled study of 808 patients with HER2-positive metastatic breast cancer. Breast tumor specimens showing HER2 overexpression defined as 3+ IHC or a FISH amplification ratio ≥2.0 (as determined in a central laboratory) were required. Patients were randomized 1:1 to receive either placebo plus trastuzumab and docetaxel or pertuzumab plus trastuzumab and docetaxel. Randomization was stratified by prior therapy (with or without adjuvant/neoadjuvant anti-HER2 therapy or chemotherapy) and geographic region (Europe, North America, South America, and Asia). Patients receiving adjuvant or neoadjuvant therapy were required to have a disease-free interval of more than 12 months prior to trial enrollment. Pertuzumab was administered as an initial dose of 840 mg followed by 420 mg intravenously every 3 weeks. Trastuzumab was administered as an initial dose of 8 mg/kg followed by 6 mg/kg intravenously every 3 weeks. Patients were treated with pertuzumab and trastuzumab until disease progression, consent to discontinuation, or unacceptable toxicity. Docetaxel was administered intravenously at a starting dose of 75 mg/ ^m² every 3 weeks for at least 6 cycles. The docetaxel dose may be increased to 100 mg/ ^m² at the investigator's discretion if the starting dose was well tolerated.

At the primary analysis, the mean number of study treatment cycles administered was 16.2 in the placebo group and 19.9 in the pertuzumab group.

The primary endpoint of randomized trials is progression-free survival (PFS) as assessed by an independent review body (IRF). PFS is defined as the time from the randomization date to the date of disease progression or death (from any cause) (if death occurs within 18 weeks of the last oncology assessment). Other endpoints include overall survival (OS), PFS (investigator-assessed), objective response rate (ORR), and duration of response.

Patient demographics and baseline characteristics were balanced across the treatment arms. The median age was 54 (range 22–89 years), 59% were white, 32% were Asian, and 4% were Black. All were female, with two exceptions. 17% of patients were recruited in North America, 14% in South America, 38% in Europe, and 31% in Asia. Tumor prognostic characteristics, including hormone receptor status (positive 48%, negative 50%), presence of visceral disease only (78%), and presence of non-visceral disease (22%), were similar across the study arms. Approximately half of the patients had received preadjuvant or neoadjuvant anti-HER2 therapy or chemotherapy (placebo 47%, pertuzumab 46%). Among patients with hormone receptor-positive tumors, 45% had received preadjuvant hormone therapy, while 11% had received hormone therapy for metastatic disease. 11% of patients had received preadjuvant or neoadjuvant trastuzumab.

This randomized trial demonstrated a statistically significant improvement in IRF-assessed PFS in the pertuzumab treatment group compared to the placebo group [hazard ratio (HR) = 0.62 (95% CI: 0.51, 0.75), p < 0.0001] and a 6.1-month increase in median PFS (median PFS was 18.5 months in the pertuzumab group versus 12.4 months in the placebo group) (see Figure 8). The investigator-assessed PFS results were comparable to those observed for IRF-assessed PFS. Consistent results were observed in several patient subgroups, including age (<65 or ≥65 years), race, geographic region, prior adjuvant/neoadjuvant anti-HER2 therapy or chemotherapy (with or without), and prior adjuvant/neoadjuvant trastuzumab (with or without). 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 non-visceral metastases (n=178), the hazard ratio was 0.96 (95% CI: 0.61, 1.52).

During the PFS analysis, 165 patients died. More deaths occurred in the placebo group (23.6%) compared to the pertuzumab group (17.2%). During the OS analysis, results were immature and did not reach the pre-specified stop boundary for statistical significance. See Table 5 and Figure 10.

Table 5: Summary of power from randomized trials

*The HR and p-values for the overall survival period analysis did not reach the predefined stopping boundaries (HR≤0.603, p≤0.0012).

16. How to supply/store and operate

16.1 How to supply

Pertuzumab is supplied in single-use tubular vials containing a preservative-free solution of 420 mg/14 mL (30 mg/mL). NDC 50242-145-01. Store the vials in a refrigerator at 2°C to 8°C (36°F to 46°F) until use. Keep the vials in their outer cardboard box to protect them from light.

Do not freeze. Do not shake.

17 Patient Advice

Inform pregnant women and women of childbearing potential that pertuzumab exposure may cause fetal harm, including embryo-fetal death or birth defects [see Warnings and Precautions (5.1) and Use in Specific Populations (8.1)].

Inform women of childbearing potential to use effective contraception during pertuzumab treatment and for 6 months following the last dose of pertuzumab. [See Warnings and Prevention (5.1) and Use in Specific Populations (8.6)]

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

Women exposed to pertuzumab during pregnancy are encouraged to participate in the MotHER Pregnancy Registry by contacting 1.800-690-6720 [see Warnings and Prevention (5.1) and Use in Specific Populations (8.1)].

Thus, the comprehensive Phase III safety and efficacy data, as described in Example 3, are provided for the product described in this example. This product can be used to ensure the safe and effective use of pertuzumab in treating patients.

Example 5: Early Breast Cancer Therapy Using Pertuzumab

Anthracycline antibiotics (usually used in combination with 5-FU and cyclophosphamide) play an important role in the management of breast cancer. Romond et al. NEJM353(16):1673-1684 (2005) and Poole et al. NEJM355(18):1851-1852 (2006).

Taxane is also part of the standard regimen for the treatment of breast cancer, in combination with anthracycline antibiotics in a regimen called TAC (Martin et al. NEJM 352(22):2302-2313(2005)) or in sequence with anthracycline antibiotics in a regimen called AC->T (Romond et al., see above; Joensuu et al. NEJM 354(8):809-820(2006)).

Carboplatin is an active and well-tolerated chemotherapeutic agent, and studies have shown its significant efficacy in breast cancer in combination with taxane 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)).

A previous neoadjuvant study (NeoSphere) evaluated pertuzumab in combination with docetaxel and trastuzumab (Gianni et al. Cancer Research 70(24)(Suppl.2)(December 2010)), rather than in combination with anthracycline-based or carboplatin-based chemotherapy.

In this embodiment, the following chemotherapy regimens are evaluated:

FEC breast cancer therapy consists of 5-fluorouracil, epirubicin, and cyclophosphamide.

FEC->T sequential chemotherapy consists of an FEC chemotherapy process followed by a docetaxel process.

Chemotherapy regimens for TCH HER2-positive breast cancer, including combination of taxane (docetaxel), carboplatin, and trastuzumab.

The treatment arm in this study was:

Arm A

The process of 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) followed by docetaxel (T) (FEC->T), along with trastuzumab and pertuzumab administered at the start of the chemotherapy regimen (i.e., concurrently with anthracycline antibiotics).

The treatment regimen consisted of 5-fluorouracil (500 mg/ ^m² ), epirubicin (100 mg/ ^m² ), followed by cyclophosphamide (600 mg/ ^m² ) for three cycles, then docetaxel for three cycles, along with trastuzumab (8 mg/kg on day 1 of the first treatment with epirubicin and 6 mg/kg every 3 weeks thereafter) and pertuzumab (840 mg on day 1 of the first treatment with FEC and 420 mg every 3 weeks thereafter). The starting dose of docetaxel was 75 mg/ ^m² for cycle 4 (the first docetaxel cycle), and then 100 mg/ ^m² for cycles 5-6, provided no dose-limiting toxicities occurred. All drugs were administered via IV.

or

Arm B

FEC->T, along with trastuzumab and pertuzumab administered at the start of taxane treatment (i.e., after anthracycline antibiotics).

The treatment regimen consisted of 5-fluorouracil (500 mg/ ^m² ), epirubicin (100 mg/ ^m² ), followed by cyclophosphamide (600 mg/ ^m² ) for three cycles, then docetaxel for three cycles, along with trastuzumab (8 mg/kg on day 1 of the first docetaxel treatment and 6 mg/kg every 3 weeks thereafter) and pertuzumab (840 mg on day 1 of the first docetaxel treatment and 420 mg every 3 weeks thereafter). The starting dose of docetaxel for cycle 4 (the first docetaxel cycle) was 75 mg/ ^m² , and then 100 mg/ ^m² for cycles 5-6, provided no dose-limiting toxicities occurred. All drugs were administered via IV.

or

Arm C

Taxane (docetaxel), carboplatin, and trastuzumab (TCH), along with pertuzumab, were administered at the start of chemotherapy.

On day 1, carboplatin (using the Calvert formula with AUC6) was followed by docetaxel, along with trastuzumab (8 mg/kg on day 1 and 6 mg/kg every 3 weeks for the first treatment with carboplatin and docetaxel) and pertuzumab (840 mg on day 1 and 420 mg every 3 weeks) for a total of 6 cycles. The dose of docetaxel was 75 mg/ ^m² for all cycles. All drugs were administered via IV.

All patients received trastuzumab every 3 weeks for up to one year from the start of treatment (cycles 1-17 for patients in arms A and C, and cycles 4-20 for patients in arm B), regardless of whether they received additional chemotherapy.

Main purpose

The primary objective is assessed when all patients have received 6 cycles of neoadjuvant therapy, undergone surgery and retrieved all necessary samples, or withdrawn from the study (whichever comes first).

Secondary objective

Preliminary assessment of the activity associated with each regimen, such as as indicated by the complete pathological response rate.

The safety profile of each treatment regimen, including preoperative (neoadjuvant) and postoperative (adjuvant) treatment, was assessed.

The study investigated overall survival, time to clinical response, time to response, disease-free survival, and progression-free survival for each treatment arm.

The study may identify biomarkers that are relevant to the primary and secondary efficacy endpoints for each treatment arm.

The study investigated the rate of breast-conserving surgery in all patients with T2-3 tumors who were scheduled for mastectomy at the time of diagnosis.

An overall assessment of the risks and benefits of each option will be conducted.

Research Design Overview

This is a phase II open-label, randomized, multicenter trial evaluating the tolerability and activity of trastuzumab and pertuzumab as neoadjuvant therapy in patients with early-stage, >2 cm in diameter, or locally advanced or inflammatory HER2-positive breast cancer, in addition to anthracycline-based or carboplatin-based chemotherapy regimens (see Figure 11).

Six cycles of active chemotherapy were administered. However, if further postoperative treatment is considered necessary, CMF (cyclophosphamide, methotrexate, and 5-fluorouracil) is recommended for patients who have received FEC->T, while FEC (5-fluorouracil, epirubicin, and cyclophosphamide) is recommended for patients who have received TCH but are deemed to require further chemotherapy.

After surgery (and, if necessary, after postoperative chemotherapy), patients receive radiation therapy according to local clinical standards, and those with estrogen-positive tumors receive hormone therapy according to local clinical standards.

In summary, all patients received at least 6 cycles of active chemotherapy and two antibodies, pertuzumab and trastuzumab, plus surgery and radiotherapy (according to local standards) plus any specified hormone procedures (according to local standards), and continued to receive trastuzumab for a total of 1 year.

Patients who discontinued neoadjuvant study treatment prior to surgery were managed according to local practice. Approximately 28 days after the last dose of the study drug, patients were required to undergo a final safety assessment (referred to as a final visit).

research group

Overview

The patient was a female, aged 18 years or older, with early-stage HER2-positive breast cancer. Her primary tumor was >2cm and had no metastases.

Inclusion criteria

1. Female patients with locally advanced, inflammatory, or early-stage, unilateral, and histologically confirmed invasive breast cancer. Initial breast cancer evaluation should be performed by a physician experienced in breast cancer surgery. Patients with inflammatory breast cancer must be able to perform a core needle biopsy.

2. Primary tumor diameter > 2cm.

3. HER2-positive breast cancer confirmed by a central laboratory. The tumor must be HER23+ (via IHC) or FISH/CISH+ (FISH/CISH positive, mandatory for HER22+ tumors).

4. Availability of FFPE tissue (which will be subjected to buffered formalin fixation) for central confirmation of HER2 eligibility (FFPE tumor tissue will then be used to assess the status of biomarkers).

5. Female patients, aged ≥18 years.

6. Baseline LVEF ≥ 55% (measured by echocardiography or MUGA).

7. Performance status ECOG≤1.

8. At least 4 weeks from the time of the unrelated major surgery for full recovery.

Accompanying medications and treatments

Permitted therapies

Accompanying treatments are any prescription medications, over-the-counter preparations, herbal remedies, or radiation therapy used by patients that began 7 days prior to enrollment in the study and continued throughout the study period.

The following treatments are permitted during the study:

1. When a female patient or her male partner has not undergone surgical sterilization or does not meet the study definition of postmenopause (≥12 months without menstruation), an acceptable method of contraception must be used.

2. H1 and H2 antagonists (e.g., diphenhydramine, cimetidine)

3. Pain relievers (e.g., paracetamol, meperidine, opioids)

4. Short-term use of corticosteroids to treat or prevent allergic or infusion reactions.

5. Antiemetics (approved prophylactic serotonin antagonists, diazepam, 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 completing postoperative chemotherapy according to local practice.

Exclusion therapy

The following therapies were excluded during the treatment period of the study:

9. Other anticancer therapies besides those used in this study include cytotoxic chemotherapy, radiotherapy (unless adjuvant radiotherapy for breast cancer after chemotherapy or other adjuvant chemotherapy immediately after surgery, if deemed necessary), immunotherapy, and biological anticancer therapies.

10. Any targeted therapy.

11. Use of steroids (except for thyroid hormone replacement therapy) and short-term corticosteroids for the purpose of treating or preventing allergic or infusion reactions.

12. High doses of systemic corticosteroids. High doses are considered to be >20 mg dexamethasone (or equivalent) per day for >7 consecutive days.

13. Any research reagents, except those used in this study.

14. Initiation of Herbal Treatment. Herbal treatment may be initiated before the study begins and continued during the study and must be reported on the appropriate eCRF.

15. Any oral, injectable, or implantable hormonal contraceptive method.

result

Table 6 below provides baseline characteristics of patients with HER2-positive early breast cancer.

Table 6: Baseline characteristics in the safety population

CBE, Clinical Breast Examination; ECOG PS, Eastern Cooperative Oncology Group Status; ER, Estrogen Receptor; FEC, 5-Fluorouracil, Epirubicin, Cyclophosphamide; FISH, Fluorescence In Situ Hybridization; H, Trastuzumab; IHC, Immunohistochemistry; P, Pertuzumab; PR, Progesterone Receptor; T, Docetaxel; TCH, Docetaxel/Carboplatin/Trastuzumab

Security data are shown in Figure 12 and Tables 7 and 8 below.

Table 7: Overall Cardiac Events

FEC, 5-fluorouracil, epirubicin, cyclophosphamide; H, trastuzumab; LVEF, left ventricular ejection fraction; LVSD, left ventricular systolic dysfunction; P, pertuzumab; T, docetaxel; TCH, docetaxel/carboplatin/trastuzumab

Table 8: 10 most common grade ≥3 adverse events during neoadjuvant therapy

FEC, 5-Fluorouracil, Epirubicin, Cyclophosphamide; H, Trastuzumab; P, Pertuzumab; T, Docetaxel; TCH, Docetaxel/Carboplatin/Trastuzumab

Efficacy data are provided in Figures 13 and 14 and Tables 9 and 10 below.

Table 9: Clinical response rate during neoadjuvant therapy

FEC, 5-Fluorouracil, Epirubicin, Cyclophosphamide; H, Trastuzumab; P, Pertuzumab; T, Docetaxel; TCH, Docetaxel/Carboplatin/Trastuzumab

Table 10: Breast-conserving surgery in patients scheduled for mastectomy

CI, confidence interval; FEC, 5-fluorouracil, epirubicin, cyclophosphamide; H, trastuzumab; P, pertuzumab; T, docetaxel; TCH, docetaxel/carboplatin/trastuzumab

in conclusion

● Results from this study indicate a low incidence of symptomatic and asymptomatic LVSD across all arms.

- Concomitant administration of pertuzumab plus trastuzumab and epirubicin resulted in similar cardiac tolerance compared to sequential administration or anthracycline-free regimens.

● Neutropenia, fever, neutropenia, leukopenia, and diarrhea are the most frequently reported adverse events (≥ grade 3) across all treatment arms.

● Regardless of the type of chemotherapy used, the combination of pertuzumab and trastuzumab produced a high pathological complete response (pCR) rate (57 to 66%) in neoadjuvant settings.

●TRYPHAENA supports the use of pertuzumab and trastuzumab plus anthracycline-based or carboplatin-based chemotherapy in neoadjuvant and adjuvant settings for early-stage breast cancer.

Example 6: Co-administration of pertuzumab and trastuzumab

In the Phase III clinical trial described above, pertuzumab in a saline IV bag was administered to patients with HER2-positive metastatic breast cancer via intravenous (IV) infusion, followed by trastuzumab and the chemotherapy agent docetaxel via saline IV infusion. Each IV infusion of pertuzumab and trastuzumab took approximately 60 to 90 minutes, with a 30 to 60 minute observation period after each administration. Due to this treatment regimen for each patient, the total visit time could be up to 7.5 hours. With recent reviews of medical payments for medications and medication administration services, commercial practices are emphasizing time reduction and improved utilization of medical resources in both clinical and hospital settings. By reducing the time patients spend in each treatment cycle in the clinic, increased patient care, adherence, and treatment efficiency are expected.

As part of a Phase III clinical trial of pertuzumab, pertuzumab and trastuzumab were administered sequentially to patients via intravenous (IV) infusion, one drug after the other. Pertuzumab was administered at a fixed dose (420 mg for maintenance and 840 mg for loading), while trastuzumab was administered by weight (6 mg/kg for maintenance). To increase patient convenience and minimize in-clinical time, the feasibility of co-administering pertuzumab and trastuzumab in a single 250 mL 0.9% saline polyolefin (PO) or polyvinyl chloride (PVC) IV infusion bag was evaluated. The monoclonal antibody was shown to be stable for up to 24 hours in the infusion bag (PO and/or PVC) at 5°C and 30°C. In this study, the compatibility and stability of pertuzumab (420 mg and 840 mg) mixed with 420 mg trastuzumab (6 mg/kg for 70 kg patients) or 720 mg (6 mg/kg for 120 kg patients) in IV bags at 5°C or 30°C for up to 24 hours were evaluated. Existing pertuzumab and trastuzumab analytical methods were used to evaluate control (i.e., pertuzumab alone in IV bags, trastuzumab alone in IV bags) and monoclonal antibody (mAb) mixture samples, including color, appearance, and clarity (CAC), concentration and turbidity by UV spectroscopy, particle analysis by HIAC-Royco, size exclusion chromatography (SEC), and ion exchange chromatography (IEC). In addition, capillary zone electrophoresis (CZE), imaging capillary isoelectric focusing (iCIEF), and potency (pertuzumab-only antiproliferative assay) were used to measure mixtures of 1:1 pertuzumab:trastuzumab and their corresponding controls (420 mg pertuzumab only and 420 mg trastuzumab only) as representative cases.

The results showed no observable differences between the time 0 (T0) control and samples stored at 5°C or 30°C for up to 24 hours in the pertuzumab/trastuzumab mixture using the assays described above. The physicochemical assays listed above detected minor variants in both molecules and the drug mixture, although some overlap in monoclonal antibody species was observed in the chromatographic spectra. Furthermore, the pertuzumab-specific inhibition of the drug mixture tested by the cell proliferation assay showed comparable potency before and after storage. The results from this study indicate that the pertuzumab and trastuzumab mixture is physically and chemically stable in IV infusion bags at 5°C or 30°C for up to 24 hours and is suitable for clinical administration if necessary.

Dosage I: 840 mg pertuzumab/trastuzumab mixture (420 mg pertuzumab and 420 mg trastuzumab)

Sample preparation: All procedures were performed aseptically under a laminar flow hood. PO IV infusion bag samples with three types of drug combinations were prepared for this study: 1) a 420 mg pertuzumab/420 mg trastuzumab mixture, 2) 420 mg pertuzumab only, and 3) 420 mg trastuzumab only. Individual pertuzumab and trastuzumab samples served as controls.

Trastuzumab was reconstituted with 20 mL of sterile water for injection (BWFI) and left on the lab bench for approximately 15 minutes before use. To prepare pertuzumab/trastuzumab sample doses, 14 mL of pertuzumab (420 mg) was directly diluted at room temperature using an 18-gauge needle into an IV infusion bag containing a nominal 250 mL (±25 mL deviation) of 0.9% saline solution, without removing an equal volume of saline, followed by 20 mL of reconstituted trastuzumab (420 mg). The total concentration of the two proteins combined in the 250 mL IV bag was expected to be approximately 3 mg/mL. Similarly, individual pertuzumab (420 mg) IV bags were prepared by directly diluting 14 mL of the 30 mg/mL drug product into the IV infusion bag. The final expected concentration was approximately 1 mg/mL. Individual trastuzumab (420 mg) IV infusion bags were also prepared in the same manner, except that 20 mL of the 21 mg/mL drug product was added to the bag. The final expected concentration was approximately 1 mg/mL.

The PO IV bags were thoroughly mixed manually by gently shaking back and forth several times to ensure homogeneity. After mixing, 10 mL of sample was removed from each bag using a syringe and stored in a sterile 15 cc Falcon tube as a control for the diluted sample at time 0 (T0). The IV bags were then covered with foil and stored at 30°C for 24 hours (T24). Immediately after storage, the remaining sample was removed from each bag using 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 subjected to CAC, UV spectral scanning (concentration and turbidity), SEC, IEC, CZE, iCIEF, HIAC-Royco, and potency analysis. Product quality of the samples was tested using product-specific SEC and IEC methods for pertuzumab and trastuzumab, but only pertuzumab-specific potency methods were performed. Other assays used were non-product-specific methods. For the intended tests, all assays were qualified in their respective molecules and no further method optimization was required.

Dosage II: 1560 mg pertuzumab/trastuzumab mixture (840 mg pertuzumab and 720 mg trastuzumab)

Sample Preparation: The upper limit of the co-administration dose of mAbs was examined in PO and PVC IV infusion bag samples (1560 mg total mixture: 840 mg pertuzumab and 720 mg trastuzumab). In events where increased protein aggregation was observed, the tendency for high molecular weight species (HMWS) formation was more likely to occur at higher doses of 1560 mg total mAbs than at mixtures containing 840 mg. To mitigate the risk during use at high dose ranges, both PO and PVC IV infusion bags were investigated to ensure no interactions were observed.

Three types of drug combinations (mixtures, 840 mg pertuzumab only, and 720 mg trastuzumab only) were prepared and operated similarly to the Dose I study. The pertuzumab/trastuzumab mixture contained 28 mL of pertuzumab (840 mg) diluted directly into a PO or PVC IV infusion bag at room temperature using an 18-gauge needle, followed by 34 mL of reconstituted trastuzumab (720 mg). The total concentration of the two mAbs combined in a single 250 mL IV bag was expected to be approximately 5 mg/mL. For the control, individual pertuzumab and trastuzumab IV infusion bag samples were prepared and operated 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 each PO or PVC IV infusion bag. The final expected concentration for individual pertuzumab (840 mg) and trastuzumab (720 mg) samples was approximately 3 mg/mL. Store the bag uncovered at 5°C or 30°C for up to 24 hours. Analyze T0 and T24 samples immediately by CAC, UV spectral scanning (concentration and turbidity), SEC, IEC, and HIAC-Royco, or keep at 5°C for up to 24 to 48 hours.

Details of dosage type, IV infusion bag, dosage and preparation, storage temperature and assay are summarized in Table 11.

Table 11: IV Bag Types, Dosage, Preparation, and Study Conditions

^a n=2

^b control

P = pertuzumab; T = trastuzumab

Determination method

All samples were maintained at 5°C or analyzed immediately. Samples were typically analyzed within 24 to 48 hours of preparation and storage. The following assays were performed to ensure product quality and short-term stability of pertuzumab/trastuzumab mixtures diluted in saline IV infusion bags, pertuzumab alone, and trastuzumab alone samples. Since several assays, namely SEC, IEC, CZE, iCIEF, and potency, are not optimized for quantitative assessment of mAb mixtures, only chromatographic or electrophoretic coverage maps of these samples and their respective controls before and after storage at 5°C or 30°C are shown in this document. For consistency, no values, such as percentage peak areas, were calculated from the performed HPLC and electrophoretic assays for all three sample types.

Color, Appearance, and Clarity (CAC)

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

UV-Vis spectrophotometer scanning for concentration measurement

Concentrations prepared via volumetric quantification were determined by measuring UV absorbance on an HP8453 spectrophotometer. The instrument was used with 0.9% saline as a blank. For each sample, absorbance was measured _{at Amax} (278 nm or 279 nm) and 320 nm in a 1-cm diameter quartz cell. The absorbance at 320 nm was used to correct for background light scattering in the solution. Concentrations were calculated using the absorbance ^at 1.50 (mg/mL) ^cm⁻¹ for both pertuzumab and trastuzumab molecules.

Size exclusion chromatography (SEC: pertuzumab specificity and trastuzumab specificity)

Each sample was injected at ambient temperature into an AGILENT HPLC column G3000SWXL, 7.8 x 300 mm. The elution peak was monitored at 280 nm. Chromatographic integration was analyzed using software. The autosampler temperature was maintained between 2 and 8 °C throughout the run, and the flow rates used were 0.2 M potassium phosphate, 0.25 mM potassium chloride, pH 6.2, and 100 mM potassium phosphate, pH 6.8, respectively, for pertuzumab and trastuzumab assays. The recommended injection load and injection volume as specified in the test protocol were 200 μg and 20 μL. A diluted 420 mg sample was injected at a lower load than recommended due to the low concentration of protein after dilution in IV bags. The maximum injection volume of the HPLC sample loop is 100 μL, which limits the volume that can be injected at one time. As a result, the injection volume was modified to 160 μg protein 100 μL for pertuzumab and trastuzumab samples alone (420 mg dose group), and to 200 μg protein 73 μL for the pertuzumab/trastuzumab mixture (840 mg dose group). This modification of the injection volume has been used in previous IV bag studies and is necessary when handling low-concentration samples.

Ion exchange chromatography (IEC)

For each sample, charge heterogeneity of pertuzumab and trastuzumab digested with carboxypeptidase B (CpB) was analyzed by IEC. For pertuzumab-specific IEC, samples were tested using either the standard IEC (“Pertuzumab-Standard IEC”) or a modified “rapid” IEC for high throughput (“Pertuzumab-IEC-Fast”), the method being tailored to the purpose of these experiments. IEC assays were performed using a WCX weak cation exchange column equilibrated with solvent A (20 mM MES, ₁ mM Na₂EDTA, pH 6.00) and solvent B (250 mM sodium chloride in solvent A). Pertuzumab-Standard IEC and Pertuzumab-IEC-Fast were monitored at 280 nm, while trastuzumab was monitored at 214 nm using solvent A (10 mM sodium phosphate, pH 7.5) and solvent B (100 mM sodium chloride in solvent A) on AGILENT HPLC. The peaks were eluted at a flow rate of 0.8 mL/min, with an increasing gradient of 18%–100% solvent B over 35 min and 90 min for pertuzumab-regular-IEC and pertuzumab-IEC-fast, respectively, while a gradient of 15%–100% solvent B was observed over 55 min for trastuzumab-IEC. For pertuzumab-regular-IEC or pertuzumab-fast-IEC and trastuzumab-IEC, the column temperature was maintained at 34°C or 42°C and ambient temperature, respectively, while the autosampler temperature was maintained at 2–8°C throughout the run.

Microscopically visible HIAC-ROYCO ^™ opacity of particles

Particle counting in diluted pharmaceutical products was performed using the HIAC-ROYCO ^™ Liquid Particle Counting System Model 9703. The average cumulative number of particles ≥10 μm and ≥25 μm per milliliter in each sample was listed using PHARMSPEC v2.0 ^™ . The test procedure was modified for a small-volume method, utilizing four 1 mL readings or four 0.4 mL readings per test phase, discarding the first reading for each sample. HIAC-ROYCO ^™ samples were degassed under vacuum for approximately 10–15 minutes per sample. Particles smaller than 10 μm were not collected for this sample set.

UV-Vis spectrophotometer scanning for turbidity measurement

The optical density of the sample (1 mg/mL or 3 mg/mL) from bag IV was measured in a quartz cell with a diameter of 1 cm on an HP8453 spectrophotometer. The sample readings were recorded using purified water as a blank. Absorbance measurements were recorded at 340 nm, 345 nm, 350 nm, 355 nm, and 360 nm, and the turbidity was expressed as an average at these wavelengths.

Capillary zone electrophoresis, CZE

CZE was performed using a PROTEOMELAB PA800 ^™ capillary electrophoresis system (Beckman Coulter) and neutral-coated capillaries (50 μm x 50 cm). The buffer composition was 40 mM ε-aminocaproic acid/acetic acid, pH 4.5, 0.2% hydroxypropyl methylcellulose (HPMC). Samples were diluted in water to 0.5 mg/mL and injected into the capillaries at 1 psi for 10 seconds. Separation was performed at 30 kV for 15 minutes, and species were detected by UV at 214 nm.

CE-SDS-LIF, reducing and non-reducing

Each sample was derivatized with 5-carboxytetramethylrhodamine succinimidyl ester, a fluorescent dye. After removing free dye via gel filtration (using a NAP-5 column), non-reducing samples were prepared by adding 40 mM iodoacetamide and heating at 70 °C for 5 min. For analysis of reducing samples, the derivatized sample was mixed with SDS to a final concentration of 1% (v/v) and 10 mL of a solution containing 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 I.D. 31.2 cm fused silica capillary maintained at 20 °C throughout the analysis. The sample was introduced into the capillary via an electrokinetic injection at 10 kV for 40 seconds. Separation was performed at a constant voltage of 15 kV in reverse polarity (negative to positive) using CE-SDS run buffer as the sieving medium. An argon-ion laser running at 488 nm was used for fluorescence excitation, and the resulting emission signal was monitored at 560 nm.

iCIEF

The distribution of charge variants of pertuzumab/trastuzumab mixtures, pertuzumab alone, and trastuzumab alone was assessed via iCIEF using an iCE280 ^™ analyzer (Convergent Bioscience) and a fluorocarbon-coated capillary tube (100 μm x 5 cm). The amphoteric electrolyte solution consisted of a mixture of 0.35% methylcellulose (MC), 0.47% Pharmalyte 3-10 carrier amphoteric electrolyte, 2.66% Pharmalyte 8-10.5 carrier amphoteric electrolyte, and 0.20% pI markers 7.05 and 9.77 in purified water. The anolyte was 80 mM phosphate, 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 a 1:100 enzyme-to-substrate ratio, followed by incubation at 37°C for 20 min. The CpB-treated sample was mixed with an amphoteric electrolyte solution and then focused by applying a voltage of 1500V for 1 minute followed by 3000V for 10 minutes. Images of the focused pertuzumab charge variants were obtained by passing 280nm UV light through a capillary and into the lens of a charge-coupled digital camera. The images were then analyzed to determine the distribution of the various charge variants.

Antiproliferative efficacy assay

This assay is based on the ability of pertuzumab to inhibit the proliferation of MDA MB 175 VII human breast cancer cells. In short, cells are seeded in 96-well tissue culture microtiter plates and incubated _overnight at 37°C with 5% CO2 to allow cell attachment. The next day, the culture medium is removed and serial dilutions of each standard, control, and sample are added to the plate. The plate is then incubated at 37°C with 5% _CO2 for 4 days, and the relative number of surviving cells is indirectly quantified using redox dyes according to the manufacturer's protocol. Each sample is measured in triplicate, and the color change measured by fluorescence is directly proportional to the number of viable cells in the culture. The absorbance of each well is then measured on a fluorescent 96-well plate reader. The results, expressed as relative fluorescence units (RFU), are plotted against antibody concentration. No quantitative measurements were performed or were not possible because no pertuzumab/trastuzumab mixture was available for reference. Therefore, the results are only for comparison of dose-response curves.

Results and Discussion

Dosage I: 840 mg total pertuzumab/trastuzumab mixture (420 mg pertuzumab and 420 mg trastuzumab)

Product quality was assessed using CAC, concentration measurements (via UV spectral scanning), turbidity, and HIAC Royco for a total of 840 mg pertuzumab/trastuzumab mixture (420 mg pertuzumab and 420 mg trastuzumab), pertuzumab alone (420 mg), and trastuzumab alone (420 mg) in IV infusion bags (n=1) before and after storage at 30°C for up to 24 hours (Table 12). Pertuzumab alone and trastuzumab IV infusion bags were used as controls and were also prepared to assess the ability of this assay to pick out suitable product attributes.

Table 12: Stability data of pertuzumab/trastuzumab mixture, pertuzumab, or trastuzumab in dose I 840 mg: 0.9% saline PO IV infusion bag (n=1)

RT = room temperature

^a. Color, appearance, and clarity: CL = clear; SOPL = slightly milky white; CO = colorless

Following storage, the pertuzumab/trastuzumab mixture, pertuzumab alone, and trastuzumab alone samples appeared as clear, colorless liquids without visible particles, as observed by CAC. Concentration and turbidity measurements showed no measurable changes in any of the three sample types after 24 hours at 30°C. For the pertuzumab/trastuzumab mixture, pertuzumab alone, or trastuzumab alone samples, no more than six particles larger than or equal to 10 μm and no particles larger than 25 μm were detected by particle analysis by HIAC Royco after storage. These results are comparable to those of 0.9% saline solution only. The absence of visible precipitate or particles indicates that the mixture and control were adequately stable after dilution in 0.9% saline IV infusion bags. The pertuzumab/trastuzumab mixture diluted in saline was run on SEC (both pertuzumab- and trastuzumab-specific methods) and showed comparable peak profiles between T0 and T24 (Figures 15 and 16). No increases were observed in the high molecular weight variant (HMWS) or low molecular weight variant (LMWS). Similarly, no changes were observed in the major peak of any sample. The major peak and the peak regions of HMWS and LMWS overlapped and could not be distinguished in the pertuzumab/trastuzumab mixture due to the size similarity between pertuzumab and trastuzumab (molecular weight approximately 150 kDa). Furthermore, no changes were observed in the peak area or profile when comparing T0 and T24 for individual pertuzumab and trastuzumab samples, as detected by the two SEC methods listed above.

Two product-specific methods for pertuzumab or trastuzumab IEC were used to analyze pertuzumab/trastuzumab mixtures (Figures 17 and 18). In cation exchange chromatography, each molecule typically contains three distinct elution regions based on relative charge: an early elution of the acidic variant, followed by the major peak, and finally the late elution of the basic variant. In the chromatograms of pertuzumab and trastuzumab alone, an overview of the acidic variant, major peak, and basic variant was observed and considered comparable between starting materials and after storage at 30°C. These results are also consistent with previous studies of pertuzumab or trastuzumab alone in saline IV infusion bags. In the chromatograms of pertuzumab/trastuzumab mixtures, the pertuzumab peak eluted first, followed by the trastuzumab peak. Due to the nature of cation exchange separation and the net charge difference between pertuzumab (~pI 8.7) and trastuzumab (~pI 8.9), two major peaks or dominant charged species were observed in the pertuzumab/trastuzumab mixture. In contrast, the SEC assay, based on the hydrodynamic size separation of molecules, showed only one major peak, which is due to the size similarity between pertuzumab and trastuzumab. The charged regions of each molecule appear to overlap with each other in the pertuzumab/trastuzumab mixture. Specifically, the basic variants of pertuzumab, expected to elute at approximately 32 min and 35 min, appear to overlap with the major peak of trastuzumab (Figures 17 and 18). Furthermore, the acidic variants of trastuzumab, expected to elute before the major peak of trastuzumab, co-eluted with the basic variants of pertuzumab and the major peak. Despite overlapping peak regions, the pertuzumab/trastuzumab mixture exhibited comparable chromatographic peak profiles before and after storage in IV saline bags at 30°C for 24 hours.

Pertuzumab/trastuzumab mixtures, pertuzumab alone, and trastuzumab alone were also measured under non-reducing conditions at CE-SDS LIF after 24 hours of storage at 30°C. The pertuzumab/trastuzumab mixture showed a consistent peak profile with no observed changes compared to the starting material after storage (Figures 19 and 20). Very minor baseline level changes attributable to noise were also observed, without affecting the peak regions. Similar to SEC, the non-reducing pertuzumab/trastuzumab mixture showed only one superimposed monomer constituting the major classes of pertuzumab and trastuzumab. The pertuzumab and trastuzumab alone samples showed no change at T0 compared to T24. However, as expected, individual molecular properties, such as fragment peak levels and classes, were observed between the pertuzumab/trastuzumab mixture, pertuzumab alone, and trastuzumab alone.

When pertuzumab/trastuzumab mixtures, pertuzumab alone, and trastuzumab alone were run on CE-SDS LIF reduced with DTT, two major peaks, termed light chain (LC) and heavy chain (HC), were detected at 17 and 21.5 minutes, respectively (Figure 20). No increase in fragmentation or associated decrease in LC and HC was observed in the pertuzumab/trastuzumab mixture after storage at 30°C. Furthermore, no detectable differences in peak profiles were observed in the pertuzumab and trastuzumab alone samples after storage.

Charge separation assays CZE and iCIEF showed comparable peak profiles for the pertuzumab/trastuzumab mixture after storage at 30°C (Figures 21 and 22). Pertuzumab and trastuzumab alone also showed consistent peak profiles when compared with their respective T0 values, with no change after storage. Furthermore, the presence of various trace species was observed, although no new peaks were detected after dilution in IV bag saline solution. As seen in the charge-based IEC assay, two main peaks with smaller overlapping flanking peaks were detected, attributed to differences in molecular pI.

Potency results based on dose-response curve comparisons showed that the potency of the pertuzumab/trastuzumab mixture stored at 30°C for 24 hours was unaffected by its corresponding T0 dose-response curve (Figure 23). Trastuzumab alone showed minimal activity in the pertuzumab potency assay. The dose-response curves of the pertuzumab/trastuzumab mixture compared to those of pertuzumab or trastuzumab alone showed that a lower dose of the pertuzumab/trastuzumab mixture was required to inhibit cell growth, suggesting that the mixture may have an additive or synergistic effect on cell proliferation inhibition.

Dosage II: 1560 mg total pertuzumab/trastuzumab mixture (840 mg pertuzumab and 720 mg trastuzumab)

In addition to the dose-I study of 840 mg total mAb, a higher dose of 1560 mg mixture (840 mg pertuzumab and 720 mg trastuzumab) and their respective drug product controls (840 mg pertuzumab alone and 720 mg trastuzumab alone) were selected to investigate the effects of diluting these three mAb types into PO or PVC IV infusion bags at 5°C or 30°C for up to 24 hours. The product quality of these IV infusion bags before and after storage was assessed by CAC, UV spectral scanning (concentration and turbidity), and HIAC-ROYCO ^™ and summarized in Table 13. SEC and IEC are shown in Figures 24-27.

Table 13: Stability data for pertuzumab/trastuzumab mixture, pertuzumab, or trastuzumab in dose II 1560 mg: 0.9% saline PO IV infusion bag (n=1 for control; n=2 for mixture).

For pertuzumab/trastuzumab mixtures, two PO or PVC IV infusion bags were prepared for each condition, while for individual pertuzumab and trastuzumab samples, only one IV infusion bag was prepared.

Particulate matter from these bags was determined by visual observation, turbidity, and HIAC-Royco measurements. All samples remained clear and colorless after storage at 5°C or 30°C for up to 24 hours. No visible particulate matter was observed, and there was no significant change in turbidity after storage. For pertuzumab/trastuzumab mixtures, pertuzumab alone, and trastuzumab alone, HIAC-Royco showed comparable particle counts before and after storage, with an increase of 0 to 10 particles ≥10 μm per milliliter and 0 particles ≥25 μm per milliliter for both PO and PVC IV infusion bags stored at 5°C or 30°C. Similarly, no significant differences in particle count were observed between PO or PVC IV infusion bags before and after storage for pertuzumab and trastuzumab alone. For all three sample types, UV spectral scanning did not show any changes in protein concentration beyond the normal measurement range, indicating a lack of protein adsorption or precipitation in IV infusion bags stored at 5°C or 30°C between T0 and T24 hours.

The physical and chemical stability of pertuzumab/trastuzumab mixtures, pertuzumab alone, and trastuzumab alone was assessed using pertuzumab- or trastuzumab-specific SEC and IEC methods, as previously described. For the pertuzumab/trastuzumab mixture, no changes were observed in the SEC profiles of samples in PO or PVC IV infusion bags between T0 and T24 hours at 5°C or 30°C (Figures 24 and 25), similar to the results for the 840 mg mixture dose I. Furthermore, no increase or decrease was observed in the high molecular weight species (HMWS), major peak, or low molecular weight species (LMWS), indicating a stable dosing solution at the upper limit of protein content in 0.9% saline. Similarly, pertuzumab alone and trastuzumab alone samples also showed no changes after storage in IV infusion bags.

IEC analysis of pertuzumab/trastuzumab mixtures using pertuzumab-specific methods was employed to assess chemical stability and revealed comparable charge variant peak profiles. No changes were observed relative 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 was observed between the two mAbs, these peak types were unaffected by increases in the mAb content of the IV infusion bag. Individual pertuzumab or trastuzumab samples in PO or PVC IV infusion bags showed no changes before and after exposure to 5°C or 30°C. These results are consistent with the 840 mg dose I study.

in conclusion

All physicochemical assays indicated no significant changes in the potency of the mixture (up to 840 mg pertuzumab and 720 mg trastuzumab for a total dose of 1560 mg) or in separate pertuzumab (up to 840 mg) and trastuzumab (up to 720 mg) IV infusion bags (PO or PVC) over T0 to T24 hours at 5°C or 30°C. Furthermore, the potency of the mixture (up to 840 mg) and the individual mAbs was comparable before and after storage. During this study, no differences were observed in IV bags containing the pertuzumab and trastuzumab mixture compared to the individual mAb components in IV bags. This current study also demonstrates that many of the assays used to measure individual mAbs are sufficient to qualitatively characterize the mixture.

Example 7: Co-administration of pertuzumab and trastuzumab and combination therapy with vinorelbine

This is a randomized, two-arm, open-label, multicenter phase II trial evaluating pertuzumab in patients with HER2-positive advanced breast cancer (metastatic or locally advanced) who have not previously received systemic non-hormonal anticancer therapy. The study design is shown in Figure 28.

Patients were randomly assigned to one of the two treatment arms at a 2:1 ratio.

Pertuzumab in combination with trastuzumab and vinorelbine (arm A)

Trastuzumab and vinorelbine (control arm B)

Arm A will consist of the following two groups:

Group 1 (first 95 patients): Pertuzumab and trastuzumab were administered sequentially in separate infusion bags, followed by vinorelbine. Patients will receive pertuzumab followed by trastuzumab in separate infusion bags.

Pertuzumab (IV infusion)

The first treatment cycle was administered with a loading dose of 840 mg on day 1, followed by 420 mg on day 1 of each cycle every 3 weeks thereafter.

The initial pertuzumab infusion will be administered over 90 (±10) minutes, and the patient will be observed for at least 30 minutes from the end of the infusion for infusion-related symptoms such as fever and chills. Interrupting or slowing the infusion may reduce these symptoms. If the infusion is well tolerated, subsequent infusions may be administered over 30 (±10) minutes, with the patient observed for another 30 minutes.

Trastuzumab (IV infusion)

Administer 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 cycle every 3 weeks thereafter; administer as directed on the product label.

Changchun Rebinocin (trastuzumab followed by IV infusion)

Administer at a dose of 25 mg/ ^m² on days 1 and 8 of the first treatment cycle, followed by 30-35 mg/ ^m² on days 1 and 8 of each subsequent 3-week cycle; administer according to the product label.

Group 2: Another 95 patients will receive pertuzumab and trastuzumab administered together from a single infusion bag starting from cycle 2, followed by vinorelbine.

Cycle 1 dosing

For the first cycle of treatment, pertuzumab and trastuzumab will be administered in separate infusion bags as described for group 1.

Vinorelbine will be administered after pertuzumab and trastuzumab, as described for group 1.

Subsequent cycle dosing

If all three drugs are well tolerated during cycle 1, pertuzumab 420 mg and trastuzumab 6 mg/kg will be administered together from a single infusion bag on day 1 of each treatment cycle every 3 weeks thereafter.

The initial combination of pertuzumab and trastuzumab infusion will be administered over 90 (±10) minutes, with cardiac monitoring and close observation of infusion-related responses during this procedure, followed by a 60-minute observation period. If this initial combination infusion is well tolerated, subsequent combinations may be administered over 60 (±10) minutes, followed by a 30-minute observation period with cardiac monitoring.

Vinorelbine will be administered after pertuzumab and trastuzumab, as described for group 1.

Control arm - Arm B

A total of 95 patients will be randomized to arm B.

Trastuzumab (IV infusion)

Administer 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 cycle every 3 weeks thereafter; administer as directed on the product label.

Changchun Rebinocin (trastuzumab followed by IV infusion)

Administer at a dose of 25 mg/ ^m² on days 1 and 8 of the first treatment cycle, followed by 30-35 mg/ ^m² on days 1 and 8 of each subsequent 3-week cycle; administer according to the product label.

Efficacy Results:

main

● Compare the objective overall response rate (ORR) of pertuzumab in combination with trastuzumab and vinorelbine, as assessed by an independent review committee (IRC) in a blinded trial, relative to trastuzumab and vinorelbine alone.

secondary

● In the pertuzumab treatment group, the efficacy and safety of pertuzumab and trastuzumab administered together in a single infusion bag were compared to those routinely administered sequentially in separate infusion bags.

●Compare pertuzumab in combination with trastuzumab and vinorelbine to trastuzumab and vinorelbine in the following aspects:

ORR as assessed by researchers

○Pre-response time as assessed by IRC and investigators

○ Response duration assessed by IRC and researchers

○Progression-free survival (PFS)

○ Time to Progress (TTP)

Overall Survival (OS)

○ Safety and tolerability

Quality of life (EQ-5D and FACT-B questionnaires)

Inclusion criteria

Patients must meet the following criteria to be eligible for this study according to the timing of the assessment:

1. Female or male patients aged 18 years or older

2. Histologically or cytologically confirmed and documented breast adenocarcinoma, with metastatic or locally advanced disease that cannot be cured by resection.

3. HER2 positivity in primary or metastatic tumors as assessed by a local laboratory (limited to immunohistochemistry (IHC) 3+ or in situ hybridization (ISH) positivity) (ISH positivity is defined as a HER2 gene copy number to CEP17 signal number ratio of 2.0 or higher, or a HER2 gene count exceeding 4 for single probe testing).

4. At least one measurable lesion and/or an evaluable non-measurable disease according to the Response Assessment Criteria in Solid Tumors (RECIST) version 1.1.

5. ECOG performance status: 0 or 1

6. Left ventricular ejection fraction (LVEF) at least 50%

7. Negative pregnancy test in women of childbearing potential (premenopausal or postmenopausal amenorrhea less than 12 months old and who have not undergone surgical sterilization).

8. For women of childbearing potential and sexually active age, consent is given to use one highly effective non-hormonal contraceptive or two effective non-hormonal contraceptives during treatment and for at least 6 months after study treatment.

9. Fertility-promoting men who are willing and able to use effective non-hormonal contraception during treatment and for at least 6 months after study treatment (barrier contraception combined with spermicidal jelly or surgical sterilization).

10. Life expectancy of at least 12 weeks

Exclusion criteria

Patients who meet any of the following exclusion criteria will not be eligible for this study:

1. Prior systemic non-hormonal anticancer therapy in patients with metastatic or locally advanced breast cancer.

2. An exception to approved or investigational anti-HER2 agents previously used in any breast cancer treatment context, or trastuzumab in an adjuvant or neoadjuvant context.

3. Disease progression while receiving trastuzumab in adjuvant or neoadjuvant settings.

4. The disease-free interval from the completion of adjuvant or neoadjuvant systemic non-hormonal therapy to disease relapse is less than 6 months.

5. Experience with persistent grade 2 or higher (NCI-CTC, version 4.0) hematologic toxicity resulting from prior adjuvant or neoadjuvant therapy.

6. Radiographic evidence of central nervous system (CNS) metastasis, such as evidence assessed by CT or MRI.

Current peripheral neuropathy of grade 7.3 or higher (NCI-CTC, version 4.0)

8. Other malignant medical history within the past 5 years, excluding cervical carcinoma in situ or basal cell carcinoma.

9. Severe, uncontrolled comorbidities that would contraindicate the use of any of the investigational drugs used in this study or would place the patient at high risk for treatment-related complications.

10. Inappropriate organ function, as demonstrated by the following laboratory results:

• Absolute neutrophil count <1,500 cells/ ^mm³

• Platelet count <100,000 cells/ ^mm³

Hemoglobin < 9 g/dL

• Total bilirubin is above the upper limit of normal (ULN) (unless the patient has a history of Gilbert's syndrome).

• AST (SGOT) or ALT (SGPT) > 2.5 × ULN

• AST (SGOT) or ALT (SGPT) > 1.5 × ULN, and serum alkaline phosphatase > 2.5 × ULN; in the presence of bone metastases, serum alkaline phosphatase > 2.5 × ULN alone may be present, while AST (SGOT) and ALT (SGPT) < 1.5 × ULN.

Serum creatinine > 2.0 mg/dL or 177 μmol/L

• International Normalized Ratio (INR) and activated partial thromboplastin time (aPTT or PTT) > 1.5 × ULN (unless therapeutic coagulation is involved).

11. Uncontrolled hypertension (systolic blood pressure >150 mmHg and/or diastolic blood pressure >100 mmHg) or clinically significant (i.e., active) cardiovascular disease within 6 months prior to initial treatment: cerebrovascular event (CVA)/stroke or myocardial infarction, unstable pharyngitis, New York Heart Association (NYHA) class II or higher congestive heart failure (CHF), or severe arrhythmia requiring medication.

12. Currently known HIV, HBV, or HCV infections

13. Dyspnea at rest due to late-stage malignant complications, or other conditions requiring continuous oxygen therapy;

14. Major surgical procedures or significant trauma within 28 days prior to randomization, or major surgery anticipated during the study treatment process.

15. Received intravenous (IV) antibiotics for infection within 14 days prior to randomization.

16. Current long-term daily treatment with corticosteroids (dose equal to or greater than 10 mg/day of methylprednisolone), except for inhaled steroids.

17. Known hypersensitivity to any investigational drug or to excipients of recombinant human or humanized antibodies.

18. Experience receiving any investigational treatment within 28 days prior to randomization.

19. Simultaneously participate in any clinical trials

The treatments described in this article are expected to demonstrate the safety and efficacy of co-administration of pertuzumab and trastuzumab from the same intravenous (IV) bag in patients with HER2-positive cancer (exemplified by HER2-positive breast cancer), in accordance with any one or more of the primary or secondary efficacy results described above, as well as the safety and efficacy of pertuzumab in combination with vinorelbine.

Example 8: Pertuzumab in combination with an aromatase inhibitor

This example is a randomized, two-arm, open-label, multicenter phase II study demonstrating the efficacy and safety of pertuzumab in combination with a 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.

Main purpose

Compare the progression-free survival (PFS) of pertuzumab in combination with trastuzumab plus an aromatase inhibitor (AI) versus trastuzumab plus AI.

Secondary objective

The following aspects are compared between pertuzumab and trastuzumab plus an AI:

- Overall Survival (OS)

- Overall Response Rate (ORR)

- Clinical Benefit Rate (CBR)

- Response duration

-Time before response

- Safety and tolerability

- Quality of life (EQ-5D questionnaire)

Experimental Design

Patients will be randomly assigned to one of the two treatment arms at a 1:1 ratio:

- Pertuzumab in combination with trastuzumab plus an AI (arm A)

-Trastuzumab plus AI (control arm, arm B)

Based on the researchers' assessment, patients may also receive induction chemotherapy (taxane, docetaxel, or paclitaxel) in combination with an assigned monoclonal antibody treatment arm for up to the first 18 weeks of treatment. In patients receiving induction chemotherapy, treatment with the AI will begin after the chemotherapy induction phase.

The stratification factor used for analysis will be:

- Optional: Receive induction chemotherapy (yes/no).

- The time from 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 previously received systemic non-hormonal anticancer therapy in a metastatic background.

Inclusion criteria

1. Age 18 or older.

2. Postmenopausal status > 1 year (meeting one or more National Comprehensive Cancer Network (NCCN) guidelines, version 2.2011).

3. Histologically or cytologically confirmed and documented breast adenocarcinoma, with metastatic or locally advanced disease that cannot be cured by surgical resection.

4. HER2 positivity (defined as IHC 3+ or ISH positivity) assessed by a local laboratory on primary or metastatic tumors (ISH positivity is defined as a HER2 gene copy number to CEP17 signal number ratio of 2.0 or higher, or a HER2 gene count exceeding 4 for single probe testing).

5. Hormone receptor positivity is defined as ER positivity and/or PgR positivity as assessed locally according to institutional standards.

6. At least one measurable lesion and/or an evaluable non-measurable disease according to the Response Assessment Criteria in Solid Tumors (RECIST) version 1.1

7. ECOG performance status: 0 or 1

8. Left ventricular ejection fraction (LVEF) at least 50%

9. Life expectancy is at least 12 weeks.

Exclusion criteria

1. Prior systemic non-hormonal anticancer therapy in patients with metastatic or locally advanced breast cancer.

2. The disease-free interval from the completion of adjuvant or neoadjuvant systemic non-hormonal therapy to disease relapse is less than 6 months.

3. Exceptions include approved or investigational anti-HER2 agents previously used in any breast cancer treatment background, and trastuzumab and/or lapatinib in neoadjuvant or adjuvant settings.

4. Disease progression while receiving trastuzumab and/or lapatinib in an adjuvant setting.

5. Experience of persistent grade 2 or higher (NCI-CTC, version 4.0) hematologic toxicity resulting from prior adjuvant or neoadjuvant therapy.

6. Radiographic evidence of central nervous system (CNS) metastasis, such as evidence assessed by CT or MRI.

7. Current peripheral neuropathy of grade 3 or higher (NCI-CTC, version 4.0)

8. A history of other malignant conditions within the past 5 years, except for cervical carcinoma in situ or basal cell carcinoma.

9. Severe, uncontrolled comorbidities that would contraindicate the use of any of the investigational drugs used in this study or would place the patient at high risk for treatment-related complications.

10. Inappropriate organ function, as demonstrated by the following laboratory results:

- Absolute neutrophil count <1,500 cells/ ^mm³

- Platelet count <100,000 cells/ ^mm³

- Hemoglobin < 9 g/dL

- Total bilirubin is above the upper limit of normal (ULN) (unless the patient has a history of Gilbert's syndrome).

-AST(SGOT) or ALT(SGPT) > 2.5 × ULN

-AST (SGOT) or ALT (SGPT) > 1.5 × ULN, while serum alkaline phosphatase > 2.5 × ULN;

In the presence of bone metastases, serum alkaline phosphatase > 2.5 × ULN and AST (SGOT) and ALT (SGPT) < 1.5 × ULN are acceptable.

- Serum creatinine > 2.0 mg/dL or 177 μmol/L

- International Normalized Ratio (INR) and Activated Partial Prothrombin Time (aPTT) (or Partial Prothrombin Time (PTT) > 1.5 × ULN (unless treatment coagulation))

11. Uncontrolled hypertension (systolic blood pressure >150 mmHg and/or diastolic blood pressure >100 mmHg) or clinically significant (i.e., active) cardiovascular disease within 6 months prior to initial treatment: cerebrovascular event (CVA)/stroke or myocardial infarction, unstable pharyngitis, New York Heart Association (NYHA) class II or higher congestive heart failure (CHF), or severe arrhythmia requiring medication.

12. Currently known HIV, HBV, or HCV infections

13. Dyspnea at rest due to late-stage malignant complications, or other conditions requiring continuous oxygen therapy;

14. Major surgical procedures or significant trauma within 28 days prior to randomization, or major surgery anticipated during the study treatment process.

15. Lack of solid integrity of the upper gastrointestinal tract, clinically significant malabsorption syndrome, or inability to ingest oral medications.

16. Received intravenous antibiotics for infection within 14 days prior to randomization.

17. Current long-term daily treatment with corticosteroids (equivalent to 10 mg/day of methylprednisolone), except for inhaled steroids.

18. Known hypersensitivity to any investigational drug or to excipients of recombinant human or humanized antibodies.

19. Experience receiving any investigational treatment within 28 days prior to randomization.

20. Simultaneously participate in any clinical trials

Arm A

Pertuzumab (IV infusion)

The first treatment cycle was administered with a loading dose of 840 mg on day 1, followed by 420 mg on day 1 of each cycle every 3 weeks thereafter.

The initial pertuzumab infusion will be administered over 90 (±10) minutes, and the patient will be observed for at least 30 minutes from the end of the infusion for infusion-related symptoms such as fever and chills. Interrupting or slowing the infusion may reduce these symptoms. If the infusion is well tolerated, subsequent infusions may be administered over 30 (±10) minutes, with the patient observed for another 30 minutes.

Trastuzumab (administered via IV infusion after pertuzumab) is administered at 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 cycle every 3 weeks thereafter; administer according to the product label.

AI (oral)

Use as indicated on the product label (anastrozole: 1 mg once daily; letrozole: 2.5 mg once daily).

Induction chemotherapy

Patients receiving induction chemotherapy for the first 18 weeks of treatment will receive taxanes (docetaxel every 3 weeks or paclitaxel weekly), administered according to their respective product labels. Chemotherapy will be administered following an infusion of monoclonal antibodies (pertuzumab and/or trastuzumab).

In patients receiving induction chemotherapy, chemotherapy using AI will begin after the chemotherapy induction phase.

Control arm - Arm B

Trastuzumab (IV infusion)

Administer 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 cycle every 3 weeks thereafter; administer as directed on the product label.

AI (oral)

Use as indicated on the product label (anastrozole: 1 mg once daily; letrozole: 2.5 mg once daily).

Induction chemotherapy

Same as the research arm.

Main efficacy results

PFS (defined as the time from randomization to the first radiographic record of disease progression or death from any cause (whichever comes first)).

Secondary efficacy results

-OS

-ORR

-CBR

- Response duration

-Time before response

Security

-Incidence and severity of adverse events (AEs) and serious adverse events (SAEs)

-Incidence of CHF

-LVEF during the research process

- Abnormal laboratory test

The combination of pertuzumab, trastuzumab, and AI is expected to be safe and effective in the patient population, and adding pertuzumab to trastuzumab and AI will prolong progression-free survival (PFS) compared to trastuzumab plus AI without pertuzumab.

Example 9: Pertuzumab for improving overall survival (OS) in cancer patients

Background: In the CLEOPATRA study described in Example 3 above, 808 patients with HER2-positive first-line (1L) metastatic breast cancer (MBC) were randomized to receive either placebo plus trastuzumab plus docetaxel (Pla+T+D) or pertuzumab plus trastuzumab plus docetaxel (P+T+D). The primary endpoint of progression-free survival was significantly improved with P+T+D compared to Pla+T+D (hazard ratio (HR) = 0.62; P < 0.0001; median, 18.5 vs. 12.4 months) (Example 3 above). This example includes a second-period overall survival (OS) analysis following longer follow-up.

Methods: Overall survival (OS) analysis during this period was performed using the Lan-DeMets α-exhaustion function, with the O’Brien-Fleming (OBF) stop boundary used to maintain overall type I error within 5%. The OBF stop for statistical significance of this analysis was P ≤ 0.0138, based on the number of observed OS events. A log-rank test (stratified by pre-treatment status and geographic region) was used to compare OS between arms in the intended treatment population. The Kaplan-Meier method was used to estimate median OS in both arms; a stratified Cox proportional hazard model was used to estimate HR and 95% CI. Subgroup analyses of OS were performed for stratification factors and other key baseline characteristics.

Results: At the time of this analysis, the median follow-up was 30 months, and 267 deaths occurred (69% of the planned events used in the final analysis). Results showed a statistically significant improvement in overall survival (OS) biased towards P+T+D (HR = 0.66; 95% confidence interval (CI), 0.52–0.84; P = 0.0008). This HR represents a 34% reduction in the risk of death. This analysis reached statistical significance and was therefore considered a confirmatory OS analysis. The median OS was 37.6 months in the Pla arm and had not yet been reached in the P arm. Treatment efficacy was 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 T (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 overall survival (OS) rates showed a survival benefit at 1, 2, and 3 years with P+T+D.

Table 14: Overall survival benefit of pertuzumab

Most patients received anticancer therapy after discontinuing study treatment (64% Pla arm, 56% P arm). Subsequent therapies using HER2-targeted agents (T, lapatinib, T emtansine) were balanced between arms. Causes of death remained unchanged from the first-period OS analysis, with progressive disease being the most common cause. Adverse events leading to death were rare and balanced between arms.

Conclusion: Treatment with P+T+D in patients with HER2-positive 1L MBC was statistically and clinically significant compared to Pla+T+D. These results suggest that the combination of HER2 blockade and chemotherapy with the P+T+D regimen can be considered standard care for patients with HER2-positive MBC in a 1L background.

This data about OS can be included in a packaging insert containing prescription information about pertuzumab in a product such as, for example, Example 4 above.

Example 10: Pertuzumab and trasulimumab together with taxane as first-line therapy for patients with HER2-positive advanced breast cancer (PERUSE)

Background: Pertuzumab (P), a humanized monoclonal antibody, inhibits downstream HER2 signaling by binding to the dimerization domain of the receptor and preventing heterodimerization with other HER family members. The epitope recognized by P differs from that bound by trastuzumab (H), thus their complementary mechanism of action produces more complete HER2 blockade. Data from the phase III CLEOPATRA trial showed significantly improved progression-free survival (PFS) in patients (pt) receiving P+H+docetaxel as first-line treatment for HER2-positive metastatic breast cancer (BC) compared to H+docetaxel+placebo.

Trial Design: This is a phase IIIb, multicenter, open-label, single-arm study in patients with HER2-positive metastatic or locally recurrent BC who have not previously received systemic non-hormonal anticancer therapy for metastatic cancer. Patients will receive: P: 840 mg starting dose, 420 mg q3w IV; H: 8 mg/kg starting dose, 6 mg/kg q3w IV; taxane: docetaxel, paclitaxel, or nab-paclitaxel (according to local guidelines). Treatment will continue until disease progression or unacceptable toxicity. A protocol modification is planned to allow hormone receptor-positive patients to receive endocrine therapy along with P+H after completion of taxane therapy, in accordance with clinical practice.

Eligibility criteria: At baseline, patients must have ≥50% LVEF, ECOG PS of 0, 1, or 2, a disease-free interval of ≥6 months, and must not have received prior anti-HER2 agents for the treatment of metastatic BC. Prior anti-HER2 and/or lapatinib in a (neo)adjuvant setting is permitted, provided there is no disease progression during treatment. Patients must not have experienced other malignancies in the past 5 years, excluding cervical carcinoma in situ or basal cell carcinoma. There must be no clinical or radiographic evidence of CNS metastases or clinically significant cardiovascular disease.

Specific Objective: Because H was not widely used in (neo)adjuvant settings prior to the CLEOPATRA recruitment, a relatively low proportion of patients in CLEOPATRA had previously received H. PERUSE will evaluate the safety and tolerability of P+H+ taxane as a first-line therapy in patients with HER2-positive metastatic or locally advanced BC in a patient population likely to have experienced greater exposure to prior H therapy.

Statistical Methods: The primary endpoints of the PERUSE study were safety and tolerability. Secondary endpoints included PFS, OS, ORR, CBR, duration of response, time to response, and QoL. The final analysis will be conducted when 1500 patients have been followed for at least 12 months since their last study treatment, unless they lose follow-up, consent to withdrawal, die, or if the study is prematurely terminated by the sponsor. Safety analyses are planned after enrollment of approximately 350, 700, and 1000 patients. Additionally, the Data and Safety Monitoring Board will review safety data after enrollment of approximately 50 patients and every 6 months thereafter.

It is anticipated that, following the protocol described in this embodiment, pertuzumab and trastuzumab together with taxane as first-line therapy will be effective for patients with HER2-positive advanced breast cancer.

Example 11: Pertuzumab combined with chemotherapy in low HER3 ovarian cancer

Epithelial ovarian cancer, along with primary peritoneal cancer and fallopian tube cancer, is the fifth leading cause of cancer-related death among women in Europe (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 adding taxanes to platinum-based chemotherapy has resulted in 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 eventually die from the disease (Du Bois et al., Cancer 115:1234-1244 (2009)). Treatment options are limited after failure of platinum-based chemotherapy. Patients with platinum-sensitive disease (disease relapse more than 6 months after the last cycle of platinum-based chemotherapy) are frequently retreated with platinum-based therapies and have a progression-free survival (PFS) of approximately 9–10; however, the prognosis is quite poor for patients with primary platinum-resistant disease. Retreat with platinum-based therapies or surgery is illogical for these patients; in fact, patients with platinum resistance are often treated with single-agent chemotherapy such as topotecan, PEGylated liposomal doxorubicin (PLD), paclitaxel, and gemcitabine.

The objective response rate for patients with platinum-resistant disease ranges from 10% to 20%, while the median progression-free survival (PFS) ranges from 3.5 to 4 months. Platinum-resistant disease is incurable; treatment goals for these patients include symptom relief, prolonged survival, and improved quality of life (QoL). Overall, results from major clinical trials conducted over the last 20 years show that the median PFS for patients with advanced disease ranges from 16 to 23 months, while the median overall survival (OS) ranges from 31 to 65 months.

Most ovarian cancer cell lines and many ovarian cancer biopsy samples express all members of the HER receptor family (Campiglio et al. J. Cell Biochem 73:522-32 (1999)). EGFR and HER2 have been the most extensively studied, and various agents targeting this receptor or related intracellular tyrosine kinases have been tested.

In a recent study, quantitative HER2 protein analysis showed that malignant ovarian tumors had significantly higher levels of HER2 compared to benign ovarian tumors and normal ovaries. Furthermore, a correlation has been observed between HER2 and HER3 protein levels (Steffensen et al., Int J Oncol. 33: 195-204 (2008)). Studies in cell culture systems have shown that the heregulin-activated HER3-HER2 heterodimer elicits the strongest proliferation and transformation response 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 in: Mol Cell Biol 16: 735 (1996)). The potency of these biological responses is likely a result of the dual and efficient activation of the MAP 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 have shown that HER3, rather than EGFR, is crucial for HER2 signaling, and that HER3 inhibits growth in three-dimensional culture and induces rapid tumor regression in xenografts in vivo (Lee-Hoeflich et al. Cancer Res 68:5878-87 (2008)).

In addition, HER3 expression has been suggested as a possible risk factor in ovarian cancer (Tanner et al. J Clin Oncol 24:4317-23 (2006)).

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

The results of this study came from two randomized phase II trials in platinum-sensitive and platinum-resistant populations. In the TOC3258g study, the efficacy and safety of gemcitabine plus pertuzumab versus gemcitabine plus placebo were evaluated in patients with advanced ovarian cancer, primary peritoneal cancer, or fallopian tube cancer who were resistant to platinum-based chemotherapy (Amler et al. J Clin Oncol 26:5552 (2008)). This study allowed patients to cross over to receive pertuzumab as their disease progressed. 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 the treatment arms. Among the most common adverse events (AEs), those with an increase in patients in the pertuzumab treatment group (at least 6 additional patients) included fatigue, nausea, diarrhea, back pain, indigestion, stomatitis, headache, epistaxis, rhinorrhea, rash, and grade 3-4 neutropenia.

In the BO17931 study, 149 patients with ovarian cancer who experienced recurrence 6 months after platinum-based therapy were randomized to receive paclitaxel and either carboplatin or gemcitabine, with or without pertuzumab. After 6 cycles of treatment, chemotherapy was discontinued, and patients in the chemotherapy + pertuzumab arm continued to receive pertuzumab alone for up to 11 additional cycles (a total of 17 cycles of pertuzumab). For the entire group, there were no significant differences in PFS or OS. The median PFS for the chemotherapy + pertuzumab group was 34.1 weeks, compared to 31.3 weeks in the chemotherapy alone group; however, an exploratory subset analysis of HER3 mRNA expression with a 6–12 month treatment-free interval indicated a tendency for clinical benefit in patients expressing high levels of HER3 mRNA (Kaye et al. J Clin Oncol 26:5520 (2008)).

The mRNA expression levels of HER receptors EGFR, HER2, HER3, and two HER ligands, amphiregulin and betacellulin, in archived tissue samples from patients recruited to two randomized phase II studies were examined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).

Only tumor HER3 mRNA expression was significantly associated with PFS. For patients achieving a clinical response, partial response (PR) was observed in 9 patients on the gemcitabine + pertuzumab arm and 3 patients on the gemcitabine + placebo arm. Six of the gemcitabine + pertuzumab patients with PR had tumor HER3 mRNA levels below the median. In contrast, no patients on the gemcitabine + placebo arm experienced PR in those with tumor HER3 mRNA levels below the median of the study population. Another 6 patients achieved PR, and all of these patients had tumor HER3 mRNA levels at or above the median of the study population. Of these patients, 3 received gemcitabine + pertuzumab and 3 received gemcitabine + placebo, indicating no pertuzumab effect in this population.

Patients with low HER3 mRNA expression (below the median level in the study population) showed a PFS hazard ratio (HR) of 0.32, compared to 1.68 for patients with HER3 mRNA expression at or above the median level; i.e., the effect of adding pertuzumab tended in the opposite direction. No significant benefit was detected in OS in patients with low HER3 mRNA expression; however, a trend toward greater OS was observed in patients receiving pertuzumab. Patients with high HER3 mRNA expression showed an OS HR of 1.59.

To assess prognostic values, HER3 mRNA expression was associated with progression-free survival (PFS) and overall survival (OS) in patients in the gemcitabine + placebo arm. For patients with low HER3 mRNA expression (n=35), the median PFS was 1.4 months, compared to 5.5 months for patients with high HER3 mRNA expression (n=24). Similarly, for patients with low HER3 mRNA expression, the median OS was 8.4 months, compared to 18.2 months for patients with high HER3 mRNA expression.

In the BO17931 study, no therapeutic effect was observed in patients with low HER3 mRNA expression (below the median level in the study population). However, in an exploratory analysis of patients with a 6-12 month treatment-free interval, the combination of chemotherapy and pertuzumab tended to benefit in terms of progression-free survival (PFS).

Research Overview

This is a two-part, multicenter trial: a non-randomized safety run portion 1 and a randomized, double-blind portion 2.

Part 1 will be conducted to evaluate the safety and tolerability of pertuzumab in novel combinations with two chemotherapy agents (topotecan or paclitaxel). Part 2 of the trial is a randomized, double-blind, placebo-controlled, two-arm, multicenter prospective trial of pertuzumab in combination with chemotherapy (topotecan, paclitaxel, or gemcitabine). Patients will receive the investigational drug until disease progression as defined by RECIST version 1.1, disease progression as assessed by CA-125 in the Gynecologic Cancer Intergroup (GCIG) criteria, unacceptable toxicity, consent to withdrawal, or death. Progression-free survival (PFS) will be assessed in Part 1 of the trial, but due to the small number of patients per group and PFS events, the results will be descriptive only. The trial design for Part 1 is provided in Figure 30.

In part 2 of the trial, patients were randomized in a 1:1 ratio to receive either of the following:

- Arm A: Pertuzumab in combination with chemotherapy (topotecan, paclitaxel, or gemcitabine), or

- Arm B: Pertuzumab - placebo plus chemotherapy (topotecan, paclitaxel, or gemcitabine).

The allocation of the study drug, whether patients receive pertuzumab or pertuzumab-placebo, will be double-blind. The chemotherapy agent allocated will be based on the investigator's judgment.

The stratification factor for Experiment 2 will be:

- Selected chemotherapy group (topotecan vs. paclitaxel vs. gemcitabine).

- Previous anti-angiogenic therapy (with or without). If the patient has previously participated in a double-blind trial using an anti-angiogenic agent, then the patient will participate in the same tier as patients known to have previously received an anti-angiogenic agent.

- Treatment-free interval (TFI) from platinum therapy (strictly defined as less than 3 months prior to the first study treatment, for those including 3 to 6 months).

The experimental design for study section 2 is provided in Figure 31.

Main objective of the study:

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

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 progression-free survival (PFS).

Secondary objective of the study:

Part 1: The secondary objective of Part 1 of this study is to descriptively evaluate the progression-free survival (PFS) of pertuzumab in combination with topotecan or paclitaxel.

Part 2: The secondary objective of Part 2 of this study is to determine whether pertuzumab plus chemotherapy is superior to placebo plus chemotherapy in the following respects:

-OS.

- Objective response rate.

- Biological progression-free interval (PFI _BIO ).

- Safety and tolerability.

-QoL.

efficacy outcome measurement

The following efficacy measures will be measured in Part 1 of this experiment:

-PFS is defined as the time from randomization to Part 1 of the trial until disease progression (according to RECIST version 1.1 or GCIG criteria for assessable disease according to CA-125) or death from any cause (whichever comes first).

The efficacy results for part 2 of this experiment are measured as follows:

-PFS is defined as the time from randomization to Part 2 of the trial until disease progression (according to RECIST version 1.1 or GCIG criteria for assessable disease according to CA-125) or death from any cause (whichever comes first).

-OS is defined as the time from randomization to experimental part 2 until death for any reason.

- Objective response rate (ORR), which will be based on RECIST version 1.1 and assessed by best (confirmed) overall response (BOR); defined as the best response recorded from the start of treatment in Part 2 of the trial until disease progression/relapse (as a reference for PD, the smallest measurement recorded from the start of treatment in Part 2 of the trial). Patients need to have two consecutive partial response (PR) or complete response (CR) assessments to be considered a responder. PR or CR must be confirmed by two consecutive tumor assessments at least 4 weeks apart. Patients with measurable disease only at baseline will be included in the analysis of objective response.

- Patients with a response according to RECIST version 1.1 and using the 50% response criteria of CA-125 are defined as responders, while patients with a response only as defined by RECIST are defined as RECIST responders. Patients without a response according to RECIST but with a response as defined by the 50% response criteria of CA-125 are defined as CA-125 responders.

-PFIBIO, based on progressive serial elevation of serum CA-125 (assessed according to CGIG criteria), is defined as the time from randomization to the date in Part 2 of the trial until the first recorded CA-125 level increases to: twice the upper limit of normal (for patients with normal pre-treatment CA-125 or elevated pre-treatment CA-125 and during initial normalization on-treatment), or twice the nadir value (for patients with elevated baseline CA-125 and during non-normalized treatment).

Safety outcome measurement

In Part 1 of the study, safety and tolerability will be assessed after all patients have received three treatment cycles.

In addition, safety outcome measures of the study will be assessed in parts 1 and 2 of the study, including: - the incidence, nature and severity of all adverse events (AEs), serious adverse events (SAEs), AEs with NCI-CTCAE version 4.0 ≥ grade 3, and AEs that lead to premature withdrawal from the study drug.

- Premature withdrawal from research and research treatment.

- The occurrence of heart disease/congestive heart failure.

- Abnormal laboratory test results.

-Left ventricular ejection fraction

Inclusion criteria

1. Female patients aged 18 or older.

2. Low HER3 mRNA expression levels (equal to or less than a concentration ratio of 2.81, as assessed by qRT-PCR on a COBAS instrument).

3. Histologically or cytologically confirmed and documented epithelial ovarian cancer that is platinum-resistant or non-reactive (defined as progression within 6 months after completion of at least 4 cycles of platinum therapy or progression during platinum therapy).

4. At least one measurable lesion and/or an unmeasurable disease according to RECIST version 1.1, or a disease assessable by cancer antigen-125 (CA-125) according to Gynecologic Center Intergroup (GCIG) criteria. The following histological types are eligible:

- Adenocarcinoma not otherwise specified.

-Clear cell adenocarcinoma.

- Endometrioid adenocarcinoma.

- Malignant Brenner's tumor.

- Mixed epithelial carcinomas, including malignant mixed Müllerian tumors.

- Mucinous gland carcinoma.

- Serous adenocarcinoma.

- Transitional cell carcinoma.

- Undifferentiated carcinoma.

5. Eastern Cooperative Oncology Group (ECOG) performance status 0 to 2.

6. LVEF is greater than or equal to 55%.

Pertuzumab dosage and administration

Pertuzumab and pertuzumab-placebo will be administered intravenously as a loading dose of 840 mg on day 1 of the first treatment cycle, followed by 420 mg on day 1 of each cycle every 3 weeks thereafter. The initial pertuzumab/pertuzumab-placebo infusion will be administered over 60 minutes, followed by a 60-minute observation period in a seated position. If the infusion is well tolerated, subsequent infusions may be administered over 30 minutes, followed by a 30-minute observation period, after which the chemotherapy agent will be administered. Pre-treatment should be performed according to local practice and the selected chemotherapy.

Topotecan dosage and administration

Topotecan should be administered daily at a dose of 1.25 mg/ ^m² via intravenous infusion over 30 minutes on days 1-5 of every 3 weeks, as directed in the product characteristics summary.

Paclitaxel dosage and administration

Paclitaxel should be administered at 80 mg/m² via intravenous infusion over 1 hour on days 1, 8, 15, and 22. Pharmacists should follow the product characteristics summary regarding the preparation and administration of the 80 mg/m² dose.

Gemcitabine dosage and administration

Gemcitabine (study portion 2 only) should be administered intravenously at 1000 mg/m2 over 30 minutes on days 1 and 8 of every 3 weeks, as directed in the product feature summary.

HER3 mRNA expression

Before patients consent to participate in the trial, they will be specifically asked for their consent to have primary tumor tissue samples collected and tested to assess HER3 mRNA levels, including mRNA and protein levels of other HER family receptors such as HER2. Patients with tumors expressing only low levels of HER3 mRNA will be eligible to participate in this trial.

During the initial screening of HER3 mRNA levels, other receptors in the HER family (e.g., EGFR, HER2, or HER4) will be assessed at both the mRNA and/or protein levels in parallel with HER3 assessment, thereby obtaining a more complete description of the status of HER family receptors at the mRNA level.

The cutoff value for eligibility in this study was defined as a concentration ratio ≤ 2.81, as assessed by qRT-PCR using the “HER2 & HER3 (qRT-PCR) mRNA Expression Assay” provided by Roche Molecular Diagnostics. The cutoff value definition is based on cutoff value modeling from previous studies and the transformation function that must be introduced due to the assay being switched to the new COBAS instrument. It is anticipated that 40–50% of screened patients will have HER3 mRNA levels below the cutoff value of 2.81, while 30% of patients expressing low levels of HER3 mRNA will be ineligible (due to other inclusion/exclusion criteria).

All patients will be required to submit formalin-fixed, paraffin-embedded tumor specimens from the primary tumor of the initial surgery prior to screening; cytological specimens are not an acceptable substitute. Patients' HER3 mRNA expression levels, as well as the mRNA and protein expression levels of other HER family receptors, will be assessed using qRT-PCR and IHC. These assessments of HER receptor mRNA/protein expression will be performed at any time after the primary surgery and prior to screening, with informed consent from the patient.

It is expected that pertuzumab in combination with topotecan or paclitaxel will be safe and effective in patients with epithelial ovarian cancer, primary peritoneal cancer, and fallopian tube cancer.

In addition, pertuzumab plus chemotherapy (topotecan, paclitaxel, or gemcitabine) is expected to be superior to placebo plus chemotherapy in patients with epithelial ovarian cancer, primary peritoneal cancer, and fallopian tube cancer, with efficacy measured by progression-free survival (PFS).

Claims (3)

1. Use of pertuzumab in the preparation of a drug for neoadjuvant therapy in patients with early HER2-positive, estrogen receptor and progesterone receptor-negative breast cancer in combination with trastuzumab and anthracycline-based chemotherapy drugs containing 5-FU, epirubicin and cyclophosphamide, and docetaxel, in patients without metastasis. Pertuzumab is provided for administration on day 1 of three 3-week cycles in concomitant with trastuzumab and 5-FU, epirubicin, and cyclophosphamide, followed by three cycles of docetaxel, trastuzumab, and pertuzumab. Furthermore, the neoadjuvant therapy resulted in a complete pathological response in the patient and did not cause symptomatic left ventricular systolic dysfunction. 2. The use of claim 1, wherein the early HER2-positive, estrogen receptor and progesterone receptor-negative breast cancer has a diameter >2 cm. 3. The use of claim 1, wherein the early HER2-positive, estrogen receptor and progesterone receptor-negative breast cancer is locally advanced or inflammatory breast cancer.