HK40012625B - Combinations of an anti-her2 antibody-drug conjugate and chemotherapeutic agents, and methods of use - Google Patents

Description

Combination of anti-HER2 antibody-drug conjugates and chemotherapy agents, and methods of administration.

This application is a divisional application of a divisional application filed on March 10, 2009, with application number 201510170572.9 and invention title "Combination of anti-HER2 antibody-drug conjugate and chemotherapeutic agent, and method of use" (original application number 200980117906.9).

Cross-reference to related applications

This non-provisional application, filed pursuant to 37 CFR §1.53(b), claims the benefit of U.S. Provisional Application No. 61/037,410, filed March 18, 2008 (included herein in full reference), pursuant to 35 USC §119(e).

Invention Field

This invention generally relates to pharmaceutical combinations having activity against highly proliferative diseases such as cancer. The invention also relates to methods for diagnosing or treating mammalian cells, or related pathological conditions, using said compound combinations in vitro, in situ, and in vivo.

Background of the Invention

The HER2 (ErbB2) receptor tyrosine is a member of the epidermal growth factor receptor (EGFR) transmembrane receptor family. HER2 overexpression is observed in approximately 20% of breast cancers, and HER2 overexpression is associated with invasive growth and poor clinical outcomes in these tumors (Slamon et al. (1987) Science 235:177-182).

Trastuzumab (CAS 180288-69-1, huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived humanized IgG1κ monoclonal antibody against mouse HER2. In cell-based assays, it selectively binds with high affinity (Kd = 5 nM) to the extracellular domain of human epidermal growth factor receptor 2 (HER2) protein (US 5677171; US 5821337; US 605). 4297; US 6165464; US 6339142; US6407213; US 6639055; US 6719971; US 6800738; US 7074404; Coussens et al. (1985) Science 230: 1132-9; Slamon et al. (1989) Science 244: 707-12; Slamon et al. (2001) New Engl. J. Med. 344: 783-792). Trastuzumab contains a human framework region and a complementarity-determining region for a HER2-binding mouse antibody (4D5). Trastuzumab binds to the HER2 antigen, thereby inhibiting the growth of cancer cells. Trastuzumab has been shown in in vitro assays and in animals to inhibit the proliferation of human tumor cells overexpressing HER2 (Hudziak et al. (1989) Mol Cell Biol 9: 1165-72; Lewis et al. (1993) Cancer Immunol Immunother; 37: 255-63; Baselga et al. (1998) Cancer Res. 58: 2825-2831). Trastuzumab is an antibody-dependent cell cytotoxic agent and a mediator of ADCC (Lewis et al. (1993) Cancer Immunol Immunother 37(4): 255-263; Hotaling et al. (1996) [abstract]. Proc. Annual Meeting Am Assoc Cancer Res; 37: 471; Pegram MD et al. (1997) [abstract]. Proc Am Assoc Cancer Res; 38: 602; Sliwkowski et al. (1999) Seminars in Oncology 26(4), Suppl 12: 60-70; Yarden Y. and Sliwkowski, M. (2001) Nature Reviews: Molecular Cell Biology, Macmillan Magazines, Ltd., Vol. 2: 127-137).

Approved in 1998 for the treatment of patients with metastatic breast cancer overexpressing ErbB2 who have received extensive prior anticancer therapy (Baselga et al., (1996) J. Clin. Oncol. 14: 737-744), and has been used in over 300,000 patients (Slamon DJ et al., N Engl J Med 2001; 344: 783-92; Vogel CL et al., J Clin Oncol 2002; 20: 719-26; Marty). M et al., J Clin Oncol 2005;23:4265-74; Romond EH et al., T N Engl J Med 2005;353:1673-84; Piccart-Gebhart MJ et al., N Engl J Med 2005;353:1659-72; Slamon D et al., [abstract]. Breast Cancer Res Treat 2006, 100(Suppl 1):52). In 2006, the FDA approved trastuzumab (Genentech Inc.) as part of a treatment regimen containing doxorubicin, cyclophosphamide, and palitaxetine for adjuvant therapy in patients with HER2-positive, nodular-positive breast cancer. While the development of trastuzumab has provided significantly better outcomes for patients with HER2-positive tumors than chemotherapy alone, virtually all HER2-positive metastatic breast cancer (MBC) patients eventually progress on existing therapies. There is still an opportunity to improve outcomes for patients with MBC. Despite trastuzumab's diverse mechanisms of action, many patients treated with trastuzumab do not show a response or cease responding after a period of treatment benefit. Some HER2+ (HER2-positive) tumors do not respond, and most patients whose tumors do respond eventually progress. There is a significant clinical need to develop alternative HER2-targeting cancer therapies for patients with unresponsive or poorly responsive tumors, tumors that overexpress HER2, or other diseases related to HER2 expression.

An alternative to antibody-targeted therapy is to use antibodies to specifically deliver cytotoxic drugs to cancer cells expressing antigens. Maytansine alkaloids, derivatives of the antimitotic drug maytansine, bind to microtubules in a manner similar to that of vinca alkaloids (Issell BF et al. (1978) Cancer Treatment. Rev. 5: 199-207; Cabanillas F et al. (1979) Cancer Treatment Rep. 63: 507-9). Antibody-drug conjugates (ADCs) consisting of maytansine alkaloid DM1 linked to trastuzumab have shown potent antitumor activity in HER2-overexpressing trastuzumab-sensitive and trastuzumab-resistant tumor cell lines, and in xenograft models of human breast cancer. Conjugates of maytansine alkaloids linked to the anti-HER2 mouse breast cancer antibody TA.1 via an MCC linker are 200-fold less potent than corresponding conjugates with disulfide linkers (Chari et al. (1992) Cancer Res. 127-133). Antibody-drug conjugates (ADCs) composed of maytansine alkaloid DM1 linked to trastuzumab have shown potent antitumor activity in HER2-overexpressing trastuzumab-sensitive and resistant tumor cell lines and xenograft models of human cancer. Trastuzumab-MCC-DM1 (T-DM1) is currently being evaluated in a phase II clinical trial in patients whose disease does not respond to HER2-targeted therapy (Beeram et al. (2007) "A phase I study of trastuzumab-MCC-DM1 (T-DM1), a first-in-class HER2 antibody-drug conjugate (ADC), in patients (pts) with HER2+ metastatic breast cancer (BC)", American Society of Clinical Oncology 43rd: June 02 (Abs 1042; Krop et al., European Cancer Conference ECCO, Poster 2118, September 23-27, 2007, Barcelona; US 7097840; US 2005/0276812; US 2005/0166993).

Combination therapy using two or more drugs in some dosage regimens or administration methods typically has one or more of the following features: (i) reducing the frequency of acquired resistance by combining drugs with minimal cross-resistance; (ii) reducing the dosage of drugs with non-overlapping toxicities and similar therapeutic properties to achieve efficacy and fewer side effects, i.e., improving the therapeutic index; (iii) sensitizing cells to the action of another drug by using one drug, such as altering cell cycle phases or growth properties; and (iv) achieving enhanced potency by exploring the additive effect of the two drugs in their biological activity, or an effect greater than the additive effect (Pegram, M. et al. (1)). 999) Oncogene18: 2241-2251; Konecny, G. et al. (2001) Breast Cancer Res. and Treatment 67: 223-233; Pegram, M. et al. (2004) J. of the Nat.Cance r Inst.96(10):739-749; Fitzgerald et al. (2006) Nature Chem.Biol.2(9):458-466; Borisy et al. (2003) Proc.Natl.Acad.Sci 100(13):7977-7982).

The Loewe additive effect (Chou, T.C. and Talalay, P. (1977) J. Biol. Chem. 252: 6438-6442; Chou, T.C. and Talalay, P. (1984) Adv. Enzyme Regul. 22: 27-55; Berenbaum, M.C. (1989) Pharmacol. Rev. 41: 93-141) and the Bliss independence/synergism (Bliss, C.I. (1956) Bacteriol. Rev. 20: 243-258; Greco et al. (1995) Pharmacol. Rev. 47: 331-385) are methods for calculating the expected dose-response relationship for combination therapy based on parameters such as IC50, i.e., the drug dose required to achieve 50% target inhibition, which in the simplest case is equal to Ki, compared to monotherapy.

HER2 dimerization inhibitor antibodies and EGFR inhibitors have been reported for combination therapy against cancer (US2007/0020261). trastuzumab-MCC-DM1 (T-DM1) and pertuzumab have shown activity in MBC patients individually, and the combination of pertuzumab and trastuzumab has shown activity in HER2-positive MBC patients (Baselga J et al., “A Phase II trial of trastuzumab and pertuzumab in patients with HER2-positive metastatic breast cancer that had progressed during trastuzumab therapy: full response data”, European Society of Medical Oncology, Stockholm, Sweden, September 12-16, 2008).

Invention Overview

This invention generally relates to an anti-HER2 antibody-drug conjugate, trastuzumab-MCC-DM1, administered in combination with one or more chemotherapeutic agents to inhibit cancer cell growth. Certain combinations of trastuzumab-MCC-DM1 and chemotherapeutic agents have shown synergistic effects in inhibiting cancer cell growth both in vitro and in vivo. The combinations and methods of this invention can be used to treat highly proliferative diseases such as cancer. These combinations can inhibit tumor growth in mammals and can also be used to treat human cancer patients.

On one hand, the present invention includes a method for treating a hyperproliferative disease, comprising administering a combination of therapeutic agents to a mammal in the form of a combination formulation or alternately, wherein the combination of therapeutic agents comprises a therapeutically effective amount of trastuzumab-MCC-DM1 and a therapeutically effective amount of a chemotherapeutic agent selected from HER2 dimerization inhibitor antibodies, anti-VEGF antibodies, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941, and GNE-390.

The therapeutically effective amount of trastuzumab-MCC-DM1 and the therapeutically effective amount of the chemotherapeutic agent may be administered in combination or alternately.

The present invention also relates to methods for diagnosing or treating mammalian cells, organisms, or related pathological conditions using the said composition in vitro, in situ, and in vivo.

The present invention also relates to a method for administering the aforementioned combination of therapeutic agents to achieve a synergistic effect.

Another aspect of the invention is a pharmaceutical composition comprising trastuzumab-MCC-DM1, a chemotherapeutic agent selected from HER2 dimerization inhibitor antibodies, anti-VEGF antibodies, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941, and GNE-390; and one or more pharmaceutically acceptable carriers, gliding agents, diluents, or excipients.

Another aspect of the invention provides a method of treating a highly proliferative disease or condition, comprising administering an effective amount of trastuzumab-MCC-DM1 and a chemotherapeutic agent to a mammal requiring such treatment. Trastuzumab-MCC-DM1 and the chemotherapeutic agent may be co-formulated for administration as a pharmaceutical preparation combination, or they may be administered separately as a therapeutic combination, alternatingly (sequentially). In one embodiment, T-DM1 is delivered by infusion, while the chemotherapeutic agent is delivered orally.

Another aspect of the invention provides a method for predicting the efficacy of effective drug combinations in vivo, wherein the combination comprises trastuzumab-MCC-DM1 and a standard anticancer chemotherapeutic agent. Qualitative and quantitative analyses are performed on efficacy data from in vitro cell proliferation and in vivo tumor xenograft experiments. Quantitative analysis methods may be based on the median effect of Chou and Talalay and equivalent line plots to generate a Combination Index (CI) value to represent synergy, antagonism, or additive effects, or based on deviations from Bliss independence band plots.

Another aspect of the invention is a method of treating diseases or conditions in mammals, such as cancer, including those regulated by HER2 or KDR9 (VEGF receptor 1), using the therapeutic combination of the invention.

Another aspect of the invention is the use of the therapeutic agents of the invention in the preparation of medicaments for treating diseases or conditions in mammals, such as cancer, including those regulated by HER2 or KDR9 (VEGF receptor 1).

Another aspect of the invention includes an article or kit containing trastuzumab-MCC-DM1, a chemotherapeutic agent, a container, and optionally a packaging insert or label indicating the treatment method.

Another aspect of the invention includes a method for determining compounds to be used in combination for cancer treatment, comprising: a) administering trastuzumab-MCC-DM1 to an in vitro tumor cell line, and a therapeutic combination of a chemotherapeutic agent selected from HER2 dimerization inhibitor antibody, anti-VEGF antibody, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941, and GNE-390, and b) measuring synergistic or non-synergistic effects.

Further advantages and novel features of the invention are set forth in the description below, and in part will be apparent to those skilled in the art upon examination of the following specification or may be learned by practice of the invention. The advantages of the invention can be recognized and obtained by means/tools, combinations, compositions, and methods particularly pointed out in the appended claims.

This invention relates to the following:

1. A method for treating a hyperproliferative disease, comprising administering a combination of therapeutic agents to a mammal in the form of a combination formulation or alternately, wherein the combination comprises a therapeutically effective amount of trastuzumab-MCC-DM1 and a therapeutically effective amount of a chemotherapeutic agent selected from HER2 dimerization inhibitor antibodies, anti-VEGF antibodies, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941, and GNE-390.

2. The method of item 1, wherein the HER2 dimerization inhibitor antibody is pertuzumab.

3. The method of item 1, wherein the anti-VEGF antibody is bevacizumab.

4. The method of item 1, wherein the chemotherapeutic agent is 5-FU.

5. The method of item 1, wherein the chemotherapeutic agent is carboplatin.

6. The method of item 1, wherein the chemotherapeutic agent is lapatinib.

7. The method of item 1, wherein the chemotherapeutic agent is ABT-869.

8. The method of item 1, wherein the chemotherapeutic agent is docetaxel.

9. The method of item 1, wherein the chemotherapeutic agent is GDC-0941.

10. The method of item 1, wherein the chemotherapeutic agent is GNE-390.

11. The method of claim 1, wherein the therapeutically effective amount of trastuzumab-MCC-DM1 and the therapeutically effective amount of the chemotherapeutic agent are administered in a combination formulation.

12. The method of item 1, wherein the therapeutically effective amount of trastuzumab-MCC-DM1 and the therapeutically effective amount of the chemotherapeutic agent are administered alternately.

13. The method of item 12, wherein the chemotherapeutic agent is administered to the mammal, followed by the administration of trastuzumab-MCC-DM1.

14. The method of item 12, wherein the combination of therapeutic agents is administered to a person with a hyperproliferative disease at intervals of approximately 3 weeks.

15. The method of item 12, wherein trastuzumab-MCC-DM1 is administered to individuals with hyperproliferative disease at intervals of approximately 1 to 3 weeks.

16. The method of item 1, wherein the administration of the combination of therapeutic agents results in a synergistic effect.

17. The method of item 1, wherein the hyperproliferative disease is cancer.

18. The method of item 17, wherein the hyperproliferative disease is a cancer expressing ErbB2.

19. The method of item 17, wherein the cancer is breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, genitourinary tract cancer, esophageal cancer, laryngeal cancer, glioblastoma, neuroblastoma, gastric cancer, skin cancer, keratoacanthoma, lung cancer, epidermoid carcinoma, large cell carcinoma, non-small cell lung cancer (NSCLC), small cell carcinoma, adenocarcinoma of the lung, bone cancer, colon cancer, adenoma, pancreatic cancer, adenocarcinoma, thyroid cancer, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver and bile duct cancer, kidney cancer, pancreatic cancer, medullary disease, lymphoma, pilosarcoma, oral cancer, nasopharyngeal carcinoma, pharyngeal cancer, lip cancer, tongue cancer, oral cancer, small bowel cancer, colorectal cancer, colon cancer, rectal cancer, brain and central nervous system cancer, Hodgkin's disease, or leukemia.

20. The method of item 1, wherein the amount of trastuzumab-MCC-DM1 and the amount of the chemotherapeutic agent are each about 1 mg to about 1000 mg.

21. The method of item 1, wherein the weight ratio of the amount of trastuzumab-MCC-DM1 to the amount of the chemotherapeutic agent is about 1:10 to about 10:1.

22. The method of item 1, wherein the mammal is a HER2-positive patient.

23. The method of item 1, wherein the HER2-positive patient has received trastuzumab or lapatinib therapy.

24. A pharmaceutical composition comprising trastuzumab-MCC-DM1, a chemotherapeutic agent selected from HER2 dimerization inhibitor antibodies, anti-VEGF antibodies, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941, and GNE-390; and one or more pharmaceutically acceptable carriers, gliding agents, diluents, or excipients.

25. The pharmaceutical composition of item 24, comprising a pharmaceutically acceptable gliding agent selected from silica, powdered cellulose, microcrystalline cellulose, metal stearate, sodium aluminosilicate, sodium benzoate, calcium carbonate, calcium silicate, corn starch, magnesium carbonate, asbestos-free talc, Stearot C, starch, starch 1500, magnesium lauryl sulfate, magnesium oxide, and combinations thereof.

26. The pharmaceutical composition of item 24, wherein the amount of trastuzumab-MCC-DM1 and the amount of the chemotherapeutic agent are each present in an amount of about 1 mg to about 1000 mg.

27. The pharmaceutical composition of item 24, wherein the amount of trastuzumab-MCC-DM1 and the amount of the chemotherapeutic agent are present in a weight ratio of about 1:10 to about 10:1.

28. Therapeutic agents are manufactured for the treatment of cancers selected from breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, genitourinary tract cancer, esophageal cancer, laryngeal cancer, glioblastoma, neuroblastoma, gastric cancer, skin cancer, keratoacanthoma, lung cancer, epidermoid carcinoma, large cell carcinoma, non-small cell lung cancer (NSCLC), small cell carcinoma, adenocarcinoma of the lung, bone cancer, colon cancer, adenoma, pancreatic cancer, adenocarcinoma, thyroid cancer, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver and bile duct cancer, kidney cancer, pancreatic cancer, medullary lesions, lymphoma, pilocytic carcinoma, oral cancer, and nasopharyngeal carcinoma. Use in medicines for cancers of the pharynx, lip, tongue, mouth, small intestine, colorectal, rectal, brain and central nervous system, Hodgkin's disease, or leukemia, wherein the combination of therapeutic agents is administered to mammals in the form of a combination formulation or alternately, the combination of therapeutic agents comprising a therapeutically effective amount of trastuzumab-MCC-DM1 and a therapeutically effective amount of a chemotherapeutic agent selected from HER2 dimerization inhibitor antibodies, anti-VEGF antibodies, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941, and GNE-390.

29. An article for treating hyperproliferative disorders, comprising:

(a) A combination of therapeutic agents administered to mammals, either in combination formulation or alternately, comprising a therapeutically effective amount of trastuzumab-MCC-DM1 and a therapeutically effective amount of a chemotherapeutic agent selected from HER2 dimerization inhibitor antibodies, anti-VEGF antibodies, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941, and GNE-390; and

(b) Instructions for use.

30. A method for determining compounds to be used in combination for cancer treatment, comprising:

(a) A combination of trastuzumab-MCC-DM1 and a chemotherapy agent selected from HER2-amplified breast cancer cells, including HER2 dimerization inhibitor antibodies, anti-VEGF antibodies, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941, and GNE-390.

(b) Measurement of inhibition of cell proliferation, wherein non-malignant and malignant breast cells are distinguished by cell viability.

31. The method of item 30, wherein the HER2-amplified breast cancer cells are BT-474.

32. A method for determining a combination of therapeutic agents to be used for cancer treatment, comprising:

(a) In vitro tumor cell lines were treated with a combination of trastuzumab-MCC-DM1 and a chemotherapy agent selected from HER2 dimerization inhibitor antibodies, anti-VEGF antibodies, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941, and GNE-390, and

(b) Measuring synergistic or non-synergistic effects;

This leads to the identification of synergistic therapeutic combinations for cancer treatment.

Brief description of the attached diagram

Figure 1 shows the in vitro cell viability of SK-BR-3 cells at 3 days relative to IC50 concentrations of trastuzumab, trastuzumab-MCC-DM1 (T-DM1), and the combination of trastuzumab and T-DM1.

Figure 2 shows the in vitro cell viability of BT-474EEI at 3 days relative to IC50 concentrations of trastuzumab, trastuzumab-MCC-DM1 (T-DM1), and the combination of trastuzumab and T-DM1.

Figure 3 shows the 5-day in vitro cell viability of MDA-MB-175 relative to IC50 concentrations of pertuzumab, trastuzumab-MCC-DM1 (T-DM1), and the combination of pertuzumab and T-DM1.

Figure 3a shows the 5-day in vitro cell viability of MDA-MB-175 relative to IC50 concentrations of pertuzumab, trastuzumab-MCC-DM1 (T-DM1), and the combination of pertuzumab and T-DM1.

Figure 4 shows the dose response of 5-day BT-474 in vitro cell viability relative to various fixed doses of pertuzumab combined with trastuzumab-MCC-DM1 (T-DM1) and various doses of T-DM1 alone.

Figure 5 shows the dose response of 5-day BT-474 in vitro cell viability relative to various fixed doses of trastuzumab-MCC-DM1 (T-DM1) in combination with pertuzumab, and various doses of pertuzumab alone.

Figure 6 shows the 5-day in vitro cell viability of BT-474 relative to IC50 concentrations of pertuzumab, trastuzumab-MCC-DM1 (T-DM1), and the combination of pertuzumab and T-DM1.

Figure 7 shows the in vitro cell viability of SK-BR-3 cells after 3 days relative to different doses of T-DM1 combined with fixed doses of lapatinib (4.5 nM, 14 nM, 41 nM, 123 nM) and different doses of T-DM1 alone (0-1000 ng/ml).

Figure 7a shows the in vitro cell viability of SK-BR-3 cells at 3 days relative to T-DM1, lapatinib, and a fixed dose ratio of T-DM1 and lapatinib combination.

Figure 8a shows the in vitro cell viability of BT-474 at 3 days relative to T-DM1, lapatinib, and a fixed-dose combination of T-DM1 and lapatinib.

Figure 8 shows the in vitro cell viability of BT-474 after 3 days relative to different doses of T-DM1 combined with fixed doses of lapatinib (1.5 nM, 4.5 nM, 14 nM, 41 nM, 123 nM) and different doses of T-DM1 alone (0-1000 ng/ml).

Figure 9 shows the in vitro cell viability of BT-474-EEI after 3 days relative to different doses of T-DM1 combined with fixed doses of lapatinib (14 nM, 41 nM, 123 nM, 370 nM, 1111 nM) and different doses of T-DM1 alone (0-1000 ng/ml).

Figure 10 shows the mean in vivo tumor volume over time of KPL-4 tumors inoculated into the mammary fat pads of SCID beige mice after the following administrations (3 million cells per mouse in Matrigel): (1) ADC buffer, (2) pertuzumab 15 mg/kg, (3) T-DM1 0.3 mg/kg, (4) T-DM1 1 mg/kg, (5) T-DM1 3 mg/kg, (6) pertuzumab 15 mg/kg + T-DM1 0.3 mg, (7) pertuzumab 15 mg/kg + T-DM1 1 mg/kg, (8) pertuzumab 15 mg/kg + T-DM1 3 mg/kg. ADC buffer and T-DM1 were administered once on day 0. Pertuzumab was administered on days 0, 7, and 14.

Figure 11 shows the mean in vivo tumor volume over time of KPL-4 tumors inoculated into the mammary fat pads of SCID beige mice after the following administrations (3 million cells per mouse in Matrigel): (1) ADC buffer, (2) 5-FU 100 mg/kg, (3) pertuzumab 40 mg/kg, the first dose of pertuzumab (groups 5, 7, and 9) was a 2x loading dose, (4) B20-4.1, 5 mg/kg, (5) T-DM1, 5 mg/kg, (6) 5-FU, 100 mg/kg + T-DM1, 5 mg, (7) pertuzumab 40 mg/kg + T-DM1, 5 mg/kg, (8) B20-4.1, 5 mg/kg + T-DM1, 5 mg/kg, (9) B20-4.1, 5 mg/kg + pertuzumab, 40 mg/kg. ADC buffer and T-DM1 were administered once via a single IV injection on day 0. Pertuzumab was administered on days 0, 7, 14, and 21 (qwk x 4). 5-FU was administered on days 0, 7, and 14 (qwk x 3). B20-4.1 was administered on days 0, 3, 7, 10, 14, 17, 21, and 24 (2X/wk, for a total of 8 doses).

Figure 12 shows the mean in vivo tumor volume over time of MMTV-HER2Fo5 transgenic mammary tumors inoculated into the mammary fat pads of CRL nu/nu mice after the following administrations: (1) medium (ADC buffer), (2) B20-4.1, 5 mg/kg, (3) T-DM1, 3 mg/kg, (4) T-DM1, 5 mg/kg, (5) T-DM1, 10 mg/kg, (6) B20-4.1, 5 mg/kg + T-DM1 3 mg/kg, (7) B20-4.1, 5 mg/kg + T-DM1 5 mg/kg, (8) B20-4.1, 5 mg/kg + T-DM1, 10 mg/kg. ADC buffer and T-DM1 were administered on days 0 and 21. B20-4.1 was administered on days 0, 3, 7, 10, 14, 17, 21 and 24 (2X/wk x4, for a total of 8 doses).

Figure 13 shows the mean in vivo tumor volume over time of MMTV-HER2 Fo5 transgenic mammary tumors inoculated into the mammary fat pads of CRL nu/nu mice after the following administrations: (1) medium (ADC buffer), (2) T-DM1 10 mg/kg, (3) 5-FU 100 mg/kg, (4) gemcitabine 120 mg/kg, (5) carboplatin 100 mg/kg, (6) 5-FU 100 mg/kg + T-DM1 10 mg/kg, (7) gemcitabine 120 mg/kg + T-DM1 10 mg/kg, (8) carboplatin 100 mg/kg + T-DM1 10 mg/kg. ADC buffer, T-DM1, and carboplatin were administered on day 0; single injection. 5-FU was administered on days 0, 7, and 14 (qwk x3). Gemcitabine was administered on days 0, 3, 6, and 9 (q3d x4).

Figure 14 shows the mean in vivo tumor volume over time of MMTV-Her2Fo5 transgenic mammary tumors inoculated into the mammary fat pads of Harlan athymic nude mice after the following administrations: (1) Media (PBS buffer) iv, qwk x4, (2) lapatinib 101 mg/kg, po, bid x21, (3) pertuzumab 40 mg/kg, iv, qwk x4, (4) B20-4.1 5 mg/kg, ip, 2x/wk x4, (5) T-D M1 15mg/kg, IV, q3wk to end, (6) lapatinib 101mg/kg, po, bid x21+T-DM1 15mg/kg, IV, q3wk to end, (7) pertuzumab 40mg/kg, IV, qwk x4+T-DM1 15mg/kg, IV, q3wk to end, (8) B20-4.1 5mg/kg, ip, 2x/wk x4+T-DM1 15mg/kg, IV, q3wk to end.

Figure 15 shows the mean in vivo tumor volume of MMTV-Her2 Fo5 transgenic mammary tumors inoculated into the mammary fat pads of Harlan athymic nude mice after the following administrations: (1) Medium (PBS buffer) po, bid x21, (2) T-DM1, 7.5 mg/kg, iv, qd x1, (3) T-DM1, 15 mg/kg, iv, qd x1, (4) ABT-869, 5 mg/kg, po, bid x21, (5) ABT-869, 15 mg/kg, po, bid x21, (6) T -DM1, 7.5mg/kg, iv, qd x1+ABT-869, 5mg/kg, po, bid x21, (7) T-DM1 7.5mg/kg, iv, qd x1+ABT-869, 15mg/kg, po, bid x21, ( 8) T-DM1, 15mg/kg, iv, qd x1+ABT-869, 5mg/kg, po, bid x21, (9) T-DM1, 15mg/kg, iv, qd x1+ABT-869, 15mg/kg, po, bid x21.

Figure 16 shows the mean in vivo tumor volume over time of MMTV-Her2 Fo5 transgenic mammary tumors inoculated into the mammary fat pads of Harlan athymic nude mice after the following administrations: (1) mediator, iv, qwk x3, (2) T-DM1, 7.5 mg/kg, iv, q3wk x2, (3) T-DM1, 15 mg/kg, iv, q3wk x2, (4) docetaxel, 30 mg/kg, iv, qwk x3, (5) T-DM1, 7.5 mg/kg, iv, q3wk x2 + docetaxel, 30 mg/kg, iv, qwk x3, (6) T-DM1, 15 mg/kg, iv, q3wk x2 + docetaxel, 30 mg/kg, iv, qwk x3

Figure 17 shows the mean in vivo tumor volume of MMTV-Her2 Fo5 transgenic breast tumors implanted into the mammary fat pads of Harlan athymic nude mice after the following administrations: (1) mediator, po, qd x21, (2) T-DM1, 7.5 mg/kg, iv, q3wk x2, (3) T-DM1, 15 mg/kg, iv, q3wk x2, (4) lapatinib, 100 mg/kg, po, bid x21, (5) T-DM1, 7.5 mg/kg, iv, q3wk x2+lapatinib, 100 mg/kg, po, bid x21, (6) T-DM1, 15 mg/kg, iv, q3wk x2+lapatinib, 100 mg/kg, po, bid x21.

Figure 18 shows the in vitro cell viability of SK-BR-3 at 3 days relative to IC50 concentrations of 5-FU, trastuzumab-MCC-DM1 (T-DM1), and a fixed-dose ratio of 5-FU and T-DM1.

Figure 19 shows the in vitro cell viability of BT-474 at 3 days relative to IC50 concentrations of 5-FU, trastuzumab-MCC-DM1 (T-DM1), and a fixed-dose ratio of 5-FU and T-DM1.

Figure 20 shows the in vitro cell viability of SK-BR-3 cells at 3 days relative to IC50 concentrations of gemcitabine, trastuzumab-MCC-DM1 (T-DM1), and a fixed-dose ratio of gemcitabine and T-DM1.

Figure 21 shows the in vitro cell viability of MDA-MD-361 at 3 days relative to IC50 concentrations of gemcitabine, trastuzumab-MCC-DM1 (T-DM1), and a fixed-dose ratio of gemcitabine and T-DM1.

Figure 22 shows the in vitro cell viability (proliferation) of KPL4 cells after treatment with T-DM1, GDC-0941 at IC50 concentrations ranging from 0.25x to 4x, and in fixed dose ratios of T-DM1 and GDC-0941 (62.5 nM to 1 μM) at a 1:10 ratio. Bliss predictions of the additive effect are plotted as dashed lines.

Figure 23 shows the in vitro cell viability (proliferation) of KPL4 cells after treatment with T-DM1, GDC-0941 at IC50 concentrations ranging from 0.0625x to 16x, and in fixed dose ratios of T-DM1 (1.25 to 80 ng/ml) and GDC-0941 (31.25 nM to 2 μM) at a 1:25 ratio. The Bliss prediction of the additive effect is plotted as a dashed line.

Figure 24 shows the in vitro cell viability (proliferation) of Her2-amplified, drug-resistant, PIK3CA(H1047R) mutant KPL-4 cells after treatment with T-DM1, PI103, GDC-0941 at IC50 concentrations of 0 to 16x, and in fixed ratios of T-DM1+PI103 and T-DM1+GDC-0941 for 3 days.

Figure 25 shows the in vitro cell viability (proliferation) of KPL4 caspase 3/7 after treatment with T-DM1 at concentrations up to 160 ng/ml, T-DM1, GDC-0941, and a fixed ratio of T-DM1 and GDC-0941 for 24 hours.

Figure 26 shows the in vitro cell viability (proliferation) of KPL4 cells after treatment with T-DM1, GDC-0941, and a fixed ratio of T-DM1 and GDC-0941 at concentrations ranging from 0 to 200 ng/ml for 3 days.

Figure 27 shows the in vitro cell viability (proliferation) of MDA-0MB-361 cells after treatment with T-DM1, GDC-0941 at IC50 concentrations of 0.125x to 8x, and in fixed dose ratios of T-DM1 (3.125 to 50 ng/ml) and GDC-0941 (62.5 nM to 1 μM) at a 1:20 ratio. Bliss predictions of the additive effect are plotted as dashed lines.

Figure 28 shows the in vitro cell viability (proliferation) of MDA-0MB-361 cells after treatment with T-DM1, GDC-0941 at IC50 concentrations ranging from 0.125x to 8x, and in fixed dose ratios of T-DM1 (3.125 to 100 ng/ml) and GDC-0941 (62.5 nM to 2 μM) at a 1:20 ratio. Bliss predictions of the additive effect are plotted as dashed lines.

Figure 29 shows the in vitro cell viability (proliferation) of BT-474 cells after treatment with T-DM1, GDC-0941 at IC50 concentrations ranging from 0.125x to 4x, and in fixed dose ratios of T-DM1 (3.125 to 100 ng/ml) and GDC-0941 (31.25 nM to 1 μM) at a 1:10 ratio. Bliss predictions of the additive effect are plotted as dashed lines.

Figure 30 shows the in vitro cell viability (proliferation) of BT-474 cells after treatment with T-DM1, GDC-0941 at IC50 concentrations of 0.25x to 4x, and in fixed dose ratios of T-DM1 (6.25 to 100 ng/ml) and GDC-0941 (62.5 nM to 1 μM) at a 1:10 ratio. Bliss predictions of the additive effect are plotted as dashed lines.

Figure 31 shows the in vitro cell viability (proliferation) of Her2-amplified, non-PI3K-mutant AU565 cells after treatment with T-DM1, PI103, GDC-0941 at IC50 concentrations of 0 to 16x, and in fixed dose ratios of T-DM1+PI103 and T-DM1+GDC-0941 for 3 days.

Figure 32 shows the in vitro cell viability (proliferation) of Her2-amplified, PIK3CA(C420R) mutant EFM192A cells after treatment with T-DM1, PI103, GDC-0941 at IC50 concentrations of 0 to 16x, and at fixed dose ratios of T-DM1+PI103 and T-DM1+GDC-0941 for 3 days.

Figure 33 shows the in vitro cell viability (proliferation) of Her2-amplified, drug-resistant, PIK3CA(H1047R) mutant HCC1954 cells after treatment with T-DM1, PI103, GDC-0941 at IC50 concentrations of 0 to 16x, and in fixed ratios of T-DM1+PI103 and T-DM1+GDC-0941.

Figure 34 shows the mean in vivo tumor volume of MMTV-Her2Fo5 transgenic breast tumors inoculated into CRL nu/nu mice after the following administrations: (1) mediator, po, qd x21, (2) T-DM1, 10 mg/kg, iv, q3wk, (3) 5-FU, 100 mg/kg, po, qwk x2, (4) T-DM1, 5 mg/kg, iv, q3wk+5-FU, 100 mg/kg, po, qwk x2.

Figure 35 shows the mean in vivo tumor volume of MMTV-Her2Fo5 transgenic breast tumors inoculated into CRL nu/nu mice after the following administrations: (1) mediator, po, qd x21, (2) T-DM1, 5 mg/kg, iv, qd x1, (3) GDC-0941, 100 mg/kg, po, qd x21, (4) GDC-0152, 50 mg/kg, po, qwk x3, (5) T-DM1, 5 mg/kg, iv, qd x1 + GDC-0941, 100 mg/kg, po, qd x21, (6) T-DM1, 5 mg/kg, iv, qd x1 + GDC-0152, 50 mg/kg, po, qwk x3.

Figure 36 shows the mean in vivo tumor volume over time of MDA-MB-361.1 breast tumors in CRL nu/nu mice after the following administrations: (1) mediator, po, qd x21, (2) GDC-0941, 25 mg/kg, po, qd x21, (3) GDC-0941, 50 mg/kg, po, qd x21, (4) GDC-0941, 100 mg/kg, po, qd x21, (5) T-DM1, 3 mg/kg, iv, qd x1, (6) T-DM1, 10 mg/kg, iv, qd x1, (7) GDC-0941, 25 mg/kg, po, qd x21 + T-DM1, 3 mg/kg, iv, qd x1 , (8)GDC-0941, 50mg/kg, po, qd x21+T-DM1, 3mg/kg, iv, qd x1, (9) GDC-0941, 100mg/kg, po, qd x21+T-DM1, 3mg/kg, iv, qd x1, (10) GDC-0941, 25mg/kg, po, qd x 21+T-DM1,10mg/kg,iv,qd x1,(11)GDC-0941,50mg/kg,po,qd x21+T-DM1,10mg/kg,iv,qd x1,(12)GDC-0941,100mg/kg,po,qd

Figure 37 shows the mean in vivo tumor volume over time of MDA-MB-361.1 breast tumors in CRL nu/nu mice after the following administrations: (1) Media [MCT (0.5% methylcellulose/0.2% TWEEN 80 ^™ ) + succinate buffer (100 mM sodium succinate, 100 mg/ml trehalose, 0.1% TWEEN 80, pH 5.0)], po + IV, qd x21 and qd, (2) GNE-390, 1.0 mg/kg, po, qd x21, (3) GNE-390, 2.5 mg/kg, po, qd x21, (4) T-DM1, 3 mg/kg, iv, qd, (5) GNE-390, 1.0 mg/kg, po, qd x21+T-DM1, 3mg/kg, iv, qd, (6) GNE-390, 2.5mg/kg, po, qdx21+T-DM1, 3mg/kg, iv, qd.

Invention Details

Certain embodiments of the invention will now be described in detail, with examples of them illustrated in the appended structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it should be understood that they are not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined in the claims. Those skilled in the art will appreciate that many methods and materials similar to or equivalent to those described herein can be used to practice the invention. The invention is by no means limited to the methods and materials described. In the event that any included documents, patents, and similar materials differ from or contradict this application, including but not limited to defined terminology, usage of terms, described techniques, and the like, this application shall prevail.

definition

The terms “comprising,” “containing,” “including,” and “having” are used in this specification and claims to describe the presence of the stated feature, integer, ingredient, or step, but do not exclude the presence or addition of one or more other features, integers, ingredients, steps, or combinations thereof.

The terms “treatment” or “management” refer to both therapeutic treatment and preventative or preventative measures, wherein the goal is to prevent or slow (alleviate) unwanted physiological changes or conditions, such as the growth, formation, or spread of highly proliferative conditions such as cancer. For the purposes of this invention, advantageous or desired clinical outcomes include, but are not limited to: symptom relief, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and recovery (whether partial or complete), whether detectable or undetectable. “Treatment” or “management” can also refer to prolonged survival compared to expected survival without treatment. Subjects requiring treatment include those who already have the disease or condition, those predisposed to developing the disease or condition, or those seeking to prevent the condition or condition.

The phrase "therapeutic effective amount" refers to an amount of the compound of the present invention that (i) treats the specific disease, condition, or symptom described herein, (ii) alleviates, improves, or eliminates one or more symptoms of the specific disease, condition, or symptom, or (iii) prevents or delays the onset of one or more symptoms of the specific disease, condition, or symptom. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; shrink the tumor volume; inhibit (i.e., slow down, preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down, preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more symptoms associated with cancer to some extent. In terms of the extent to which the drug can prevent cancer cell growth and/or kill existing cancer cells, it may be cellularly inhibitory and/or cellularly toxic. For cancer therapies, efficacy may be measured, for example, by evaluating time to disease progression (TTP) and/or measuring the response rate (RR).

"Hyperproliferative disorders" refer to tumors, cancers, and neoplasms, including pre-malignant and non-neoplastic stages, as well as psoriasis, endometriosis, polyps, and fibroadenomas.

The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals characterized by unregulated cell growth. A “tumor” includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

"Chemotherapy agents" refer to chemical compounds that can be used to treat cancer, regardless of their mechanism of action. Categories of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapy agents include compounds used in "targeted therapy" and conventional chemotherapy. Examples of chemotherapy agents include: erlotinib (Genentech/OSI Pharm.), docetaxel (Sanofi-Aventis), 5-FU (fluorouracil, CAS No. 51-21-8), gemcitabine (Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatin(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), and paclitaxel (Bristol). -Myers Squibb Oncology, Princeton, N.J.), trastuzumab (Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazonobicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethylamine), doxorubicin Akti-1/2, HPPD, and rapamycin.

More examples of chemotherapy agents include: oxaliplatin (Sanofi), bortezomib (Millennium Pharm.), sutent (SU11248, Pfizer), letrozole (Novartis), imatinib mesylate (Novartis), XL-518 (MEK inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), and SF-1126 (PI3K inhibitor, Semafore). Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, Wyeth), lapatinib (GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR ^™ , SCH 66336, Schering Plough), sorafenib (BAY43-9006, Bayer Labs), gefitinib (AstraZeneca), irinotecan (CPT-11, Pfizer), tipifarnib (ZARNESTRA ^™) Johnson & Johnson), ABRAXANE ^™ is cremophor-free, albumin-modified nanoparticle formulation paclitaxel (American Pharmaceutical Partners, Schaumberg, Il), vandetanib (rINN, ZD6474, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (Telik), thiotepa, and alkyl sulfonates of cyclophosphamide. Sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; acetogenides. ns)(especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard Mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, and uracil. Mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediynes (e.g., calicheamicin, calicheamicin γ1I, calicheamicin ωI1). Chem. Intl. Ed. Engl., (1994) 33: 183-186); anthracycline antibiotics (dynemicin), dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromogenic proteins (ene diyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin C, car Abicin, carminomycin, carzinophilin, chromomycin, dactinomycin D, daunorubicin, detorubicin, 6-diazol-5-oxo-L-leucine, morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrroledoxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid Drugs containing lysine, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); and folic acid analogues, such as denopterin, methotrexate, and pteroprolol. Trimetrexate; purine analogs, such as fludarabine, mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; and androgens, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergics, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; 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; podophyllinic acid; ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural) Products, Eugene, OR); Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid acid); triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (T-2 toxin, verrucarin A, roridin A, and anguidin); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; thioguanine; mercaptopurine Mercaptopurine; Methotrexate; Platinum analogs, such as cisplatin and carboplatin; Vinblastine; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Teniposide; Edatraxate; Daunomycin; Aminopterin; Capecitabine (Roche); Ibandronate; CPT-11; Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; and pharmaceutically acceptable salts, acids, and derivatives of any of the above substances.

The definition of "chemotherapeutic agents" also includes: (i) anti-hormonal agents, which regulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene citrate. (ii) Aromatase inhibitors, which inhibit aromatase that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole, Novartis, and anastrozole. e); AstraZeneca; (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxane cytosine analog); (iv) protein kinase inhibitors, such as MEK inhibitors (WO2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those inhibiting involved... Antisense oligonucleotides, such as PKC-α, Raf, and H-Ras, in signaling pathways that aberrantly proliferate genes, such as oblimersen (Genta Inc.); (vii) ribozymes, such as VEGF expression inhibitors (e.g.) and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines, such as rIL-2; topoisomerase 1 inhibitors, such as rmRH; (ix) anti-angiogenic agents, such as bevacizumab (Genentech); and pharmaceutically acceptable salts, acids, and derivatives of any of the above substances.

The definition of "chemotherapeutic agents" also includes therapeutic antibodies, such as alemtuzumab (Campath), bevacizumab (Genentech), cetuximab (Imclone), panitumumab (Amgen), rituximab (Genentech/Biogen Idec), pertuzumab (OMNITARG ^™ , 2C4, Genentech), trastuzumab (Genentech), tositumomab (Bexxar, Corixia), and antibody-drug conjugates, such as gemtuzumab ozogamicin (Wyeth).

Humanized monoclonal antibodies with therapeutic potential as chemotherapy agents in combination with trastuzumab-MCC-DM1 include: alemtuzumab, apolizumab, asilizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumabozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natali zumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, r uplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, and visilizumab.

"Metabolite" refers to a product generated in the body through metabolism of a particular compound or its salt. Metabolites of compounds can be identified using conventional techniques known in the art, and their activity can be determined using tests such as those described herein. Such products may originate from, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc., of the applied compound. Therefore, the present invention includes metabolites of the compounds of the present invention, including compounds generated by a process comprising exposing the compounds of the present invention to a mammal for a period of time sufficient to generate their metabolites.

The term "packaging insert" is used to refer to the instructions for use typically included in the commercial packaging of therapeutic products, which contain information about the indications, usage, dosage, administration, contraindications, and/or warnings related to the application of such therapeutic products.

The phrase "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of the compounds of the present invention. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannins, pantothenates, bitartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucurons, glycosides, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pyrates (i.e., 1,1'-methylenebis(2-hydroxy-3-naphthylcarbamate)). A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, succinate ion, or other counterion. The counterion can be any organic or inorganic portion that stabilizes the charge of the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one type of charged atom in their structure. In cases where multiple charged atoms are components of a pharmaceutically acceptable salt, multiple counterions may be present. Therefore, pharmaceutically acceptable salts may have one or more charged atoms and/or one or more counterions.

If the compound of the present invention is a base, then the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, such as treating the free base with an inorganic acid (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, etc.) or with an organic acid (such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid/fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid (such as glucuronic acid or galacturonic acid), α-hydroxy acid (such as citric acid or tartaric acid), amino acid (such as aspartic acid or glutamic acid), aromatic acid (such as benzoic acid or cinnamic acid), sulfonic acid (such as p-toluenesulfonic acid or ethanesulfonic acid), etc.). The literature discusses acids generally considered suitable for forming pharmaceutically useful or acceptable salts from basic pharmaceutical compounds, such as P. Stahl et al., Camille G. (ed.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al., Journal of Pharmaceutical Sciences (1977) 66(1) 1 19; P. Gould, International Journal of Pharmaceutical Sciences. utics (1986) 33 201 217; Anderson et al., The Practice of Medicinal Chemistry (1996), Academic Press, New York; Remington’s Pharmaceutical Sciences, 18th edition, (1995) Mack Publishing Co., Easton PA; and The Orange Book (Food & Drug Administration, Washington, D.C., their website). These publications are incorporated herein by reference.

If the compounds of the present invention are acids, then the desired pharmaceutically acceptable salts can be prepared by any suitable method, such as treating the free acid with inorganic or organic bases (such as amines (primary, secondary, or tertiary)), alkali metal hydroxides, or alkaline earth metal hydroxides, etc. Exemplary examples of suitable salts include, but are not limited to, organic salts derived from amino acids (such as glycine and arginine), ammonium, primary/secondary/tertiary amines, and cyclic amines (such as piperidine, morpholine, and piperazine), and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

The phrase “pharmaceutically acceptable” indicates that the substance or composition must be chemically and/or toxicologically compatible with the other components constituting the formulation and/or the mammals to which it is used for treatment.

"Solvate" refers to a physical association or complex of one or more solvent molecules with a compound of the present invention. The compounds of the present invention can exist in both unsolvated and solvated forms. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" refers to a complex in which the solvent molecules are water. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, solvates can be separated, for example when one or more solvent molecules are incorporated into the lattice of a crystalline solid. The preparation of solvates is generally known, for example, M. Caira et al., J. Pharmaceutical Sci., 93(3), 601 611 (2004). Similar preparations of solvates, hemisolates, hydrates, etc., are described in E.C. van Tonder et al., AAPSPharmSciTech., 5(1), article 12 (2004); and A.L. Bingham et al., Chem. Commun., 603 604 (2001). A typical non-limiting process involves dissolving the compound of the invention in a desired amount of a desired solvent (organic or water or a mixture thereof) at a temperature higher than ambient, cooling the solution at a rate sufficient to form crystals, and then separating the crystals by standard methods. Analytical techniques such as, for example, I.R. spectroscopy show the presence of the solvent (or water) in the crystals as solvates (or hydrates).

As used herein, the term "synergistic" refers to a combination of therapeutic agents that is more effective than the sum of two or more single agents. The determination of synergistic interactions between trastuzumab-MCC-DM1 and one or more chemotherapeutic agents can be based on results obtained from the assays described herein. The results of these assays were analyzed using CalcuSyn software with the Chou and Talalay combination method and dose-effect analysis to obtain a combination index "CI" (Chou and Talalay (1984) Adv. Enzyme Regul. 22: 27-55). The combinations provided by this invention have been evaluated in several assay systems, and data can be analyzed using standard procedures to quantify synergistic, additive, and antagonistic effects among anticancer agents. The preferred procedure is described in Chou and Talalay, "New Avenues in Developmental Cancer Chemotherapy," Academic Press, 1987, Chapter 2. A combination index (CI) less than 0.8 indicates synergy, greater than 1.2 indicates antagonism, and values between 0.8 and 1.2 indicate an additive effect. Combination therapies can provide “synergy” and demonstrate that they are “synergistic,” meaning that the effect achieved when the active ingredients are used together is greater than the sum of the effects achieved when they are used separately. Synergistic effects are achieved when the active ingredients are: (1) co-formulated and administered or delivered in unit doses of the combination; (2) delivered alternately as separate formulations; or (3) provided with some other regimen. When delivered in alternating therapies, synergistic effects are achieved when the compounds are administered or delivered sequentially (e.g., by different injections in separate syringes). Generally, during alternating therapies, each active ingredient is administered sequentially (i.e., sequentially in time).

TRASTUZUMAB-MCC-DM1

This invention includes a therapeutic combination comprising trastuzumab-MCC-DM1 (T-DM1), an antibody-drug conjugate having the following structure (CAS Reg. No. 139504-50-0):

Where Tr refers to trastuzumab, which is linked to the maytansine alkaloid drug moiety DM1 (US 5208020; US 6441163) via the linker portion MCC. The drug-to-antibody ratio or drug loading is represented by p in the above structure of trastuzumab-MCC-DM1 and ranges from 1 to an integer value of approximately 8. The drug loading value p is 1 to 8. trastuzumab-MCC-DM1 comprises all mixtures of various loaded and attached antibody-drug conjugates, wherein drug moieties 1, 2, 3, 4, 5, 6, 7, and 8 are covalently attached to the antibody trastuzumab (US 7097840; US 2005/0276812; US 2005/0166993). trastuzumab-MCC-DM1 can be prepared according to Example 1.

Trastuzumab is produced from mammalian cell (Chinese hamster ovary, CHO) suspension cultures. The HER2 (or c-erbB2) proto-oncogene encodes a 185 kDa transmembrane receptor protein that is structurally associated with the epidermal growth factor receptor. HER2 protein overexpression has been observed in 25%–30% of primary breast cancers and can be identified using immunohistochemical assessment of fixed tumor masses (Press MF et al. (1993) Cancer Res 53:4960–70). Trastuzumab is a mouse 4D5 antibody (ATCC CRL 10463, deposited at the American Center for Type Culture Collection on May 24, 1990, under the Budapest Treaty, 12301). Antibodies derived from mouse 4D5 antibodies (Parklawn Drive, Rockville, Md. 20852) or antigen-binding residues. Exemplary humanized 4D5 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8, as described in US 5821337.

In the Phase I study, the maximum tolerated dose (MTD) of T-DM1 administered via IV infusion every 3 weeks was 3.6 mg/kg. DLT (dose-limiting toxicity) included grade 4 thrombocytopenia in 2 of the 3 patients treated with 4.8 mg/kg. Grade ≥2 adverse events associated with 3.6 mg/kg were rare and manageable. As previously stated, this treatment schedule was well tolerated and associated with significant clinical activity. The Phase II study has shown similar tolerability with a 3.6 mg/kg dose level every 3 weeks, with only a small percentage of patients (3 out of 112 patients) requiring a dose reduction. Thus, the 3.6 mg/kg T-DM1 dose was chosen for testing in this study based on the following points: 1) the proven efficacy and safety of 3.6 mg/kg T-DM1 every 3 weeks, and 2) the convenience of the 3-week regimen for this patient population.

Chemotherapy

Some chemotherapeutic agents have demonstrated surprising and unexpected properties in inhibiting cell proliferation, both in vitro and in vivo, when combined with trastuzumab-MCC-DM1. These agents include HER2 dimerization inhibitor antibodies, anti-VEGF antibodies, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941, and GNE-390.

Pertuzumab (CAS Reg. No. 380610-27-5, 2C4, Genentech) is a recombinant, humanized monoclonal antibody that inhibits HER2 dimerization (US 6054297; US6407213; US 6800738; US6627196, US 6949245; US 7041292). Pertuzumab and trastuzumab target different extracellular regions of the HER-2 tyrosine kinase receptor (Nahta et al. (2004) Cancer Res. 64: 2343-2346). A hybridoma cell line expressing 2C4 (pertuzumab) was deposited on April 8, 1999, as ATCC HB-12697 at the American Center for Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, USA. Pertuzumab blocks the ability of the HER2 receptor to cooperate with other members of the HER2 receptor family, namely HER1/EGFR, HER3, and HER4 (Agus et al. (2002) Cancer Cell 2: 127-37; Jackson et al. (2004) Cancer Res 64: 2601-9; Takai et al. (2005) Cancer 104: 2701-8; US 6949245). In cancer cells, interference with the ability of HER2 to cooperate with other HER family receptors blocks cell signaling and ultimately leads to cancer cell growth inhibition and cancer cell death. Due to their unique mode of action, HDI has the potential to operate in a wide range of tumors, including those that do not express HER2 (Mullen et al. (2007) Molecular Cancer Therapeutics 6: 93-100).

Pertuzumab is based on the human IgG1(κ) framework sequence. It consists of two heavy chains and two light chains. Like trastuzumab, pertuzumab targets the extracellular domain of HER2. However, it differs from trastuzumab in the epitope binding regions of the light and heavy chains. Therefore, pertuzumab binds to epitopes within subdomain 2 of HER2, while the epitopes of trastuzumab are located in subdomain 4 (Cho et al. 2003; Franklin et al. 2004). Pertuzumab works by blocking the binding of HER2 to other members of the HER family, including HER1 (epidermal growth factor receptor; EGFR), HER3, and HER4. This binding is required in the presence of ligands and via signaling by MAP-kinase and PI3-kinase. Therefore, pertuzumab inhibits ligand-initiated intracellular signaling. Inhibition of these signaling pathways leads to growth arrest and apoptosis, respectively (Hanahan and Weinberg 2000). Because pertuzumab and trastuzumab bind to different epitopes on the HER2 receptor, downstream signaling ligand activation is blocked by pertuzumab but not by trastuzumab. Therefore, pertuzumab may not require HER2 overexpression to exert its antitumor activity. Furthermore, due to their complementary modes of action, the combination of pertuzumab and T-DM1 may have potential applications in diseases with HER2 overexpression.

Pertuzumab has been evaluated as a single agent in five phase II studies across various cancer types, including MBC with low HER2 expression, non-small cell lung cancer, hormone-insensitive prostate cancer, and ovarian cancer. One phase II trial evaluated pertuzumab as a single agent in second- or third-line treatment of patients with metastatic breast cancer (MBC) with normal HER2 expression (Cortes et al. (2005) J. Clin. Oncol. 23: 3068). Pertuzumab has been evaluated in combination with trastuzumab in two phase II studies (Baselga J et al., “A Phase II trial of trastuzumab and pertuzumab in patients with HER2-positive metastatic breast cancer that had progressed during trastuzumab therapy: full response data”, European Society of Medical Oncology, Stockholm, Sweden, September 12-16, 2008; Gelmon et al. (2008) J. Clin. Oncol. 26: 1026). The first study enrolled 11 patients with HER2-positive MBC who had previously received up to three prior trastuzumab-containing regimens (Portera et al. 2007). Bevacizumab (CAS Reg. No. 216974-75-3, Genentech) is an anti-VEGF monoclonal antibody targeting vascular endothelial growth factor (US 7227004; US 6884879; US 7060269; US 7169901; US 7297334) in cancer treatment, where it inhibits tumor growth by blocking angiogenesis. Bevacizumab was the first clinically available angiogenesis inhibitor in the United States and was approved by the FDA in 2004 in combination with standard chemotherapy for the treatment of metastatic colon cancer and most forms of metastatic non-small cell lung cancer. Several advanced clinical studies are currently determining its safety and efficacy in patients with the following cancers: adjuvant/non-metastatic colon cancer, metastatic breast cancer, metastatic renal cell carcinoma, metastatic glioblastoma multiforme, metastatic ovarian cancer, metastatic hormone-insensitive prostate cancer, and metastatic or unresectable locally advanced pancreatic cancer.

Anti-VEGF antibodies typically do not bind to other VEGF homologues, such as VEGF-B or VEGF-C, nor to other growth factors, such as PlGF, PDGF, or bFGF. Preferred anti-VEGF antibodies include monoclonal antibodies that bind to the same epitopes as monoclonal anti-VEGF antibody A4.6.1 generated from hybridoma ATCC HB 10709; and recombinant humanized anti-VEGF monoclonal antibodies generated according to Presta et al. (1997) Cancer Res. 57: 4593-4599, including but not limited to bevacizumab. bevacizumab comprises a mutated human IgG1 framework region and an antigen-binding complementarity-determining region derived from mouse anti-hVEGF monoclonal antibody A.4.6.1, which blocks human VEGF from binding to its receptor. Approximately 93% of the amino acid sequence of bevacizumab (including most of the framework region) is derived from human IgG1, while approximately 7% of the sequence is derived from mouse antibody A4.6.1. bevacizumab has a molecular weight of approximately 149,000 Daltons and is glycosylated. bevacizumab and other humanized anti-VEGF antibodies are further described in US6884879. Other anti-VEGF antibodies include G6 or B20 series antibodies (e.g., G6-31, B20-4.1), as shown in any of Figures 27-29 in WO2005/012359. In one embodiment, the B20 series antibodies bind to functional epitopes on human VEGF containing residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103, and C104.

Hybridoma cell lines expressing anti-VEGF A 4.6.1 (ATCC HB 10709) and B 2.6.2 (ATCC HB 10710) have been deposited and maintained at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, USA. A clone (US 6391311) expressing a VEGF-E polypeptide encoded by a nucleotide sequence insert identified as DNA 29101-1276 from the ATCC collection was deposited on March 5, 1998, at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA, and is maintained as ATCC 209653.

5-FU (fluorouracil, CAS Reg. No. 51-21-8) is a thymidylate synthase inhibitor and has been used for decades in the treatment of cancers, including colorectal and pancreatic cancer (US 2802005, US 2885396; Barton et al. (1972) Jour. Org. Chem. 37: 329; Hansen, R.M. (1991) Cancer Invest. 9: 637-642). The name of 5-FU is 5-fluoro-1H-pyrimidine-2,4-dione, and it has the following structure:

Carboplatin (CAS Reg. No. 41575-94-4) is a chemotherapy drug used for ovarian cancer, lung cancer, and head and neck cancer (US 4140707). Carboplatin's name is azanide; cyclobutane-1,1-dicarboxylic acid platinum, and it has the following structure:

Lapatinib (CAS Reg. No. 388082-78-8, GW572016, GlaxoSmithKline) has been approved in combination with capecitabine (Roche) for the treatment of patients with advanced or metastatic breast cancer whose tumors overexpress HER2 (ErbB2) and who have received prior therapy (including anthracyclines, taxanes, and trastuzumab). Lapatinib is an ATP-competitive dual tyrosine kinase inhibitor of epidermal growth factor (EGFR) and HER2/neu (ErbB-2) (US 6727256; US 6713485; US 7109333; US 6933299; US7084147; US 7157466; US 7141576), which inhibits receptor autophosphorylation and activation by binding to the ATP-binding pocket of the EGFR/HER2 protein kinase domain. The name of lapatinib is N-(3-chloro-4-(3-fluorobenzyloxy)phenyl)-6-(5-((2-(methanesulfonyl)ethylamino)methyl)furan-2-yl)quinazolin-4-amine, and it has the following structure:

ABT-869 (Abbott and Genentech) is a multi-target inhibitor of VEGF and PDGF family receptor tyrosine kinases, with potential oral therapeutic applications in cancer (US 7297709; US 2004/235892; US 2007/104780). Clinical trials have been initiated for the treatment of non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), and renal cell carcinoma (RCC). ABT-869 is named 1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea (CAS No. 796967-16-3) and has the following structure:

Docetaxel (Sanofi-Aventis) is used to treat breast cancer, ovarian cancer, and NSCLC (US 4814470; US 5438072; US 5698582; US 5714512; US 5750561). The name of docetaxel is (2R,3S)-N-carboxy-3-phenylisoserine N-tert-butyl ester with 5,20-epoxy-1,2,4,7,10,13-hexahydroxytaxane-11-en-9-one 4-acetate 2-benzoate ester 13-ester, trihydrate (US 4814470; EP 253738; CAS Reg. No. 114977-28-5), and it has the following structure:

GDC-0941 (Genentech Inc.) is a selective, orally bioavailable thienopyrimidine inhibitor of PI3K, with promising pharmacokinetic and pharmaceutical properties (Folkes et al. (2008) Jour. of Med. Chem. 51(18): 5522-5532; US 2008/0076768; US 2008/0207611; Belvin et al., American Association for Cancer Research Annual Meeting 2008, 99th: A). April 15, Abstract 4004; Folkes et al., American Association for Cancer Research Annual Meeting 2008, 99th: April 14, Abstract LB-146; Friedman et al., American Association for Cancer Research Annual Meeting 2008, 99th: April 14, Abstract LB-110). GDC-0941 showed synergistic activity against solid tumor cell lines in vitro and in vivo in combination with certain chemotherapeutic agents (US Ser. No. 12/208, 227, Belvin et al., “Combinations of Phosphoinositide 3-Kinase Inhibitor Compounds and Chemootherapeutic Agents, and Methods of Use”, filed 10 Sept 2008). GDC-0941 is named 4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine (CAS Reg. No. 957054-30-7), and has the following structure:

GNE-390 (Genentech Inc.) is a selective, orally bioavailable thiophene-3-pyrimidine inhibitor of PI3K with promising pharmacokinetic and pharmaceutical properties (US 2008/0242665; WO 2008/070740). GNE-390 has shown synergistic activity against solid tumor cell lines in vitro and in vivo in combination with certain chemotherapeutic agents (US Ser. No. 12/208,227, Belvin et al., “Combinations of Phosphoinositide 3-Kinase Inhibitor Compounds and Chemotherapeutic Agents, and Methods of Use”, filed 10 Sept 2008). GNE-390 is named (S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxyprop-1-one, and has the following structure:

Biological assessment

In vitro cell culture studies were conducted on various HER2-amplified cell lines using trastuzumab-MCC DM1 (T-DM1) in combination with different chemotherapeutic agents or biological targets. Data were analyzed using the Chou & Talalay method, determining the combination index (CI) for each combination set at an IC50 fold for each drug. A CI value less than 0.7 indicated synergy; a CI value between 0.7 and 1.3 indicated additive; and a CI value greater than 1.3 indicated antagonism. For combinations with chemotherapeutic agents, T-DM1 in combination with docetaxel or 5-FU resulted in additive or synergistic antiproliferative activity, while combinations with gemcitabine or carboplatin had no effect or antagonized T-DM1. Similar results were observed in mouse xenograft studies, where T-DM1 in combination with docetaxel or 5-FU resulted in significantly enhanced antitumor efficacy compared to treatment with either drug alone. T-DM1 in combination with carboplatin resulted in enhanced efficacy compared to either drug alone, while the combination of T-DM1 and gemcitabine was not more effective than T-DM1 alone. Compared to treatment with the single agent, T-DM1 in combination with any of pertuzumab, lapatinib, or GDC-0941 resulted in additive or synergistic antiproliferative activity in cell culture assays and significantly enhanced antitumor efficacy in vivo. Conversely, uncoupled trastuzumab antagonized T-DM1 activity due to binding to the same epitope on HER2. In in vivo studies using T-DM1 in combination with antiangiogenic agents such as antibody B20-4.1 or small molecule inhibitor ABT-869, all tested combinations resulted in enhanced antitumor efficacy, except for the highest dose of T-DM1 (10 or 15 mg/kg) administered with B20-4.1.

The combination of trastuzumab-MCC-DM1 (T-DM1) with various anticancer drugs was investigated by measuring its in vitro antiproliferative activity in breast tumor cells overexpressing HER2 and its in vivo antitumor efficacy in a breast cancer xenograft model. In these studies, trastuzumab-MCC-DM1 was added to cytotoxic chemotherapeutic agents, antibodies, or small molecule kinase inhibitors.

Anti-VEGF mouse antibody B20-4.1 (Liang et al. (2006) Jour. Biol. Chem. 281: 951-961), a bevacizumab alternative, combined with trastuzumab-MCC-DM1, resulted in greater antitumor activity in a mouse xenograft model of breast cancer than B20-4.1 alone. The results of these studies provide a predictive basis for synergistic effects and a fundamental principle for future clinical evaluation of treatment regimens including trastuzumab-MCC-DM1 in combination with different antitumor therapies in HER2-positive breast cancer.

Synergistic drug effects have been observed with HER2-targeting agents such as trastuzumab-DM1 plus lapatinib, or trastuzumab-DM1 combined with the HER2 antibody pertuzumab (a HER2 dimerization inhibitor).

Trastuzumab-MCC-DM1 in combination with carboplatin or 5-FU showed enhanced activity compared to treatment with each drug alone, while combination therapy with gemcitabine did not result in increased antitumor activity.

The activity of trastuzumab-DM1 was enhanced by blocking the PI3 kinase pathway with a small molecule pan-inhibitor of the p110 isoform (WO 2007/129161) that is GDC-0941.

T-DM1 in combination with the PI3K inhibitor GDC-0941 enhanced antitumor activity in HER2-amplified breast cancer lines with mutant PI3K: BT-474 (K111N), MDA-361.1 (E545K), and KPL4 (H1047R). In vitro, the combination therapy resulted in additive or synergistic inhibition of cell proliferation and increased apoptosis. Similarly, in xenograft models of MDA-MB-361.1 and Fo5HER2 amplification, the combination therapy enhanced in vivo efficacy compared to the activity of the single agent. Biochemical analysis of biomarkers for each agent revealed inhibition of phosphate-Akt and phosphate-ERK by both T-DM1 and GDC-0941, a decrease in Rb and PRAS40 phosphorylation by GDC-0941, and decreased levels of mitotic markers phosphate-histone H3 and cyclin B1 after treatment with T-DM1. Furthermore, T-DM1 treatment induced apoptosis in these breast cancer models, as determined by the appearance of 23kDa PARP cleavage fragments, decreased Bcl-XL levels, and activation of caspases 3 and 7. Adding GDC-0941 to T-DM1 further enhanced apoptosis induction. These studies provide evidence for the rational use of drug combinations in HER2-amplified breast cancer and offer alternative treatment options for patients whose disease has progressed on trastuzumab or lapatinib-based therapies.

In vitro cell proliferation assay

The cell proliferation assay described in Example 2, CellTiter-luminescent cell viability assay, available from Promega Corp., Madison, WI, was used to measure the in vitro efficacy of the combination of trastuzumab-MCC-DM1 with a chemotherapeutic agent. This homogeneous assay is based on recombinant expression of Coleoptera luciferase (US 5583024; US5674713; US 5700670) and determines the number of surviving cells in the culture based on the amount of ATP present, an indicator of metabolically active cells (Crouch et al. (1993) J. Immunol. Meth. 160: 81-88; US 6602677). The CellTiter assay was performed in 96 or 384-well configurations to accommodate automated high-throughput screening (HTS) (Cree et al. (1995) AntiCancer Drugs 6: 398-404). The homogenization assay involves the direct addition of a single reagent (CellTiter reagent) to cells cultured in serum-supplemented medium. No cell washing, medium removal, or multiple pipetting steps are required. The system can detect as few as 15 cells/well in a 384-well configuration within 10 minutes of reagent addition and mixing.

The homogenized "addition-mixing-measurement" process results in cell lysis and the generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is proportional to the number of cells present in the culture. The CellTiter assay generates a "glow-type" luminescent signal, produced by a luciferase reaction with a half-life generally greater than 5 hours (depending on the cell type and culture medium used). Viable cells are represented as relative light units (RLU). The substrate beetle luciferin is oxidatively decarboxylated by recombinant firefly luciferase, accompanied by the conversion of ATP to AMP and the generation of photons. The extended half-life eliminates the need for reagent syringes and provides flexibility for processing multiple plates in continuous or batch modes. This cell proliferation assay can be used with various well formats, such as 96- or 384-well formats. Data can be recorded using a photometer or CCD camera imaging device. The luminescence output is expressed as relative light units (RLU) measured over time.

The antiproliferative effects of trastuzumab-MCC-DM1 and its combination with chemotherapeutic agents on the tumor cell lines in Figures 1-9 and 18-33 were measured by CellTiter assay (Example 2).

An exemplary implementation includes a method for determining compounds to be used in combination for cancer treatment, comprising: a) administering a combination of trastuzumab-MCC-DM1 (T-DM1) and a chemotherapeutic agent to an in vitro tumor cell line, and b) measuring synergistic or non-synergistic effects. A combination index (CI) value greater than 1.3 indicates antagonism; a CI value between 0.7 and 1.3 indicates additivity; and a CI value less than 0.7 indicates synergistic drug interaction.

Figure 1 shows the antagonistic effects of trastuzumab in combination with trastuzumab-MCC-DM1 (T-DM1) at various concentrations (Table 1) at different IC50 values in trastuzumab-sensitive SK-BR-3 cells. The number of surviving cells is plotted relative to the IC50 value. A combination index (CI) greater than 2 at _IC10 to _IC90 for each combination indicates in vitro antagonism. However, the T-DM1+trastuzumab combination did not show an antagonistic effect in vivo.

Table 1 SK-BR-3 proliferation – 3 days

IC50 multiple trastuzumab ng/ml T-DM1ng/ml Effect(%) CI 0.5X 20.57 2.28 5.1 >2 1X 61.72 6.86 26.2 >2 2X 185.19 20.58 36.3 >2 4X 555.56 61.73 43.6 >2 8X 1666.67 185.19 45.0 >2 16X 5000 555.56 41.7 >2

Figure 2 shows the antagonistic effects of trastuzumab combined with trastuzumab-MCC-DM1 (T-DM1) at various concentrations (Table 2) at different IC50 folds in trastuzumab-resistant BT-474EEI cells. Survival cell numbers are plotted relative to IC50 folds. A combination index (CI) greater than 2 at _IC10 to _IC90 for each combination indicates antagonism.

Table 2 BT-474-EEI Proliferation – 3 Days

IC50 multiple trastuzumab ng/ml T-DM1ng/ml Effect(%) CI 0.125X 1.52 1.52 9.5 >2 0.25X 4.57 4.57 4.5 >2 0.5X 13.71 13.71 3.1 >2 1X 41.15 41.15 12.1 >2 2X 123.46 123.46 10.8 >2 4X 370.4 370.4 11.6 >2 8X 1111.1 1111.1 18.4 >2

Figure 3 shows the synergistic effect of pertuzumab combined with trastuzumab-MCC-DM1 (T-DM1) at various concentrations (Table 3) at different IC50 folds in MDA-MB-175 cells. Survival cell numbers are plotted relative to IC50 folds. Combination indices (CI) of less than 1 for each combination at _IC10 to _IC90 , with a mean CI of 0.387, indicate synergy (Table 3).

Table 3 MDA-MB-175 proliferation – 5 days

IC50 multiple pertuzumab ng/ml T-DM1ng/ml Effect(%) CI 0.0625X 39.06 31.25 21.1 0.2 0.125X 78.13 62.5 33.3 0.107 0.25X 156.3 125 21.9 .766 0.5X 312.5 250 33.6 0.597 1X 625 500 50.7 0.391 2X 1250 1000 67.7 0.259

Figure 3a shows the 5-day in vitro cell viability of MDA-MB-175 relative to IC50 concentrations of pertuzumab, trastuzumab-MCC-DM1 (T-DM1), and the combination of pertuzumab and T-DM1. The number of surviving cells is plotted relative to IC50 values. Each combination had a combination index (CI) below 1 at IC10 to IC90, with a mean CI of 0.096, indicating synergy (Table 3a).

Table 3a MDA-MB-175 proliferation – 5 days

IC50 multiple Effect(%) CI 0.0625x 21.3 0.093 0.125x 37.5 0.037 0.25x 40.1 0.060 0.5x 50.3 0.052 1x 53.9 0.078 2x 57.0 0.120 4x 65.5 0.117 8x 66.8 0.208

Figure 4 shows the dose-response of BT-474 in vitro cell viability at 5 days to various fixed doses of pertuzumab in combination with trastuzumab-MCC-DM1 (T-DM1) and various doses of T-DM1 alone. Figure 4 illustrates the effect of a fixed dose of T-DM1 in combination with various doses of pertuzumab. Adding pertuzumab to T-DM1 resulted in slightly greater antiproliferative activity than T-DM1 alone.

Figure 5 shows the dose-response of BT-474 cells at 5 days in vitro to various fixed doses of trastuzumab-MCC-DM1 (T-DM1) combined with pertuzumab, and various doses of pertuzumab alone. Figure 5 shows the effect of a fixed dose of pertuzumab combined with various doses of T-DM1 on BT-474 cell proliferation. Adding T-DM1 to pertuzumab enhances the effect of pertuzumab alone.

Figure 6 shows the synergistic effect of pertuzumab combined with trastuzumab-MCC-DM1 (T-DM1) at various concentrations (Table 4) at different IC50 folds in BT-474 cells. The number of surviving cells is plotted relative to the IC50 fold. The combination index (CI) values for _IC10 to _IC90 range from 0.198 to 1.328. The mean CI for this range is 0.658, indicating synergy.

Table 4 BT-474 Proliferation – 5 Days

IC50 multiple pertuzumab ng/ml T-DM1ng/ml Effect(%) CI 0.25X 34.29 11.43 3.9 >2 0.5X 102.88 34.29 2.0 >2 1X 308.64 102.88 58.9 0.198 2X 925.93 308.64 64.6 0.449 4X 2777 926 64.9 1.328

Figure 7 shows the in vitro cell viability of SK-BR-3 cells at 3 days compared to different doses of T-DM1 combined with fixed doses of lapatinib (4.5 nM, 14 nM, 41 nM, 123 nM) and different doses of T-DM1 alone (0-1000 ng/ml). Adding lapatinib to T-DM1 resulted in slightly greater antiproliferative activity than T-DM1 alone.

Figure 7a shows the in vitro cell viability of SK-BR-3 cells at 3 days against T-DM1, lapatinib, and a fixed-dose combination of T-DM1 and lapatinib (as shown in Table 7a). The mean CI value between IC10 and IC90 = 0.793, indicating an additive effect.

Table 7a SK-BR-3 proliferation – 3 days

IC50 multiple lapatinib nM T-DM1ng/ml Effect(%) CI 0.25x 4.57 1.52 3.5 >2 0.5x 13.72 4.57 22.0 1.384 1x 41.15 13.72 52.5 0.596 2x 123.44 41.15 75.9 0.406 4x 370.33 123.44 81.1 0.787 8x 1111 370.33 80.1 >2

Figure 8a shows the in vitro cell viability of BT-474 at 3 days versus T-DM1, lapatinib, and a fixed-dose ratio of T-DM1 and lapatinib (as shown in Table 8a). The mean CI value between IC10 and IC90 was 0.403, indicating synergy.

Table 8a BT-474 proliferation – 3 days

IC50 multiple lapatinib nM T-DM1ng/ml Effect(%) CI 0.125x 0.51 1.52 1.4 >2 0.25x 1.52 4.57 1.2 >2 0.5x 4.57 13.72 26.8 0.493 1x 13.72 41.15 62.2 0.201 2x 41.15 123.44 73.9 0.293 4x 123.44 370.33 84.1 0.390 8x 370.33 1111 89.3 0.638

Figure 8 shows the in vitro cell viability of BT-474 at 3 days compared to different doses of T-DM1 combined with fixed doses of lapatinib (1.5 nM, 4.5 nM, 14 nM, 41 nM, 123 nM) and different doses of T-DM1 alone (0-1000 ng/ml). Adding lapatinib to T-DM1 resulted in greater antiproliferative activity compared to either drug alone.

Figure 9 shows the in vitro cell viability of BT-474-EEI after 3 days compared to different doses of T-DM1 combined with fixed doses of lapatinib (14 nM, 41 nM, 123 nM, 370 nM, 1111 nM) and different doses of T-DM1 alone (0-1000 ng/ml). Adding lapatinib to T-DM1 resulted in greater antiproliferative activity than either drug alone.

Figure 18 shows the in vitro cell viability of SK-BR-3 cells at 3 days relative to IC50 concentrations of 5-FU, trastuzumab-MCC-DM1 (T-DM1), and a fixed-dose ratio of 5-FU and T-DM1 (Table 18). The 5-FU and T-DM1 combination was additive on SK-BR-3 cells, with a mean CI of 0.952, between IC10 and IC90.

Table 18 5-FU+T-DM1: SK-BR-3 proliferation – 3 days

IC50 multiple 5-FU (μM) T-DM1ng/ml Effect(%) CI 0.5x 62.5 1.95 38.9 1.035 1x 125 3.91 60.3 0.647 2x 250 7.81 69.2 0.835 4x 500 15.625 74.3 1.292

Figure 19 shows the 3-day in vitro cell viability of BT-474 cells relative to IC50 concentrations of 5-FU, trastuzumab-MCC-DM1 (T-DM1), and a fixed-dose ratio of 5-FU and T-DM1 (Table 19). The 5-FU and T-DM1 combination was synergistic in BT-474 cells, with a mean CI of 0.623.

Table 19 5-FU+T-DM1: BT-474 proliferation – 3 days

IC50 multiple 5-FU (μM) T-DM1ng/ml Effect(%) CI 0.25x 0.488 3.90 17.1 0.508 0.5x 0.976 7.81 26.8 0.494 1x 1.95 15.62 38.2 0.513 2x 3.91 31.25 46.8 0.661 4x 7.81 62.5 53.6 0.941

Figure 20 shows the 3-day in vitro cell viability of SK-BR-3 cells relative to IC50 concentrations of gemcitabine, trastuzumab-MCC-DM1 (T-DM1), and a fixed-dose ratio of gemcitabine and T-DM1 (Table 20). Gemcitabine combined with T-DM1 resulted in antagonistic drug interactions, with CI values >1.3 for all tested combinations.

Table 20 Gemcitabine (GEM) + T-DM1: SK-BR-3 proliferation – 3 days

IC50 multiple GEM(nM) T-DM1ng/ml Effect(%) CI 0.5x 3.12 6.25 28.7 1.308 1x 6.25 12.5 61.4 1.500 2x 12.5 25 69.9 2.588 4x 25 50 72.2 4.957

Figure 21 shows the in vitro cell viability of MDA-MD-361 at 3 days relative to IC50 concentrations of gemcitabine, trastuzumab-MCC-DM1 (T-DM1), and a fixed-dose ratio of gemcitabine and T-DM1 (Table 21). The drug combinations showed antagonistic effects, with a mean CI of 1.706.

Table 21 Gemcitabine (GEM) + T-DM1: MDA-MD-361 proliferation – 3 days

IC50 multiple GEM(nM) T-DM1ng/ml Effect(%) CI 0.125x 0.39 3.12 4.5 1.420 0.25x 0.78 6.25 10.3 1.584 0.5x 1.56 12.5 30.7 1.336 1x 3.12 25 59.2 1.280 2x 6.25 50 76.3 1.581 4x 12.5 100 80.3 2.747

Figure 22 shows the in vitro cell viability (proliferation) of KPL4 cells after 3 days of treatment with T-DM1, GDC-0941 at IC50 concentrations of 0.25x to 4x, and in fixed ratios of T-DM1 (6.25 to 100 ng/ml) and GDC-0941 (62.5 nM to 1 μM). Table 22 shows the effects in the 10-90% inhibition range and the calculated CI values, with a mean CI of 1.111.

The additivity Bliss prediction is plotted as a dashed line in Figure 22. The Bliss independence plot shows the additivity response calculated from the combination of the two single compounds.

Table 22 GDC-0941+T-DM1: KPL4 proliferation – 3 days

IC50 multiple GDC-0941(nM) T-DM1ng/ml Effect(%) CI 0.25x 62.5 6.25 1.0 6.319 0.5x 125 12.5 33.9 1.229 1x 250 25 71.8 1.053 2x 500 50 91.1 1.051 4x 1000 100 93.7 1.753

Figure 23 shows the in vitro cell viability (proliferation) of KPL4 cells after 3 days of treatment with T-DM1, GDC-0941 at IC50 concentrations ranging from 0.0625x to 16x, and in fixed ratios of T-DM1 (1.25 to 80 ng/ml) and GDC-0941 (31.25 nM to 2 μM). Additive Bliss predictions are plotted as dashed lines. Table 23 shows the effects in the 10–90% inhibition range and the calculated CI values, with a mean CI of 0.802. The combination of T-DM1 and GDC-0941 was additive in the KPL4 cell line.

Table 23 GDC-0941+T-DM1: KPL4 proliferation – 3 days

IC50 multiple GDC-0941(nM) T-DM1ng/ml Effect(%) CI 0.125x 31.25 1.25 12.6 1.100 0.25x 62.5 2.5 20.6 1.344 0.5x 125 5 39.2 1.263 1x 250 10 84.5 0.452 2x 500 20 94.9 0.350 4x 1000 40 97.1 0.440 8x 2000 80 97.9 0.668

Figure 24 shows the in vitro cell viability (proliferation) of Her2-amplified, resistant, PIK3CA(H1047R) mutant KPL-4 cells after treatment with T-DM1, PI103, GDC-0941 at IC50 concentrations ranging from 0 to 16x, and with fixed ratios of T-DM1+PI103 and T-DM1+GDC-0941. Table 24 shows the combination index values. The results suggest moderate in vitro synergy between T-DM-1 and GDC-0941 (since CI values ranged between 0.5 and 1) and additivity between T-DM-1 and PI103 (since CI values were close to 1).

Table 24 Combinations: KPL4 Proliferation

CI: T-DM1+GDC-0941 T-DM1+PI103 ED50 0.74303 1.04069 ED75 0.63448 0.9721 ED90 0.54179 0.91094

The selective inhibitor of PI3K, PI103 (Hyakawa et al. (2007) Bioorg. Med. Chem. Lett. 17: 2438-2442; Raynaud et al. (2007) Cancer Res. 67: 5840-5850; Fan et al. (2006) Cancer Cell 9: 341-349; US 6608053), has the following structure:

Figure 25 shows the in vitro apoptosis (programmed cell death) of KPL4 caspase 3/7 cells 24 hours after treatment with T-DM1, GDC-0941, and a fixed-dose combination of T-DM1 and GDC-0941. The combination of T-DM1 and GDC-0941 resulted in significantly enhanced apoptosis compared to either agent alone.

Figure 26 shows the in vitro apoptosis (programmed cell death) of KPL4 cells 3 days after treatment with T-DM1, GDC-0941, and a fixed-dose combination of T-DM1 and GDC-0941. The combination of T-DM1 and GDC-0941 resulted in significantly enhanced apoptosis compared to either agent alone.

Figure 27 shows the in vitro cell viability (proliferation) of MDA-MB-361 cells after treatment with T-DM1, GDC-0941 at IC50 concentrations of 0.125x to 8x, and at fixed ratios of T-DM1 (3.125 to 50 ng/ml) and GDC-0941 (62.5 nM to 1 μM). Additive Bliss prediction is plotted as a dashed line. Table 27 shows the effects in the 10-90% inhibition range and the calculated CI values, with a mean CI of 0.888. T-DM1 combined with GDC-0941 resulted in additive antiproliferative activity in MDA-MB-361 cells, with a mean CI of 0.889.

Table 27 GDC-0941+T-DM1: MDA-MB-361 proliferation – 3 days

Figure 28 shows the in vitro cell viability (proliferation) of MDA-MB-361 cells after treatment with T-DM1, GDC-0941 at IC50 concentrations of 0.125x to 8x, and at fixed ratios of T-DM1 (3.125 to 100 ng/ml) and GDC-0941 (62.5 nM to 2 μM). Additive Bliss prediction is plotted as a dashed line. Table 28 shows the results in the 10-90% inhibition range and the calculated CI values with a mean CI of 0.813. T-DM1 combined with GDC-0941 resulted in additive antiproliferative activity in MDA-MB-361 cells, with a mean CI of 0.813.

Table 28 GDC-0941+T-DM1: MDA-MB-361 proliferation – 3 days

IC50 multiple GDC-0941(nM) T-DM1ng/ml Effect(%) CI 0.25x 62.5 3.125 28.6 0.785 0.5x 125 6.25 36.7 0.960 1x 250 12.5 48.5 1.026 2x 500 25 66.6 0.807 4x 1000 50 82.2 0.590 8x 2000 100 87.7 0.709

Figure 29 shows the in vitro cell viability (proliferation) of BT-474 cells after treatment with T-DM1, GDC-0941 at IC50 levels of 0.125x to 4x, and at fixed dose ratios of T-DM1 (3.125 to 100 ng/ml) and GDC-0941 (31.25 nM to 1 μM). Additive Bliss predictions are plotted as dashed lines. Table 29 shows the results in the 10–90% inhibition range, along with calculated CI values and a mean CI of 1.2122. Using these dose ratios, GDC-0941 and T-DM1 did not have a combined effect on BT-474 cells.

Table 29 GDC-0941+T-DM1: BT-474 proliferation – 3 days

IC50 multiple GDC-0941(nM) T-DM1ng/ml Effect(%) CI 0.125x 31.25 3.125 8.0 >2 0.25x 62.5 6.25 22.7 1.032 0.5x 125 12.5 31.4 1.178 1x 250 25 43.9 1.207 2x 500 50 53.9 1.473 4x 1000 100 71.5 1.171

Figure 30 shows the in vitro cell viability (proliferation) of BT-474 cells after treatment with T-DM1, GDC-0941 at IC50 concentrations of 0.25x to 4x, and at fixed ratios of T-DM1 (6.25 to 100 ng/ml) and GDC-0941 (62.5 nM to 1 μM). Additive Bliss predictions are plotted as dashed lines. Table 30 shows the results in the 10–90% inhibition range, along with calculated CI values and a mean CI of 0.997, indicating additive effect.

Table 30 GDC-0941+T-DM1: BT-474 proliferation – 3 days

IC50 multiple GDC-0941(nM) T-DM1ng/ml Effect(%) CI 0.25x 62.5 6.25 19.7 1.338 0.5x 125 12.5 31.5 1.167 1x 250 25 49.0 0.886 2x 500 50 66.0 0.708 4x 1000 100 73.9 0.886

Figure 31 shows the in vitro cell viability (proliferation) of Her2-expanded, non-PI3K-mutant AU565 cells after treatment with T-DM1, PI103, GDC-0941 at IC50 concentrations ranging from 0 to 16x, and in fixed ratios of T-DM1+PI103 and T-DM1+GDC-0941 for 3 days. Table 31 shows the combination index values. The results suggest in vitro antagonism between T-DM-1 and GDC-0941 (because CI values are >1), and additive or slight antagonism between T-DM-1 and PI103 (because CI values are close to or slightly greater than 1).

Table 31 Combination: AU565 Proliferation

CI: T-DM1+GDC-0941 T-DM1+PI103 ED50 1.19123 1.12269 ED75 1.36342 0.97338 ED90 1.56063 0.84956

Figure 32 shows the in vitro cell viability (proliferation) of Her2-amplified PIK3CA(C420R) mutant EFM192A cells after treatment with T-DM1, PI103, GDC-0941 at IC50 concentrations of 0 to 16x, and fixed doses of T-DM1+PI103 and T-DM1+GDC-0941 for 3 days. Table 32 shows the combination index values. The results suggest moderate in vitro synergy between T-DM-1 and GDC-0941 (since the CI values are between 0.5 and 1), and synergy between T-DM-1 and PI103 (since the CI values are close to 0.5).

Table 32 Combinations: EFM192A Proliferation

CI at: T-DM1+GDC-0941 T-DM1+PI103 ED50 0.80379 0.53861 ED75 0.66352 0.52087 ED90 0.5485 0.52001

Figure 33 shows the in vitro cell viability (proliferation) of Her2-amplified, resistant, PIK3CA(H1047R) mutant HCC1954 cells after treatment with T-DM1, PI103, GDC-0941 at IC50 concentrations of 0 to 16x, and at fixed ratios of T-DM1+PI103 and T-DM1+GDC-0941 for 3 days. Table 33 shows the combination index values. The results suggest an additive or slightly synergistic relationship between T-DM-1 and GDC-0941 (because the CI values are close to 1), and a slightly synergistic relationship between T-DM-1 and PI103 (because the CI values are <1).

Table 33 Combinations: Proliferation of HCC1954

CI at: T-DM1+GDC-0941 T-DM1+PI103 ED50 1.15864 0.78902 ED75 0.92365 0.78684 ED90 0.74198 0.80771

Efficacy of xenografts for tumors in vivo

The efficacy of the combination of the present invention can be measured in vivo by implanting allogeneic or xenografts of cancer cells into rodents and treating the tumor with the combination. Depending on the cell line, the presence or absence of certain mutations in the tumor cells, the order of administration of trastuzumab-MCC-DM1 and the chemotherapeutic agent, the dosage regimen, and other factors, the expected results are variable. Test mice were treated with the drug or a control (medium) and monitored for several weeks or longer to measure time to tumor doubling, logarithmic cell killing, and tumor suppression (Example 3). Figures 10-17 and 34-37 demonstrate the efficacy of trastuzumab-MCC-DM1 in combination with the chemotherapeutic agent through xenograft tumor suppression in mice.

Figure 10 shows the mean in vivo tumor volume over time of KPL-4 tumors inoculated into the mammary fat pads of SCID beige mice after the following administrations: (1) ADC buffer, (2) pertuzumab 15 mg/kg, (3) T-DM1 0.3 mg/kg, (4) T-DM1 1 mg/kg, (5) T-DM1 3 mg/kg, (6) pertuzumab 15 mg/kg + T-DM1 0.3 mg, (7) pertuzumab 15 mg/kg + T-DM1 1 mg/kg, (8) pertuzumab 15 mg/kg + T-DM1 3 mg/kg. Animals administered ADC buffer (1) showed 0 PRs and 0 CRs. Animals administered 15 mg/kg pertuzumab (2) showed 0 PRs and 0 CRs. Animals administered 0.3 mg/kg T-DM1 (3) alone showed 0 PRs and 0 CRs. One animal that received 1 mg/kg T-DM1 alone (4) showed a partial response (PR) and 0 animals that received 0 complete responses (CR). Seven animals that received 3 mg/kg T-DM1 alone (5) showed a PR and 0 animals that received CR. Five animals that received a combination of 15 mg/kg pertuzumab and 0.3 mg/kg T-DM1 (6) showed a PR and 0 animals that received CR. Eight animals that received a combination of 15 mg/kg pertuzumab and 1 mg/kg T-DM1 (7) showed a PR and 0 animals that received CR. Eight animals that received a combination of 15 mg/kg pertuzumab and 3 mg/kg T-DM1 (8) showed a PR and 0 animals that received CR. The combination of pertuzumab and T-DM1 resulted in greater antitumor activity in KPL4 xenografts than either agent alone.

Figure 11 shows the mean in vivo tumor volume of KPL-4 tumors inoculated into the mammary fat pads of SCID beige mice after the following administrations: (1) ADC buffer, (2) 5-FU 100 mg/kg, (3) pertuzumab 40 mg/kg, (4) B20-4.1 5 mg/kg, (5) T-DM1 5 mg/kg, (6) 5-FU 100 mg/kg + T-DM1 5 mg, (7) pertuzumab 40 mg/kg + T-DM1 5 mg/kg, (8) -4.1 5 mg/kg + T-DM1 5 mg/kg, (9) B20-4.1 5 mg/kg + pertuzumab 40 mg/kg. At the end of the study, histological evaluation was performed on all remaining tumors with a volume of less than 50 ^mm³ , and no evidence of surviving tumor cells was found in 8 samples of the single agent (5) T-DM1, 5 mg/kg, 5 samples of the combination group (6) 5-FU, 100 mg/kg + T-DM1, 5 mg, and 8 samples of the combination group (7) pertuzumab, 40 mg/kg + T-DM1, 5 mg/kg.

Figure 12 shows the mean in vivo tumor volume over time of MMTV-HER2Fo5 transgenic breast tumors inoculated into the mammary fat pads of CRL nu/nu mice after the following administrations: (1) medium (ADC buffer), (2) B20-4.1, 5 mg/kg, (3) T-DM1, 3 mg/kg, (4) T-DM1, 5 mg/kg, (5) T-DM1, 10 mg/kg, (6) B20-4.1, 5 mg/kg + T-DM1, 3 mg/kg, (7) B20-4.1, 5 mg/kg + T-DM1, 5 mg/kg, (8) B20-4.1, 5 mg/kg + T-DM1, 10 mg/kg. For T-DM1, 3 mg/kg and 5 mg/kg, instead of 10 mg/kg, the combination of T-DM1 and B20-4.1 resulted in enhanced tumor growth inhibition.

Figure 13 shows the mean in vivo tumor volume over time of MMTV-HER2Fo5 transgenic mammary tumors inoculated into the mammary fat pads of CRL nu/nu mice after the following drug administration: (1) medium (ADC buffer), (2) T-DM1, 10 mg/kg, (3) 5-FU, 100 mg/kg, (4) gemcitabine, 120 mg/kg, (5) carboplatin, 100 mg/kg, (6) 5-FU, 100 mg/kg + T-DM1, 10 mg/kg, (7) gemcitabine, 120 mg/kg + T-DM1, 10 mg/kg, (8) carboplatin, 100 mg/kg + T-DM1, 10 mg/kg. T-DM1 combined with 5-FU, carboplatin or gemcitabine resulted in enhanced antitumor efficacy compared to single-agent treatment.

Figure 14 shows the mean in vivo tumor volume of MMTV-Her2Fo5 transgenic breast tumor xenografts inoculated into the mammary fat pads of Harlan athymic nude mice after the following administrations: (1) Media (PBS buffer) iv, qwk x4, (2) lapatinib, 101 mg/kg, po, bid x21, (3) pertuzumab, 40 mg/kg, iv, qwk x4, (4) B20-4.1, 5 mg/kg, ip, 2x/wk x4, (5) T -DM1, 15mg/kg, iv, q3wk to end, (6) lapatinib, 101mg/kg, po, bid x21+T-DM1, 15mg/kg, iv, q3wk to end, (7) pertuzumab, 40mg/kg, iv, qwk x4+T-DM1, 15mg/kg, iv, q3wk to end, (8) B20-4.1, 5mg/kg, ip, 2x/wk x4+T-DM1, 15mg/kg, iv, q3wk to end.

There was no significant difference between the 15 mg/kg dose of T-DM1 as a single agent (5) and the combination of 15 mg/kg T-DM1 and 5 mg/kg B20-4.1 (8). In this study, lapatinib and pertuzumab showed no difference compared to the medium. B20-4.1 showed a trend toward increased efficacy compared to the medium. T-DM1 was effective as a single agent (p<0.01). The combination of T-DM1 and lapatinib was significantly better than lapatinib alone (p<0.01), but not different from T-DM1 alone. The combination of T-DM1 and pertuzumab was significantly better than pertuzumab alone (p<0.01), but not different from T-DM1 alone. The combination of T-DM1 and B20-4.1 was significantly better than B20-4.1 alone (p<0.01), but not different from T-DM1 alone.

Figure 15 shows the in vivo efficacy of the mean tumor volume of MMTV-Her2Fo5 transgenic breast tumor xenografts inoculated into the mammary fat pads of Harlan athymic nude mice over time after the following administrations: (1) Media (PBS buffer) po, bid x21, (2) T-DM1, 7.5 mg/kg, iv, qd x1, (3) T-DM1, 15 mg/kg, iv, qd x1, (4) ABT-869, 5 mg/kg, po, bid x21, (5) ABT-869, 15 mg/kg, po, bid x21, (6)T-DM1, 7.5mg/kg, iv, qdx1+ABT-869, 5mg/kg, po, bid x21, (7) T-DM1 7.5mg/kg, iv, qd x1+ABT-869, 15mg/kg, po, bid x21 , (8)T-DM1, 15mg/kg, iv, qd x1+ABT-869, 5mg/kg, po, bid x21, (9) T-DM1, 15mg/kg, iv, qd x1+ABT-869, 15mg/kg, po, bid x21.

The combination of T-DM1 and ABT-869, 5 mg/kg, showed two partial responses (8), which were not significantly more effective than ABT-869, 5 mg/kg alone (4). The combination of T-DM1 and ABT-869, 15 mg/kg (9) was slightly more effective than ABT-869, 15 mg/kg alone (5). For time to endpoint, ABT-869 at 5 mg/kg was significantly better than the medium (p<0.01), but there was no difference in time to tumor doubling. For both time to tumor doubling and time to tumor endpoint, ABT-869 at 15 mg/kg and T-DM1 at 7.5 or 15 mg/kg were significantly better than the medium (p<0.01). The combination of 7.5 mg/kg T-DM1 and 5 mg/kg ABT-869 was not different from 7.5 mg/kg T-DM1 alone. Compared to 5 mg/kg ABT-869 alone, the combination of 7.5 mg/kg T-DM1 + 5 mg/kg ABT-869 showed a significantly better time to tumor doubling (p<0.01), but no difference in time to the endpoint. The combination of 7.5 mg/kg T-DM1 and 15 mg/kg ABT-869 was significantly better than either single agent (p<0.01). The combination of 15 mg/kg T-DM1 + 5 mg/kg ABT-869 was not different from 15 mg/kg T-DM1 alone. Compared to 5 mg/kg ABT-869 alone, the combination of 15 mg/kg T-DM1 and 5 mg/kg ABT-869 showed no difference in time to the endpoint, but a significant difference in time to tumor doubling (p<0.01). Regarding the time to tumor doubling, the combination of 15 mg/kg T-DM1 and 15 mg/kg ABT-869 was significantly better than 15 mg/kg ABT-869 alone, and also better than 15 mg/kg T-DM1 alone (p<0.01). There was no difference in the time to reach the endpoint between 15 mg/kg T-DM1 and 15 mg/kg T-DM1 + 15 mg/kg ABT-869.

Figure 16 shows the mean in vivo tumor volume of MMTV-Her2Fo5 transgenic breast tumor xenografts inoculated into the mammary fat pads of Harlan athymic nude mice after the following administrations: (1) mediator, iv, qwk x3, (2) T-DM1, 7.5 mg/kg, iv, q3wk x2, (3) T-DM1, 15 mg/kg, iv, q3wk x2, (4) docetaxel, 30 mg/kg, iv, qwk x3, (5) T-DM1, 7.5 mg/kg, iv, q3wk x2 + docetaxel, 30 mg/kg, iv, qwk x3, (6) T-DM1, 15 mg/kg, iv, q3wk x2 + docetaxel, 30 mg/kg, iv, qwk x3.

Six animals administered 15 mg/kg T-DM1 alone (3) showed partial response (PR) and one animal showed complete response (CR). Two animals administered 30 mg/kg docetaxel alone (4) showed PR. Ten animals administered a combination of 7.5 mg/kg T-DM1 and 30 mg/kg docetaxel (5) showed PR. Seven animals administered a combination of 15 mg/kg T-DM1 and 30 mg/kg docetaxel (6) showed dose response and three animals showed PR. All single-drug groups were significantly different from the mediator groups (p<0.01). The 7.5 mg/kg T-DM1 + docetaxel combination was significantly better than either single-drug combination for both time to tumor doubling and time to endpoint (p<0.01). There was no objective response in the 7.5 mg/kg T-DM1 group, while there were two partial responses (PR) in the docetaxel single-drug group. The combination of 7.5 mg/kg T-DM1 and docetaxel resulted in 9 partial responses (PR) and 1 complete response (CR). For time to tumor doubling and time to endpoint, the 15 mg/kg T-DM1 + docetaxel combination was significantly better than either single agent (p<0.01). Single-agent treatment with 15 mg/kg T-DM1 resulted in 5 PRs and 2 CRs. The 15 mg/kg T-DM1 + docetaxel combination enhanced the objective response rate to 7 PRs and 3 CRs. All mice in this combination group showed an objective response to treatment.

Figure 17 shows the mean in vivo tumor volume of MMTV-Her2Fo5 transgenic breast tumor xenografts inoculated into the mammary fat pads of Harlan athymic nude mice after the following administrations: (1) mediator, po, qd x21, (2) T-DM1, 7.5 mg/kg, iv, q3wk x2, (3) T-DM1, 15 mg/kg, iv, q3wk x2, (4) lapatinib, 100 mg/kg, po, bid x21, (5) T-DM1, 7.5 mg/kg, iv, q3wk x2+lapatinib, 100 mg/kg, po, bid x21, (6) T-DM1, 15 mg/kg, iv, q3wk x2+lapatinib, 100 mg/kg, po, bid x21.

Six animals administered 15 mg/kg T-DM1 alone (3) showed partial response (PR) and three showed complete response (CR). Four animals administered a combination of 7.5 mg/kg T-DM1 and 100 mg/kg lapatinib (5) showed PR and five showed CR. Eight animals administered a combination of 15 mg/kg T-DM1 and 100 mg/kg lapatinib (6) showed CR. There were significant differences between the single-agent groups and the mediators in both time to tumor doubling and time to endpoint (p<0.01). Administration of 7.5 mg/kg T-DM1 in combination with lapatinib was significantly better than either lapatinib or 7.5 mg/kg T-DM1 as a single agent (p<0.01). Administration of 15 mg/kg T-DM1 in combination with lapatinib was significantly better than lapatinib as a single agent (p<0.01). This combination is no different from taking 15 mg/kg T-DM1 as a single agent.

Time to tumor doubling was measured as 2X Vo using Kaplan-Meier statistical analysis. Time to tumor doubling and survival analysis were quantified using Log-rank-p values. Time to progression was the time taken for the tumor volume to reach 1000 ^mm³ , or survival time in cases where the tumor volume did not reach 1000 ^mm³ . T-DM1 combined with lapatinib resulted in significantly enhanced antitumor efficacy compared to monotherapy.

Figure 34 shows the mean in vivo tumor volume over time in MMTV-Her2Fo5 transgenic breast tumors inoculated into CRL nu/nu mice after the following administrations: (1) Mediator, po, qd x21, (2) T-DM1, 10 mg/kg, iv, q3wk, (3) 5-FU, 100 mg/kg, po, qwk x2, (4), (5) T-DM1, 5 mg/kg, iv, q3wk + 5-FU, 100 mg/kg, po, qwk x2. Animals administered the mediator showed 0 partial responses (PR) and 0 complete responses (CR). Animals administered T-DM1 showed 1 PR and 0 CR. Animals administered 5-FU showed 0 PR and 0 CR. Animals administered the combination of T-DM1 and 5-FU showed 3 PR and 0 CR at day 42. Treatment with T-DM1 and 5-FU resulted in enhanced antitumor activity compared to either agent alone.

Figure 35 shows the mean in vivo tumor volume of MMTV-Her2Fo5 transgenic breast tumors inoculated into CRL nu/nu mice after the following administrations: (1) mediator, po, qd x21, (2) T-DM1, 5 mg/kg, iv, q3wk, (3) GDC-0941, 100 mg/kg, po, bid x21, (4) GDC-0152, 50 mg/kg, po, qwk x2, (5) T-DM1, 5 mg/kg, iv, q3wk+GDC-0941, 100 mg/kg, po, bid x21, (6) T-DM1, 5 mg/kg, iv, q3wk+GDC-0152, 50 mg/kg, po, qwk x2. Treatment with T-DM1 and GDC-0941 resulted in enhanced antitumor activity compared to single-agent therapy, while the combination of T-DM1 and GDC-0152 was not more effective than T-DM1 alone.

GDC-0152 is an inhibitor of caspase, which is an inhibitor of apoptosis protease (Call et al. (2008) The Lancet Oncology, 9(10): 1002-1011; Deveraux et al. (1999) J Clin Immunol 19: 388-398).

Figure 36 shows the mean in vivo tumor volume of MDA-MB-361.1 breast tumors in CRL nu/nu mice after the following administrations: (1) mediator, po, qd x21, (2) GDC-0941, 25 mg/kg, po, qd x21, (3) GDC-0941, 50 mg/kg, po, qd x21, (4) GDC-0941, 100 mg/kg, po, qd x21, (5) T-DM1, 3 mg/kg, iv, q3wk, (6) T-DM1, 10 mg/kg, iv, q3wk, (7) GDC-0941, 25 mg/kg, po, qd x21 + T-DM1, 3 mg/kg, iv, q3wk , (8) GDC-0941, 50 mg/kg, po, qd x21+T-DM1, 3 mg/kg, iv, q3wk, (9) GDC-0941, 100 mg/kg, po, qd 21+T-DM1, 10 mg/kg, iv, q3wk, (11) GDC-0941, 50 mg/kg, po, qd x21+T-DM1, 10 mg/kg, iv, q3wk, (12) GDC-0941, 100 mg/kg, po, qd

Animals administered the medium (1) showed 0 partial responses (PR) and 0 complete responses (CR). Animals administered 25 mg/kg GDC-0941 alone (2) showed 0 PR and 0 CR. Animals administered 50 mg/kg GDC-0941 alone (3) showed 1 PR and 0 CR. Animals administered 100 mg/kg GDC-0941 alone (4) showed 0 PR and 0 CR. Animals administered 3 mg/kg T-DM1 alone (5) showed 1 PR and 1 CR. Animals administered 10 mg/kg T-DM1 alone (6) showed 8 PR and 1 CR. Animals administered a combination of 3 mg/kg T-DM1 and 25 mg/kg GDC-0941 (7) showed 5 PR and 0 CR. Animals administered a combination of 3 mg/kg T-DM1 and 50 mg/kg GDC-0941 (8) showed 3 PR and 0 CR. Three animals that received 3 mg/kg T-DM1 and 100 mg/kg GDC-0941 (9) showed partial remission (PR) and one animal showed complete remission (CR). Nine animals that received a combination of 10 mg/kg T-DM1 and 50 mg/kg GDC-0941 (10) showed PR and none showed CR. Seven animals that received a combination of 10 mg/kg T-DM1 and 50 mg/kg GDC-0941 (11) showed PR and two showed CR. Nine animals that received a combination of 10 mg/kg T-DM1 and 100 mg/kg GDC-0941 (12) showed PR and one showed CR.

Figure 37 shows the mean in vivo tumor volume over time of MDA-MB-361.1 breast tumors in CRL nu/nu mice after the following administration: (1) Media [MCT (0.5% methylcellulose/0.2% TWEEN 80) + succinate buffer (100mM sodium succinate, 100mg/ml trehalose, 0.1% TWEEN 80, pH 5.0)], po+IV, qd x21 and qd, (2) GNE-39 0, 1.0mg/kg, po, qd x21, (3) GNE-390, 2.5mg/kg, po, qd x21, (4) T-DM1, 3mg/kg, iv, qd, (5) GNE-390, 1 .0mg/kg,po,qd x21+T-DM1,3mg/kg,iv,qd,(6)GNE-390,2.5mg/kg,po,qdx21+T-DM1,3mg/kg,iv,qd.

Animals administered the medium (1) showed 0 partial responses (PR) and 0 complete responses (CR). Animals administered 1.0 mg/kg GNE-390 alone (2) showed 0 PR and 0 CR. Animals administered 2.5 mg/kg GNE-390 alone (3) showed 1 PR and 0 CR. Animals administered 3 mg/kg T-DM1 alone (5) showed 1 PR and 1 CR. Animals administered 3 mg/kg T-DM1 alone (4) showed 0 PR and 0 CR. Animals administered a combination of 3 mg/kg T-DM1 and 25 mg/kg GNE-390 (5) showed 3 PR and 0 CR. Animals administered a combination of 3 mg/kg T-DM1 and 2.5 mg/kg GNE-390 (6) showed 5 PR and 1 CR. In the MDA-MB-361.1 breast cancer xenograft model, the combination of GNE-390 and T-DM1 significantly increased the number of partial and complete antitumor responses compared to GNE-390 or T-DM1 alone.

Pharmaceutical Composition

The pharmaceutical compositions or formulations of the present invention include trastuzumab-MCC-DM1, a chemotherapeutic agent, and one or more pharmaceutically acceptable carriers, gliding agents, diluents, or excipients.

The trastuzumab-MCC-DM1 and chemotherapeutic agents of the present invention can exist in unsolvated and solvent-solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc., and the present invention covers both solvent-solvated and unsolvated forms.

The trastuzumab-MCC-DM1 and chemotherapeutic agents of the present invention can also exist in different tautomer forms, and all such forms are covered within the scope of the present invention. The term "tautomer" or "tautomer form" refers to structural isomers with different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also called proton-transfer tautomers) include tautomerization via proton transfer, such as keto-enol and imine-enamine isomerization. Valence tautomers include tautomerization by rearranging some of the bonding electrons.

Pharmaceutical compositions encompass both bulk compositions and individual dosage units, which contain more than one (e.g., two) pharmaceutically active agents (including trastuzumab-MCC-DM1 and chemotherapeutic agents selected from the other pharmaceutical lists described herein) and any pharmaceutically inactive excipients, diluents, carriers, or gliding agents. Both bulk compositions and individual dosage units may contain a fixed amount of the aforementioned pharmaceutically active agents. A bulk composition refers to a substance that has not yet been formed into individual dosage units. An illustrative dosage unit is an oral dosage unit, such as tablets, pills, capsules, etc. Similarly, the methods of treating a patient by administering the pharmaceutical compositions of the present invention described herein are also intended to cover the administration of bulk compositions and individual dosage units.

The pharmaceutical compositions also encompass isotopically labeled compounds of the present invention, which are identical to the compounds described herein except that one or more atoms are replaced by atoms having atomic masses or mass numbers different from those commonly found in nature. All isotopes of any specific atom or element specified are covered within the scope of the compounds of the present invention and their uses. Exemplary isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as ^2H , ^3H , ^11C , ^13C , ^14C , ^13N , ^15N , ^15O , ^17O , ^18O , ^32P , ^33P , ^35S , ^18F , ^36Cl , ^123I , and ^125I . Certain isotopically labeled compounds of the present invention (e.g., those labeled with ^3H and ^14C ) are useful in compound and/or substrate tissue distribution assays. Tritium ( ^3H ) and carbon-14 ( ^14C ) isotopes are useful due to their ease of preparation and detectability. Additionally, substitution with heavier isotopes such as deuterium ( ^2H ) can provide certain therapeutic advantages stemming from greater metabolic stability (e.g., prolonged in vivo half-life or reduced dose requirement), and is therefore preferred in some cases. Positron-emitting isotopes, such as ^15O , ^13N , ^11C , and ^18F , can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of the present invention can generally be prepared by substituting the non-isotopically labeled reagent with an isotopically labeled reagent, following procedures similar to those disclosed in the schemes and/or examples below.

Trastuzumab-MCC-DM1 and chemotherapeutic agents can be formulated according to standard pharmaceutical practice for use in therapeutic combinations for the treatment (including prophylactic treatment) of highly proliferative diseases in mammals, including humans. This invention provides pharmaceutical compositions comprising trastuzumab-MCC-DM1 in combination with one or more pharmaceutically acceptable carriers, gliding agents, diluents, or excipients.

Suitable carriers, diluents, and excipients are well known to those skilled in the art and include substances such as carbohydrates, waxes, water-soluble and/or expandable polymers, hydrophilic or hydrophobic substances, gelatin, oils, solvents, water, etc. The specific carrier, diluent, or excipient used will depend on the means and purpose of applying the compounds of this invention. Solvents are generally selected based on those generally recognized by those skilled in the art as being safe for mammalian administration (GRAS). Generally, safe solvents are non-toxic aqueous solvents, such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG 400, PEG 300), etc., and mixtures thereof. The formulation may also include one or more buffers, stabilizers, surfactants, humectants, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, flow aids, processing aids, colorants, sweeteners, flavorings, tasters, and other known additives to provide a refined appearance for the medicine (i.e., the compound of the present invention or a pharmaceutical composition thereof) or to aid in the manufacture of a pharmaceutical product (i.e., the drug).

Formulations can be prepared using conventional dissolution and mixing procedures. For example, bulk pharmaceutical substances (i.e., compounds of the present invention or stabilized forms of compounds (e.g., complexed with cyclodextrin derivatives or other known complexing agents) can be dissolved in a suitable solvent in the presence of one or more of the excipients described above. The compounds of the present invention are typically formulated into pharmaceutically sized dosage forms to provide a drug with easily controllable dosage and to ensure patient compliance with prescription regimens.

Pharmaceutical compositions (or formulations) for supply can be packaged in various ways, depending on the mode of administration. Generally, dispensed items include containers in which the pharmaceutical formulation, in a suitable form, is deposited. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), capsules, ampoules, plastic bags, metal tubes, etc. Containers may also include anti-interference devices to prevent accidental access to the contents. Additionally, a label describing the contents of the container is deposited on the container. The label may also include appropriate warnings.

Pharmaceutical formulations of the compounds of the present invention can be formulated in the form of lyophilized formulations, ground powders, or aqueous solutions using pharmaceutically acceptable diluents, carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1995) 18th edition, Mack Publ. Co., Easton, PA) for administration via various routes and types. Formulation can be carried out by mixing with a physiologically acceptable carrier (i.e., a carrier that is non-toxic to the recipient at the dose and concentration used) at ambient temperature and a suitable pH, and to the desired purity. The pH of the formulation depends primarily on the specific application and the concentration of the compound, but can range from about 3 to about 8.

Pharmaceutical formulations are preferably sterile. Specifically, formulations intended for in vivo administration must be sterile. This sterility is easily achieved through filtration using sterile membrane filters.

Pharmaceutical formulations are typically stored as solid compositions, lyophilized formulations, or aqueous solutions.

The pharmaceutical formulations of this invention will be administered and dosed in a manner consistent with good medical practice, namely, in terms of dosage, concentration, schedule, course of treatment, medium, and route of administration. Factors to be considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the drug, the method of administration, the schedule of administration, and other factors known to a medical practitioner. The "therapeuticly effective amount" of the compound to be administered will be determined by such considerations and is the minimum amount necessary to prevent, improve, or treat clotting factor-mediated conditions. Such an amount is preferably below levels that are toxic to the host or below levels that would significantly increase the host's susceptibility to bleeding.

As a general recommendation, the initial pharmaceutically effective dose of trastuzumab-MCC-DM1 administered per dose will be in the range of approximately 0.01-100 mg/kg, or approximately 0.1 to 20 mg/kg of patient body weight per day, with a typical initial range of 0.3-15 mg/kg/day for the compound used.

Acceptable diluents, carriers, excipients, and stabilizers are non-toxic to the recipient at the dosage and concentration used, and include: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; chlorhexidine diammonium chloride; benzalkonium chloride, benzyl chloride; phenols, butanol, or benzyl alcohol; hydroxybenzoic acid esters such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (below about 10 residual...) The active pharmaceutical ingredient may be encapsulated in the following ways: 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; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, such as TWEEN ^™ (including Tween 80), PLURONICS ^™ , or polyethylene glycol (PEG) (including PEG400). The active pharmaceutical ingredient may also be encapsulated in microcapsules prepared, for example, by coagulation techniques or by interfacial polymerization (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in coarse-drop emulsions. This technology was disclosed in Remington's Pharmaceutical Sciences, 18th edition, (1995) Mack Publ. Co., Easton, PA.

Pharmaceutical formulations include those suitable for the routes of administration detailed herein. Formulations can be readily available in unit dose form and can be prepared by any method known in the pharmaceutical field. Techniques and formulations are generally found in Remington's Pharmaceutical Sciences, 18th edition (1995), Mack Publishing Co., Easton, PA. Such methods involve the step of combining the active ingredient with a carrier constituting one or more auxiliary components. Generally, formulations are prepared by uniformly and tightly combining the active ingredient with a liquid carrier or a pulverized solid carrier, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration of chemotherapeutic agents can be prepared in discrete units, such as pills, hard or soft forms like gelatin capsules, flat capsules, lozenges, tablets, aqueous or oily suspensions, dispersible powders or granules, emulsions, syrups, or acetaminophen, each containing a predetermined amount of the trastuzumab-MCC-DM1 compound and/or the chemotherapeutic agent. Such formulations can be prepared according to any method known in the art for manufacturing pharmaceutical compositions, and such compositions can contain one or more pharmaceutical agents, including sweeteners, flavoring agents, coloring agents, and preservatives, to provide a palatable article. Compressed tablets can be prepared by pressing an active component in a free-flow form (such as powder or granules) in a suitable machine, optionally mixed with binders, lubricants, inert diluents, preservatives, surfactants, or dispersants. Cast tablets can be prepared by casting a mixture of powdered active components moistened with an inert liquid diluent in a suitable machine. Tablets can optionally be coated or scored, and optionally formulated to provide a slow or controlled release of the active component from them.

The tablet excipients of the pharmaceutical formulations of the present invention may include: fillers (or diluents) to increase the bulk volume of the powdered drug constituting the tablet; disintegrants, which, when ingested, promote the disintegration of the tablet into small fragments, ideally individual drug particles, and promote rapid dissolution and absorption of the drug; binders to ensure the formation of particulate granules and tablets with the required mechanical strength and to keep the tablets intact after compression, preventing them from disintegrating into their constituent powders during packaging, transportation, and routine operations; flow aids to improve the flowability of the powder constituting the tablet during production; lubricants to ensure that the powder forming the tablet does not adhere to the equipment used for tablet compression during manufacturing. They improve the flow of the powder mixture through the tablet press and minimize friction and breakage of the formed tablets as they are ejected from the equipment; anti-adhesion agents, which function similarly to flow aids, reducing adhesion between the powder constituting the tablet and the machine used to extrude the tablet shape during manufacturing; flavoring agents incorporated into the tablet to give the tablet a more pleasant taste or mask unpleasant tastes; and coloring agents to aid identification and patient compliance.

Tablets containing an active ingredient mixed with a non-toxic, pharmaceutically acceptable excipient suitable for tablet preparation are acceptable. These excipients may be, for example, inert diluents such as calcium carbonate or sodium carbonate, lactose, calcium phosphate or sodium carbonate; granulating and disintegrants such as corn starch or alginate; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be pre-coated, or can be coated using known techniques, including microencapsulation, to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period. For example, time-delaying materials such as glyceryl monostearate or glyceryl distearate may be used alone or in combination with waxes.

For the treatment of the eyes or other external tissues, such as the mouth and skin, the formulation is preferably applied as a topical ointment or cream containing the active ingredient in an amount, for example, 0.075 to 20% w/w. When formulated as an ointment, the active ingredient can be used together with a paraffin or water-miscible ointment base or in combination with the active ingredient. Alternatively, the active ingredient can be formulated in a cream together with an oil-in-water cream base.

If desired, the aqueous phase of the cream base may include polyhydroxy alcohols, i.e., alcohols having two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerin, and polyethylene glycol (including PEG 400) and mixtures thereof. Surface formulations may include, as needed, compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogues.

The oil phase of the emulsion of the present invention can be constructed from known components in a known manner, comprising at least one emulsifier with fats or oils, or with a mixture of both fats and oils. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. In summary, with or without a stabilizer, the emulsifier constitutes an emulsified wax, and the wax, together with the oils and fats, constitutes an emulsified ointment base, which forms the oily dispersed phase of the ointment formulation. Suitable emulsifiers and emulsion stabilizers for use in the formulations of the present invention include 60, 80, a mixture of cetyl alcohol and octadecyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate.

The aqueous suspension of the pharmaceutical formulation of the present invention contains an active material mixed with excipients suitable for preparing the aqueous suspension. Such excipients include: suspending agents, such as sodium carboxymethyl cellulose, croscarmellose, povidone, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, astragalus gum, and gum arabic; and dispersing or wetting agents, such as naturally occurring phospholipids (e.g., lecithin), condensation products of olefin oxides and fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide and long-chain aliphatic alcohols (e.g., heptadecanethoxycetyl alcohol), and condensation products of ethylene oxide and metaesters derived from fatty acids and hexadiol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives, such as ethylparaben or n-propylparaben; one or more colorants; one or more flavoring agents; and one or more sweeteners, such as sucrose or saccharin.

The pharmaceutical composition may be in the form of a sterile injectable formulation, such as a sterile injectable aqueous or oily suspension. Such suspensions may be formulated using suitable dispersants or wetting agents and suspending agents described above, in accordance with known techniques. The sterile injectable formulation may be a sterile injectable solution or suspension in a non-toxic, parenteral acceptable diluent or solvent, such as in 1,3-butanediol or a solution prepared from a lyophilized powder. Water, Ringer's solution, and isotonic sodium chloride solution are among the acceptable media and solvents that may be used. Additionally, sterile non-volatile oils may be conveniently used as solvents or suspension media. For this purpose, any mild non-volatile oil may be used, including synthetic monoglycerides or diesters. Furthermore, fatty acids such as oleic acid may also be used in the preparation of injectable formulations.

The amount of active ingredient that can be combined with a carrier material to produce a single dose varies depending on the host being treated and the specific mode of administration. For example, a time-release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of the active material, combined with an appropriate and convenient amount of carrier material, which may vary from approximately 5% to approximately 95% (by weight) of the total composition. Pharmaceutical compositions can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain approximately 3 to 500 μg of the active ingredient per milliliter of solution to allow for the infusion of an appropriate volume at a rate of approximately 30 mL/hour.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickeners.

Formulations suitable for topical application to the eye also include eye drops, wherein the active ingredient is dissolved or suspended in a carrier (especially an aqueous solvent) suitable for the active ingredient. The active ingredient is preferably present at a concentration of about 0.5 to 20% w/w, for example about 0.5 to 10% w/w, for example about 1.5% w/w.

Formulations suitable for oral surface application include tablets containing the active ingredient in a flavoring base (typically sucrose and gum arabic or astragalus gum); soft tablets containing the active ingredient in an inert base (such as gelatin and glycerin, or sucrose and gum arabic); and mouthwashes containing the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may exist as suppositories with suitable base materials, including, for example, cocoa butter or salicylates.

Formulations suitable for intrapulmonary or nasal administration have particle sizes ranging from, for example, 0.1 to 500 micrometers (including particle sizes in increments of a few micrometers between 0.1 and 500 micrometers, such as 0.5, 1, 30 micrometers, 35 micrometers, etc.), and are administered by rapid inhalation through the nasal passages or by oral inhalation to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration can be prepared using conventional methods and can be delivered together with other therapeutic agents, such as compounds previously used to treat or prevent the conditions described below.

Formulations suitable for vaginal application may exist as vaginal suppositories, tampons, creams, gels, pastes, foams, or sprays containing, in addition to the active ingredient, a carrier known in the art.

Formulations can be packaged in unit-dose or multi-dose containers, such as sealed ampoules and tubular vials, and can be stored under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, such as water, for immediate injection before use. Solutions and suspensions for immediate injection can be prepared from sterile powders, granules, and tablets of the types described above. Preferred unit-dose formulations are those containing the active ingredient at the daily dose or a unit daily sub-dose, or a suitable portion thereof, as described above.

The present invention further provides veterinary compositions comprising at least one active ingredient as defined above and a veterinary carrier thereof. The veterinary carrier is a material suitable for the purpose of administering the composition and may be a solid, liquid, or gaseous substance, or otherwise inert or a substance acceptable in the veterinary pharmaceutical field and compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally, or via any other desired route.

Combination therapy

Trastuzumab-MCC-DM1 can be used in combination with other chemotherapeutic agents to treat hyperproliferative diseases or conditions, including tumors, cancers, and neoplasms, as well as pre-malignant and non-neoplastic or non-malignant hyperproliferative conditions. In some embodiments, trastuzumab-MCC-DM1 is used as a combination therapy in a pharmaceutical formulation or dosing regimen in combination with a second compound having anti-hyperproliferative properties or suitable for treating hyperproliferative conditions. The second compound in the pharmaceutical formulation or dosing regimen preferably has an activity complementary to trastuzumab-MCC-DM1, such that they do not adversely affect each other. Such compounds are suitably combined in amounts effective for the intended purpose. In one embodiment, the composition of the present invention comprises trastuzumab-MCC-DM1 in combination with chemotherapeutic agents such as those described herein. Examples 4 and 5 are clinical regimens of T-DM1+pertuzumab and T-DM1+GDC-0941, respectively.

The therapeutic combination of the present invention includes formulations, dosage regimens, or other treatment procedures comprising the administration of trastuzumab-MCC-DM1 and a chemotherapeutic agent selected from HER2 dimerization inhibitor antibodies, anti-VEGF antibodies, 5-FU, carboplatin, lapatinib, ABT-869, and docetaxel, as a combination preparation for separate, simultaneous, or sequential use in the treatment of hyperproliferative disorders.

Combination therapy can be administered as a simultaneous or sequential regimen. When administered sequentially, the combination can be used in one or more administrations. Combination administration includes co-administration using separate formulations or single-drug formulations, and sequential administration in any order, wherein preferably there is a period during which all active agents exert their biological activity simultaneously.

The appropriate dose of any of the above co-administered drugs is that which is currently in use and can be reduced due to the combined (synergistic) effects of newly identified drugs and other chemotherapeutic agents or treatments.

In one specific implementation of anticancer therapy, trastuzumab-MCC-DM1 can be combined with chemotherapy agents, including hormones or antibody agents such as those described herein, as well as in combination with surgical therapy and radiotherapy. The relative timing and amount of trastuzumab-MCC-DM1 and other pharmaceutically active chemotherapy agents will be selected to achieve the desired combination therapy effect.

Administration of the pharmaceutical composition

The compounds of the present invention can be administered via any route suitable for the condition to be treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intra-arterial, inhalation, intradermal, intrathecal, epidural, and infusion techniques), transdermal, rectal, nasal, surface (including oral and sublingual), vaginal, intraperitoneal, intrapulmonary, and intranasal administration. Surface administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. A discussion of pharmaceutical formulations can be found in Remington's Pharmaceutical Sciences, 18th edition, (1995) Mack Publishing Co., Easton, PA. Other examples of pharmaceutical formulations can be found in Liberman, H.A. and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, Vol. 3, 2nd edition, New York, NY. For local immunosuppressive therapy, the compounds can be administered via intralesional administration, including perfusion or otherwise exposing the graft to the inhibitor prior to transplantation. It should be understood that the preferred route can vary depending on, for example, the recipient's condition. If the compound is to be administered orally, it can be formulated into pills, capsules, tablets, etc., together with a pharmaceutically acceptable carrier, gliding agent, or excipient. If the compound is to be administered parenterally, it can be formulated into a single-dose injectable form together with a pharmaceutically acceptable parenteral medium or diluent, as detailed below.

The dosage range for treating human patients with trastuzumab-MCC-DM1 can be from about 100 mg to about 500 mg. Depending on pharmacokinetic (PK) and pharmacodynamic (PD) characteristics, including absorption, distribution, metabolism, and excretion, trastuzumab-MCC-DM1 can be administered every six weeks, every three weeks, weekly, or more frequently. The dosage range for chemotherapy agents used in combination with trastuzumab-MCC-DM1 can be from about 10 mg to about 1000 mg. Chemotherapy agents can be administered every six weeks, every three weeks, weekly, or more frequently, such as once or twice daily. Additionally, toxic factors may affect the dosage and administration regimen. When administered orally, pills, capsules, or tablets can be taken once daily or less frequently for a prescribed period. The regimen can be repeated for multiple treatment cycles.

Treatment

The therapeutic combination of (1) trastuzumab-MCC-DM1 and (2) chemotherapeutic agents can be used to treat diseases, conditions, and/or ailments, including but not limited to those characterized by HER2 pathway activation. Alternatively, another aspect of the invention includes methods for treating diseases or conditions that can be treated by targeting HER2 or VEGFR receptor 1. The therapeutic combination of (1) trastuzumab-MCC-DM1 and (2) chemotherapeutic agents can be used to treat highly proliferative diseases or ailments, including tumors, cancers, and neoplasms, as well as pre-malignant and non-neoplastic or non-malignant highly proliferative ailments.

Cancers treatable according to the methods of this invention include, but are not limited to, breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, genitourinary tract cancer, esophageal cancer, laryngeal cancer, glioblastoma, neuroblastoma, gastric cancer, skin cancer, keratoacanthoma, lung cancer, epidermoid carcinoma, large cell carcinoma, non-small cell lung cancer (NSCLC), small cell carcinoma, adenocarcinoma of the lung, bone cancer, colon cancer, adenoma, pancreatic cancer, adenocarcinoma, thyroid cancer, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver cancer and bile duct cancer, kidney cancer, medullary cancer, lymphoid cancer, pilocarcinoma, oral and pharyngeal cancer (oral cavity), lip cancer, tongue cancer, oral cancer, pharyngeal cancer, small bowel cancer, colorectal cancer, colon cancer, rectal cancer, brain and central nervous system cancers, Hodgkin's disease and leukemia.

Another aspect of the invention provides pharmaceutical compositions or therapeutic combinations for use in the treatment of mammals, such as humans, suffering from the diseases or conditions described herein. Use of the pharmaceutical compositions in the preparation of medicaments for treating warm-blooded animals, such as mammals, such as humans, suffering from the diseases and conditions described herein is also provided.

Products

In another embodiment of the invention, an article, or “kit,” containing trastuzumab-MCC-DM1 for the treatment of the diseases and conditions described above is provided. In one embodiment, the kit comprises a container filled with trastuzumab-MCC-DM1. The kit may further include a label or packaging insert on or related to the container. The term “packaging insert” is used to refer to instructions typically included in the commercial packaging of therapeutic products, which contain information regarding indications, usage, dosage, administration, contraindications, and/or warnings relating to the use of such therapeutic products. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc., which may be formed from various materials such as glass or plastic. The container may contain trastuzumab-MCC-DM1 or a formulation thereof for the effective treatment of the conditions and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierced by a hypodermic needle). At least one active ingredient in the composition is trastuzumab-MCC-DM1. The label or packaging insert indicates that the composition is used to treat a selected condition, such as cancer. In one embodiment, the label or packaging insert indicates that the composition containing trastuzumab-MCC-DM1 can be used to treat conditions arising from abnormal cell growth. The label or packaging insert may also indicate that the composition can be used to treat other conditions. Alternatively/additionally, the article may further include a second container containing pharmaceutically acceptable buffers, such as bactericidal water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

The kit may further include instructions on administering trastuzumab-MCC-DM1 and (if present) a second drug formulation. For example, if the kit includes a first composition and a second drug formulation containing trastuzumab-MCC-DM1, then the kit may further include instructions on administering the first and second drug compositions simultaneously, sequentially, or separately to patients in need.

In another embodiment, the kit is suitable for delivering a solid oral form of trastuzumab-MCC-DM1, such as tablets or capsules. Such kits preferably comprise multiple unit doses. These kits may include cards in which the doses are positioned in the order they are intended to be used. An example of such a kit is a "blister pack." Blister packs are well-known in the packaging industry and are widely used for packaging pharmaceutical dosage units. If desired, memory aids, such as numbers, letters, other markings, or calendar inserts, may be provided to indicate the days for dose administration in a treatment schedule.

According to one embodiment, the kit may include (a) a first container containing trastuzumab-MCC-DM1; and optionally (b) a second container containing a second pharmaceutical formulation, wherein the second pharmaceutical formulation comprises a second compound having anti-proliferative activity. Alternatively/additionally, the kit may further include a third container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

If the kit contains a combination of trastuzumab-MCC-DM1 and a second therapeutic agent, i.e., a chemotherapy agent, the kit may include multiple containers to hold the separate compositions, such as separate vials or separate foil packets; however, the separate compositions may also be contained in a single, non-separated container. Typically, the kit includes instructions on the administration of the separate components. This kit format is particularly advantageous when different components are preferably administered in different dosage forms (e.g., oral and parenteral), at different dose intervals, or when the prescribing physician wishes to adjust the dosage of the components in the combination.

Example

The following embodiments are provided to illustrate the present invention. However, it should be understood that these embodiments are not intended to limit the present invention, but are merely intended to illustrate methods of carrying out the present invention.

Example 1 : Preparation of trastuzumab-MCC-DM1

Self-purified trastuzumab was prepared by buffer exchange at 20 mg/mL in 50 mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5, followed by treatment with 7.5 to 10 molar equivalents of succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid ester (SMCC, Pierce Biotechnology, Inc.) in 20 mM DMSO or DMA (dimethylacetamide), 6.7 mg/mL (US 2005/0169933; US 2005/0276812). After stirring at ambient temperature under argon for 2–4 hours, the reaction mixture was filtered through a Sephadex G25 column equilibrated with 50 mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5. Alternatively, the reaction mixture was gel filtered with 30 mM citrate and 150 mM sodium chloride at pH 6. The fractions containing antibodies were pooled and determined. The recovery rate of trastuzumab-SMCC was 88%.

The drug-connector intermediate trastuzumab-MCC from above was diluted to a final concentration of 10 mg/mL with 50 mM potassium phosphate/50 mM sodium chloride/2 mM EDTA at pH 6.5 and reacted with DM1 (1.7 equivalents, assuming 5 SMCC/trastuzumab, 7.37 mg/mL) in a 10 mM solution of dimethylacetamide. DM1 can be prepared from anisole fermentation products (US 6790954; US7432088) and derivatized for conjugation (US 6333410; RE 39151). The reaction was stirred at ambient temperature under argon for 4 to approximately 16 hours. The conjugation reaction mixture was filtered through a Sephadex G25 gel filter column (1.5 x 4.9 cm) using 1x PBS at pH 6.5. Alternatively, the reaction mixture was filtered through a gel filter using 10 mM succinate and 150 mM sodium chloride at pH 5. The DM1/trastuzumab ratio (p) was 3.1, as measured by absorbance at 252 nm and 280 nm. The drug-to-antibody ratio (p) could also be measured by mass spectrometry. Conjugation could also be monitored by SDS-PAGE. Aggregation could be assessed by laser scattering analysis.

Alternatively, trastuzumab-MCC-DM1 can be prepared by forming an MCC-DM1 linker-drug reagent and then reacting it with trastuzumab.

Typically, the conjugation reaction of trastuzumab-MCC with DM1 results in a heterogeneous mixture containing antibodies with varying numbers of DM1 drugs attached and conjugated (i.e., drug loads), where p is a distribution from 1 to approximately 8. Another scale of heterogeneity exists in the different attachment sites of SMCC to trastuzumab, where many different nucleophiles on trastuzumab (e.g., terminal lysine amino groups) can react with SMCC. Thus, trastuzumab-MCC-DM1 comprises isolated, purified speciation molecules and a mixture with an average drug load of 1 to 8, wherein MCC-DM1 is attached at any site of the trastuzumab antibody.

The average number of DM1 drug moieties for each trastuzumab antibody in the trastuzumab-MCC-DM1 preparation from the conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assays, electrophoresis, and HPLC. The quantitative distribution of trastuzumab-MCC-DM1 in terms of p can also be determined. The average p value in a specific ADC preparation can be determined by ELISA (Hamblett et al. (2004) Clinical Cancer Res. 10: 7063-7070; Sanderson et al. (2005) Clinical Cancer Res. 11: 843-852). However, the detection limits of antibody-antigen binding and ELISA cannot distinguish the distribution of p (drug) values. Similarly, ELISA assays used to detect antibody-drug conjugates cannot determine where the drug moieties attach to the antibody, such as heavy or light chain fragments, or specific amino acid residues. In some cases, the separation, purification, and characterization of homogeneous trastuzumab-MCC-DM1 with a certain p value from trastuzumab-MCC-DM1 with other drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis.

Example 2 : In vitro cell proliferation assay

The efficacy of the combination of the present invention was measured using a cell proliferation assay according to the following protocol (Promega Corp. Technical Bulletin TB288; Mendoza et al. (2002) Cancer Res. 62: 5485-5488). The Cell-Titer Glo assay reagents and protocol are commercially available (Promega). The assay assesses the ability of compounds to enter cells and affect cell proliferation. The principle of the assay is to determine the number of viable cells present by quantifying cellular ATP. Cell-Titer Glo refers to the reagent used for this quantification. It is a homogeneous assay in which the addition of Cell-Titer Glo causes cell lysis and the generation of a luminescent signal via a luciferase reaction. The luminescent signal is proportional to the amount of ATP present.

DMSO and culture medium plates: 96-well conical bottom polypropylene plates (Nunc, catalog number 249946)

Cell plate: 384-well black plate with cap, transparent bottom (microscopically transparent), TC plate (Falcon, 353962).

Cell culture medium: RPMI or DMEM high glucose; Ham's F-12 (50:50), 10% fetal bovine serum, 2 ml glutamine

CellTiter-Glo: Promega (Catalogue Number G7572)

Procedure:

Day 1 – Inoculate cells into cell plates and harvest cells. Inoculate 1000-2000 cells per well (54 μl per well) into a 384-well cell plate and perform assays over 3 days. Incubate overnight (approximately 16 hours) at 37°C with 5% _CO2 .

Day 2 – Add drugs to cells, dilute compounds, and dispense DMSO (serial 1:2, 9 spots). Add 20 μL of the compound (10 mM stock solution for small molecule drugs) to column 2 of a 96-well plate. Perform a serial 1:2 (10 μL + 10 μL 100% DMSO) dispensing between plates using Precision plates, for a total of 9 spots (1:50 dilution). Add 147 μL of culture medium to all wells of each 96-well plate. Transfer 3 μL of DMSO + compound from each well in the DMSO plate to the corresponding well in the culture medium plate using a Rapidplate. For two-drug combination studies, transfer 1.5 μL of one drug (DMSO + compound) from each well in the DMSO plate to the corresponding well in the culture medium plate using a Rapidplate. Then, transfer 1.5 μL of the other drug to the culture medium plate.

The drug was added to the cells, and the cells were partitioned (1:10 dilution). 6 μl of culture medium + compound was added directly to the cells (the cells already had 54 μl of culture medium on them). The cells were incubated at 37°C and 5% CO2 for 3 days in an incubator that is not frequently opened.

Day 5 – Develop the plate and thaw the CellTiterGlo buffer at room temperature. Remove the cell plate from 37°C and equilibrate to room temperature for approximately 30 minutes. Add CellTiterGlo buffer to CellTiterGlo substrate (vial to vial). Add 30 μl of CellTiterGlo reagent to each well. Incubate on a shaker for approximately 30 minutes. Read the luminescence using a PerkinElmer Envision (0.1 seconds per well) or an Analyst HT plate reader (30 seconds per well).

Cell viability assays and combination assays: Cells were seeded at 1000-2000 cells/well in 384-well plates for 16 hours. The following day, nine consecutive 1:2 compound dilutions were performed in DMSO in 96-well plates. The compound was further diluted in growth medium using a Rapidplate robot (Zymark Corp., Hopkinton, MA). The diluted compound was then added to four wells of the 384-well cell plate and incubated at 37°C and 5% CO2. After 4 days, the relative number of surviving cells was measured by luminescence using a Cell-Titer Glo (Promega) according to the manufacturer's instructions, and read on an Envision or Wallac multi-label reader (PerkinElmer, Foster City). EC50 values were calculated using Kaleidagraph 4.0 (Synergy Software) or Prism 4.0 software (GraphPad, San Diego). In combination assays, the drug was administered at an initial dose of 8 x EC50 concentration. In cases where the EC50 of the drug is >2.5 μM, the highest concentration used is 10 μM. In all assays, trastuzumab-MCC-DM1 and the chemotherapy agent were added simultaneously or at 4-hour intervals (one before and one after).

Another illustrative in vitro cell proliferation assay includes the following steps:

1. In each well of a 384-well, opaque-walled plate, a 100 μl aliquot of cell culture containing approximately ^10⁴ cells was placed (cell lines and tumor types are shown in Figure 1).

2. Prepare control wells containing culture medium but without cells.

3. Add the compound to the experimental wells and incubate for 3-5 days.

4. Allow the plate to equilibrate to room temperature for approximately 30 minutes.

5. Add an equal volume of CellTiter-Glo reagent to the volume of cell culture medium present in each well.

6. Mix the contents on a fixed-track shaker for 2 minutes to induce cell lysis.

7. Incubate the plate at room temperature for 10 minutes to stabilize the luminescence signal.

8. Record and report luminescence as RLU = relative luminescence unit.

Alternatively, cells were seeded at the optimal density in 96-well plates and incubated for 4 days in the presence of the test compound. Alamar Blue ^™ was then added to the assay medium, and the cells were incubated for 6 hours, followed by excitation at 544 nm and emission readings at 590 nm. _EC50 values were calculated using sigmoid dose-response curve fitting.

Example 3 : In Vitro Tumor Xenografts

Animals suitable for transgenic experiments were available from standard commercial sources. Multiple groups of female CB-17SCID beige mice (Charles River Laboratory) were implanted with 3 million KPL-4 (Her2 overexpressing) breast cancer cells into the mammary fat pad using Matrigel. Multiple groups of female athymic nude mice (Charles River Laboratory or Harlan) were implanted with 2x2 ^mm fragments of MMTV-Her2Fo5 transgenic breast tumors into the mammary fat pad. Mouse xenografts were administered drugs, drug combinations, or mediators on day 0 according to the schedule prescribed for each tumor model. 5-FU, gemcitabine, carboplatin, and B20-4.1 were administered intraperitoneally; pertuzumab was administered intravenously or intraperitoneally as directed; trastuzumab-MCC-DM1 and docetaxel were administered intravenously; and lapatinib, GDC-0941, and ABT-869 were administered orally via force-feeding. Tumor size was recorded twice weekly during the study. Mouse weight was also recorded twice weekly, and mice were observed periodically. Tumor volume was measured in two dimensions (length and width) using an Ultra Cal IV caliper (model 54-10-111; Fred V. Fowler Co., Inc.; Newton, MA) and analyzed using Excel v.11.2 (Microsoft Corporation; Redmond, WA). Tumor suppression maps were plotted using KaleidaGraph, version 3.6 (Synergy Software; Reading, PA). Tumor volume was calculated using the following formula: Tumor size ( ^mm³ ) = (longer measurement x ^shorter measurement²) x 0.5

Animal weight was measured using an Adventurera Pro AV812 balance (Ohaus Corporation; Pine Brook, NJ). Plotting was performed using KaleidaGraph version 3.6. Percentage weight change was calculated using the following formula: Group percentage weight change = (1 - (initial weight / new weight)) x 100.

Mice with tumors exceeding 2000 ^mm³ or whose weight loss exceeds 20% of their initial weight should be euthanized immediately in accordance with management guidelines.

The percentage tumor growth delay (%TGD) at the end of the study (EOS) is calculated using the following formula: %TGD = 100 x (median time to end in the treatment group – median time to end in the control group) / median time to end in the control group.

Tumor incidence (TI) was determined based on the number of measurable tumors remaining in each group at the end of the study. Partial response (PR) was defined as a reduction in tumor volume of more than 50% but less than 100% compared to the initial tumor volume observed in three consecutive measurements. Complete response (CR) was defined as a reduction in tumor volume of 100% compared to the initial tumor volume observed in three consecutive measurements. Data were analyzed using Dunnett's t-test and p-values were determined using JMP statistical software, version 5.1.2 (SAS Institute; Cary, NC). Tumor volumes and mean ± SEM values of tumor volumes at the end of the study were calculated using JMP statistical software, version 5.1.2. Weight data were plotted based on the mean ± SEM percentage change relative to initial body weight.

Example 4 : Clinical study of trastuzumab-MCC-DM1 (T-DM1) in combination with pertuzumab

A phase 1b/II, open-label study was designed to determine the safety, tolerability, and efficacy of intravenous administration of trastuzumab-MCC-DM1 (T-DM1) in combination with pertuzumab in patients with HER2-positive locally advanced or metastatic breast cancer who have progressed on prior therapy. The combination was administered every 3 weeks to patients with HER2-positive locally advanced or metastatic breast cancer who had previously received trastuzumab in any line of therapy, had received chemotherapy combined with HER2-targeted therapy for advanced disease, or had progressed on their most recent therapy. Another component was to evaluate the pharmacokinetics of T-DM1 when administered in combination with pertuzumab according to this schedule. A further component was to conduct a preliminary assessment of the efficacy of the T-DM1 and pertuzumab combination according to this schedule, such as using the Modified Response Evaluation Criteria in Solid Tumors (RECIST), version 1.0, based on investigator evaluations measured by objective response rates. The secondary objectives of this study were as follows: (1) to evaluate progression-free survival (PFS) in patients receiving the combination of T-DM1 and pertuzumab administered according to this schedule; (2) to evaluate the duration of response to the combination of T-DM1 and pertuzumab administered according to this schedule; and (3) to evaluate the development of anti-therapeutic antibodies against T-DM1.

In patients with HER2-positive locally advanced or metastatic breast cancer who have previously received trastuzumab and whose disease has progressed after or at the time of their last therapy, pertuzumab, also administered intravenously (IV), is combined with T-DM1 administered intravenously (IV). Patients receive the T-DM1 and pertuzumab combination in repeated cycles at minimum 3-week intervals.

Before treating any patient at a higher dose level after receiving their first dose of the study drug, DLT (dose-limiting toxicity) will be observed in patients at a given dose level during the DLT observation period (defined as 21 days from the first dose of T-DM1). If no DLT is observed in these patients during the DLT observation period, a dose increase to the next dose level may be performed.

DLT is defined as any of the following treatment-related toxicities occurring during the DLT observation period: (1) a non-hematologic adverse event of grade ≥3 not caused by disease progression or other clearly identifiable cause, except for alopecia of any grade; (2) grade 3 diarrhea in response to standard care therapy; (3) grade 3 nausea or vomiting in response to standard care therapy without prior administration of medication under anesthesia; (4) a grade ≥3 elevation of serum bilirubin, liver transaminases (ALT or AST), or alkaline phosphatase (ALP) lasting 72 hours due to liver or bone metastases, except in patients with a grade 2 liver transaminase or ALP level at baseline (≤5 upper limit of normal [ULN]). (5) A liver transaminase or ALP level ≥10 ULN is considered DLT; (6) Grade 4 thrombocytopenia lasting 24 hours; (7) Grade 4 neutropenia lasting 4 days or accompanied by fever (oral or tympanic membrane temperature 100.4°F or 38°C) (absolute neutrophil count <500/cell/ ^mm3 ); (8) any subjective intolerable toxicity felt by the investigator in connection with any of the test compounds; (9) any treatment-related toxicity that precludes the initiation of a second round of treatment.

Once a decision is made to proceed to the next maximum dose level, intra-patient dose escalation will be permitted; enrolled patients in the study will initially receive a reduced dose of T-DM1 (3.0 mg/kg) and a full dose of pertuzumab. Once these patients' peers have completed the DLT observation period, they will be permitted to escalate to full doses of both drugs in subsequent cycles. However, the safety of the 3.6 mg/kg dose level will be assessed based on DLT. If patients (including those enrolled during the dose escalation phase of the study) remain in the study after the first follow-up tumor assessment, they will be considered evaluable for efficacy. Echocardiography (ECHO) or multigate detection (MUGA) scans should be performed at the end of cycle 1 and then every 3 cycles throughout the treatment period.

T-DM1 formulation

T-DM1 is available as a single-use lyophilized preparation in 20-mL Type I USP/European Pharmacopoeia glass vials with a 20-mm fluoropolymer laminated stopper, an aluminum seal, and a dark gray flip-off plastic cap. After reconstitution with 8.0 mL of sterile water for injection (SWFI), the resulting product contains 20 mg/mL T-DM1 in 10 mM sodium succinate, pH 5.0, 6% (w/v) sucrose, and 0.02% (w/v) polysorbate 20. Each 20-mL vial contains approximately 172 mg of T-DM1 to allow for the delivery of 160 mg of T-DM1. Draw the specified volume of T-DM1 solution from the vial and add it to an IV bag. Dilute the reconstituted T-DM1 into a PVC or latex-free, PVC-free polyolefin (PO) bag containing 0.45% or 0.9% sodium chloride (minimum volume 250 mL). Preferred containers are PVC or PO bags containing 0.45% sodium chloride. When using PVC or PO bags containing 0.9% sodium chloride, a 0.22 μm embedded filter is recommended. Gently invert the bag to mix the solution. T-DM1 solution diluted for infusion in USP PVC or latex-free PVC-free polyolefin (PO) bags containing 0.9% or 0.45% sodium chloride injection can be stored for short periods at 2°C–8°C (36°F–46°F).

pertuzumab formulations

Pertuzumab is supplied as a single-use formulation containing 30 mg/mL pertuzumab prepared in 20 mM L-histidine (pH 6.0), 120 mM sucrose, and 0.02% polysorbate 20. Each 20-cc vial contains approximately 420 mg of pertuzumab (14.0 mL/via). Dispense the specified volume of pertuzumab solution from the vial and add it to a 250-cc IV bag containing 0.9% sodium chloride solution for injection. Gently invert the bag to mix the solution and visually inspect for particles and discoloration before administration. Pertuzumab solution for infusion diluted in a polyethylene or non-PVC polyolefin bag containing 0.9% sodium chloride solution can be stored for short periods at 2°C–8°C (36°F–46°F).

Security outcome measurement

The following primary safety outcome measures will be used to assess the safety and tolerability of T-DM1 and pertuzumab: (1) the incidence, nature, and severity of adverse events; (2) adverse events or changes in physical signs and clinical laboratory results that result in dose changes, delays, or interruptions of T-DM1 and/or pertuzumab during and after administration of the study drug; and (3) changes in cardiac function (i.e., left ventricular ejection fraction [LVEF], segmental wall abnormalities), including changes in ECHO or MUGA scans.

Pharmacokinetic and pharmacodynamic results measurement

Where appropriate and where data permit, the following pharmacokinetic parameters of T-DM1 and pertuzumab will be determined in all patients receiving study treatment using non-compartmental and/or population methods: (1) serum concentrations of T-DM1 (conjugate) and total trastuzumab (free and conjugated to DM1); (2) plasma concentrations of free DM1; (3) total exposure (area under concentration-time curve [AUC]); (4) maximum serum concentration (Cmax); (5) minimum concentration (Cmin); (6) clearance; (7) volume of distribution; (8) terminal half-life; and (9) anti-therapeutic antibody against T-DM1.

Efficacy Result Measurement

The objective response rate using modified RECIST, v1.0 will be evaluated as a measure of efficacy outcome. The secondary efficacy outcome measures for this study are as follows: (1) PFS, defined as the time from the initiation of study treatment to the first occurrence of disease progression or death from any cause in the study (within 30 days after the last dose of study treatment), as determined by the investigators in review of the tumor assessment using modified RECIST, v1.0; and (2) Duration of response, defined as the time from the first recorded objective response to disease progression, as determined by the investigators in review of the tumor assessment using modified RECIST (v1.0), or death from any cause in the study (within 30 days after the last dose of study treatment).

Research on treatment

T-DM1 will be administered intravenously at a dose of 2.4, 3.0, or 3.6 mg/kg at a frequency not exceeding every 3 weeks. The T-DM1 dose may be gradually reduced in any patient, down to 2.4 mg/kg. Based on toxicity experienced in patients treated at 3.0 mg/kg, and if 3.0 mg/kg T-DM1 is proven to be tolerable, the dose may be increased to 3.6 mg/kg IV every 3 weeks in subsequent cycles. Pertuzumab will be administered intravenously at a loading dose of 840 mg on day 1 of cycle 1, followed by 420 mg IV every 3 weeks in subsequent cycles.

Statistical methods

The primary efficacy endpoint of this study was investigator-assessed objective response, defined as a complete or partial response determined at two consecutive time points ≥4 weeks apart. An estimated objective response rate and corresponding 95% confidence intervals were calculated. For objective response, patients without effective post-baseline tumor assessment were counted as non-responders. For duration of response and PFS, data from patients not followed up were processed as follows: reviewed on the last day of known progression-free status. Data from patients without post-treatment tumor assessment or who died were reviewed on day 1 of treatment initiation.

Example 5 : Clinical study of trastuzumab-MCC-DM1 (T-DM1) in combination with GDC-0941

This phase Ib, open-label study was designed to determine the safety, tolerability, pharmacokinetics, and activity of a combination of intravenous T-DM1 and oral GDC-0941 in patients with HER2-positive metastatic breast cancer who have progressed on prior trastuzumab-based therapy. The primary objectives of this study were: to evaluate the safety and tolerability of GDC-0941 administered in combination with T-DM1; to evaluate the median dose (MTD) of GDC-0941 when administered in combination with T-DM1; to identify a recommended phase II dose of GDC-0941 for combined administration with T-DM1; and to characterize any observed antitumor activity of GDC-0941 when administered in combination with T-DM1. The pharmacokinetic objectives were: to determine the pharmacokinetics of GDC-0941 in the absence and presence of T-DM1; and to determine the pharmacokinetics of T-DM1 in the absence and presence of GDC-0941.

GDC-0941 formulation

GDC-0941 is a dry powder intended for PO (potentially purported) administration. The formulated drug product is provided in two strengths (15 mg and 50 mg) of hard gelatin capsules, which are in No. 0 shells, powdered, and color-coded. The excipients included in the capsule formulation are microcrystalline cellulose NF/EP, sodium lauryl sulfate NF/DP (only in the 50 mg strength), anhydrous citrate USP/EP, croscarmellose sodium NF/EP, colloidal silica NF/EP, and magnesium stearate (non-bovine) NF/EP. GDC-0941 capsules should be stored at a refrigerated temperature between 36°F and 46°F (2°C and 8°C). Patients will be instructed to store the investigational drug at a refrigerated temperature between 36°F and 46°F (2°C and 8°C).

Outcome metrics

The results measures for safety, pharmacokinetics, pharmacodynamics, and efficacy will be determined and evaluated, including statistical methods such as those described in Example 4.

Research on treatment

The study treatment will be administered in 3-week cycles. Depending on the developmental status, drug availability, and other factors, patients who experience clinical benefit from the study treatment may be eligible for more cycles, which can occur in separate studies.

During the dose expansion phase of the study, enrolled patients received a single dose of GDC-0941 on an empty stomach on Day 1 of Cycle 1 to allow for the collection of pre- and post-administration GDC-0941 pharmacokinetic (PK) samples and to observe intra-patient variability. The starting dose of GDC-0941 was 60 mg once daily, a dose that had been established as safe as a single agent in a Phase I study with no dose-limiting toxicities. On Day 2 of Cycle 1, a full dose of T-DM1 was administered intravenously at 3.6 mg/kg IV over 90 minutes without a loading dose. This was followed by a dose of GDC-0941. Patients were observed for 90 minutes after the first T-DM1 infusion. GDC-0941 was then administered once daily for a total of 14 doses, followed by a one-week break, constituting Cycle 1.

In subsequent patients, dose escalation of GDC-0941 will continue until development or intolerance occurs. The subsequent study will involve a 3-week treatment cycle, initially with an IV infusion of 3.6 mg/kg T-DM1 over 30 minutes on day 1 of each cycle, followed by GDC-0941, for a total of 2 weeks of treatment followed by a 1-week break. Treatment will continue until development or intolerance occurs. Based on the tolerability of T-DM1 in the original study, T-DM1 will be administered as an IV infusion over 30–90 minutes (±10 minutes). If the 90-minute infusion is well tolerated, subsequent infusions can be delivered over 30 (±10) minutes.

The above description is considered merely an illustration of the principles of the invention. Furthermore, since numerous modifications and variations will be apparent to those skilled in the art, it is not intended to limit the invention to the precise construction and process described above. Therefore, all suitable modifications and equivalents are considered to fall within the scope of the invention as defined by the appended claims.

Claims (12)

1. Use of a therapeutic agent combination in the manufacture of a medicament for treating cancers expressing ErbB2, wherein the therapeutic agent combination is administered to a mammal having said ErbB2-expressing cancer in the form of a combination formulation or alternately, and said combination comprises a therapeutically effective amount of trastuzumab-MCC-DM1 and a therapeutically effective amount of a 5-FU chemotherapeutic agent, wherein administration of said therapeutic agent combination results in at least an additive effect compared to administration of trastuzumab-MCC-DM1 or 5-FU as a single agent. 2. The use of claim 1, wherein the therapeutically effective amount of trastuzumab-MCC-DM1 and the therapeutically effective amount of the chemotherapeutic agent are administered in a combination formulation. 3. The use of claim 1, wherein the therapeutically effective amount of trastuzumab-MCC-DM1 and the therapeutically effective amount of the chemotherapeutic agent are administered alternately. 4. The use according to any one of claims 1-3, wherein trastuzumab-MCC-DM1 is administered to mammals with ErbB2-expressing cancer at intervals of 1 to 3 weeks. 5. The use according to any one of claims 1-4, wherein the cancer is breast cancer. 6. The use according to any one of claims 1-5, wherein the amount of trastuzumab-MCC-DM1 and the amount of the chemotherapeutic agent are each from 1 mg to 1000 mg. 7. The use according to any one of claims 1-6, wherein the weight ratio of the amount of trastuzumab-MCC-DM1 to the amount of the chemotherapeutic agent is 1:10 to 10:1. 8. The use according to any one of claims 1-7, wherein the mammal is a HER2-positive patient. 9. A pharmaceutical composition comprising trastuzumab-MCC-DM1, a 5-FU chemotherapeutic agent, and one or more pharmaceutically acceptable carriers, gliding agents, diluents, or excipients. 10. The pharmaceutical composition of claim 9, wherein the amount of trastuzumab-MCC-DM1 and the amount of the chemotherapeutic agent are each present in amounts from 1 mg to 1000 mg. 11. The pharmaceutical composition of claim 9 or 10, wherein the amount of trastuzumab-MCC-DM1 and the amount of the chemotherapeutic agent are present in a weight ratio of 1:10 to 10:1. 12. Use of a therapeutic agent combination in the manufacture of a medicament for treating breast cancer, wherein the therapeutic agent combination is administered to a mammal in the form of a combination formulation or alternately, and said combination comprises a therapeutically effective amount of trastuzumab-MCC-DM1 and a therapeutically effective amount of a 5-FU chemotherapeutic agent, wherein administration of said therapeutic agent combination results in at least an additive effect compared to administration of trastuzumab-MCC-DM1 or 5-FU as a single agent.