U.S. patent application number 16/952670 was filed with the patent office on 2021-03-25 for subcutaneous her2 antibody formulations.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc., Hoffmann-La Roche Inc.. Invention is credited to Sreedhara Alavattam, Tanja Badovinac-Crnjevic, Jennifer Eng-Wong, Amit Garg, Sarah Heeson, Tarik Ali Khan, Whitney Kirschbrown, Jasper Lin, Christine Wurth.
Application Number | 20210085597 16/952670 |
Document ID | / |
Family ID | 1000005222896 |
Filed Date | 2021-03-25 |
View All Diagrams
United States Patent
Application |
20210085597 |
Kind Code |
A1 |
Eng-Wong; Jennifer ; et
al. |
March 25, 2021 |
SUBCUTANEOUS HER2 ANTIBODY FORMULATIONS
Abstract
Fixed dose HER2 antibody formulations for subcutaneous
administration are provided along with their use in the treatment
of cancer. The formulations include fixed dose subcutaneous
formulations of pertuzumab and subcutaneous co-formulations of
pertuzumab and trastuzumab, and their use in the treatment of
cancer.
Inventors: |
Eng-Wong; Jennifer; (South
San Francisco, CA) ; Kirschbrown; Whitney; (South San
Francisco, CA) ; Khan; Tarik Ali; (Basel, CH)
; Lin; Jasper; (South San Francisco, CA) ;
Alavattam; Sreedhara; (South San Francisco, CA) ;
Garg; Amit; (South San Francisco, CA) ; Heeson;
Sarah; (Welwyn Garden City, GB) ; Badovinac-Crnjevic;
Tanja; (Basel, CH) ; Wurth; Christine; (Basel,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc.
Hoffmann-La Roche Inc. |
South San Francisco
Little Falls |
CA
NJ |
US
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
1000005222896 |
Appl. No.: |
16/952670 |
Filed: |
November 19, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15872648 |
Jan 16, 2018 |
10849849 |
|
|
16952670 |
|
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62447359 |
Jan 17, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/56 20130101;
A61K 2039/505 20130101; A61K 47/183 20130101; A61K 38/47 20130101;
A61P 35/00 20180101; C07K 2317/24 20130101; C07K 16/28 20130101;
A61K 2039/54 20130101; A61K 47/20 20130101; A61K 47/26 20130101;
A61K 39/001106 20180801; A61K 9/08 20130101; C07K 2317/90 20130101;
A61K 2039/507 20130101; C07K 16/32 20130101; A61K 2039/545
20130101; A61K 39/39591 20130101; A61K 2039/585 20130101; C07K
16/30 20130101; A61K 9/0019 20130101; C12Y 302/01035 20130101 |
International
Class: |
A61K 9/00 20060101
A61K009/00; C07K 16/32 20060101 C07K016/32; A61K 47/18 20060101
A61K047/18; A61K 47/26 20060101 A61K047/26; A61K 39/395 20060101
A61K039/395; A61K 47/20 20060101 A61K047/20; A61K 39/00 20060101
A61K039/00; A61P 35/00 20060101 A61P035/00; A61K 9/08 20060101
A61K009/08; A61K 38/47 20060101 A61K038/47 |
Claims
1. An article of manufacture containing a single fixed dose of a
HER2 antibody comprising the variable light chain and variable
heavy chain amino acid sequences of SEQ ID Nos. 7 and 8,
respectively, wherein the fixed dose is about 600 mg or about 1200
mg.
2. The article of manufacture of claim 1, which is a single-use
vial or a syringe.
3. The article of manufacture of claim 1, wherein the HER2 antibody
is pertuzumab.
4. The article of manufacture of claim 1, comprising two single
dose vials, wherein a first vial contains a single fixed dose of
about 1200 mg of pertuzumab, and a second vial contains a single
fixed dose of about 600 mg of pertuzumab.
5. The article of manufacture of claim 1, further containing a
single fixed dose of trastuzumab.
6. The article of manufacture of claim 5, wherein the single fixed
dose of pertuzumab and the single fixed dose of trastuzumab are
contained in a single liquid formulation for subcutaneous
administration.
7. The article of manufacture of claim 6, wherein the liquid
formulation comprises a single fixed dose of about 600 mg of
pertuzumab and a single fixed dose of about 600 mg of
trastuzumab.
8. The article of manufacture of claim 6, wherein the liquid
formulation comprises a single fixed dose of about 1200 mg of
pertuzumab and a single fixed dose of about 600 mg of
trastuzumab.
9. The article of manufacture of claim 6, wherein the liquid
formulation further comprises a hyaluronidase enzyme.
10. The article of manufacture of claim 9, wherein the
hyaluronidase enzyme is recombinant human hyaluronidase PH20 enzyme
(rHuPH20).
11. The article of manufacture of claim 10, wherein the rHuPH20 is
present in the liquid formulation in an amount sufficient to result
in an increase in the dispersion of the pertuzumab and trastuzumab
contained in the same liquid formulation during subcutaneous
administration.
12. The article of manufacture of claim 11, wherein the rHuPH20 is
present in the liquid formulation at a concentration of at least
about 600 U/mL.
13. The article of manufacture of claim 12, wherein the rHuPH20 is
present in the liquid formulation at a concentration of about 1,000
U/mL or about 2,000 U/mL.
14. The article of manufacture of claim 5, further comprising a
package insert instructing the user to administer the fixed doses
of pertuzumab and trastuzumab subcutaneously to a patient with HER2
positive cancer.
15. The article of manufacture of claim 14, wherein the package
insert instructs the user to co-administer the fixed dose
pertuzumab and the fixed dose of trastuzumab subcutaneously as two
separate subcutaneous injections.
16. The article of manufacture of claim 14, wherein the package
insert instructs the user to administer the fixed dose pertuzumab
co-mixed with the fixed-dose trastuzumab as a single subcutaneous
injection.
17. The article of manufacture of any one of claim 14, wherein the
package insert instructs the user to administer a fixed dose
combination of pertuzumab and trastuzumab as a single subcutaneous
injection.
18. The article of manufacture of claim 14, wherein the HER2
positive cancer is selected from the group consisting of breast
cancer, peritoneal cancer, fallopian tube cancer, lung cancer,
colorectal cancer, biliary cancer and bladder cancer.
19. The article of manufacture of claim 14, wherein the HER2
positive cancer is early breast cancer (EBC) or metastatic breast
cancer (MBC).
20. An aqueous formulation for subcutaneous administration
comprising pertuzumab at a concentration of about 120 mg/mL,
recombinant human hyaluronidase PH20 enzyme (rHuPH20) at a
concentration of about 1000 to 2000 U/mL, a histidine buffer to
adjust to pH to about 5.5 to 5.7, sucrose, methionine, and
polysorbate 20.
21. The aqueous formulation of claim 20, wherein the rHuPH20 is
present at a concentration of about 1000 U/mL or 2000 U/mL.
22. A liquid subcutaneous pharmaceutical composition comprising a
fixed dose of pertuzumab and a fixed dose of trastuzumab
co-formulated in an aqueous solution further comprising recombinant
human hyaluronidase PH20 enzyme (rHuPH20), a buffering agent
suitable to adjust the pH to about 5.0 to 6.0, a stabilizer, and a
surfactant.
23. The liquid subcutaneous pharmaceutical composition of claim 22,
wherein the buffering agent is a histidine buffer.
24. The liquid subcutaneous pharmaceutical composition of claim 23,
wherein the buffering agent is histidine acetate.
25. The liquid subcutaneous pharmaceutical composition of claim 22,
wherein the pH is about 5.5 to 5.7.
26. The liquid pharmaceutical composition of claim 22, comprising
sucrose as a stabilizer, methionine as a stabilizer, trehalose as a
stabilizer, and polysorbate 20 as a surfactant.
27. A liquid pharmaceutical composition comprising 600 mg
pertuzumab at a concentration of 60 mg/mL, 600 mg trastuzumab at a
concentration of 60 mg/mL, 1,000 U/mL or 2,000 U/mL recombinant
human hyaluronidase PH20 enzyme (rHuPH20), 20 mM Histidine-HCl pH
5.5, 105 mM trehalose, 100 mM sucrose, 0.04% polysorbate 20, 10 mM
methionine, and sterile water for injection up to a total volume of
10 mL.
28. The liquid pharmaceutical composition of claim 27, contained in
a 15 mL vial.
29. A liquid pharmaceutical composition comprising 1200 mg
pertuzumab at a concentration of 80 mg/mL, 600 mg trastuzumab at a
concentration of 40 mg/mL, 1,000 U/mL or 2,000 U/mL recombinant
human hyaluronidase PH20 enzyme (rHuPH20), 20 mM Histidine-HCl pH
5.5, 70 mM trehalose, 133 mM sucrose, 0.04% polysorbate 20, 10 mM
methionine, and sterile water for injection up to a total volume of
15 mL.
30. The liquid pharmaceutical composition of claim 29, contained in
a 20 mL vial.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
15/872,648, filed Jan. 16, 2018 which claims the benefit of
priority under 35 USC .sctn. 119(e) of provisional Application No.
62/447,359, filed Jan. 17, 2017, the full disclosure of which is
hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 18, 2020, is named P34027-US-2_SL.txt and is 31,993 bytes
in size.
FIELD OF THE INVENTION
[0003] The invention concerns fixed dose HER2 antibody formulations
for subcutaneous administration and their use in the treatment of
cancer. In particular, the invention concerns fixed dose pertuzumab
formulations, subcutaneous formulations comprising pertuzumab and
trastuzumab, and their use in the treatment of cancer.
BACKGROUND OF THE INVENTION
[0004] HER2 Antibodies
[0005] Members of the HER family of receptor tyrosine kinases are
important mediators of cell growth, differentiation and survival.
The receptor family includes four distinct members including
epidermal growth factor receptor (EGFR, ErbB 1, or HER1), HER2
(ErbB2 or p185.sup.new), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).
Members of the receptor family have been implicated in various
types of human malignancy.
[0006] A recombinant humanized version of the murine anti-HER2
antibody 4D5 (huMAb4D5-8, rhuMAb HER2, trastuzumab or
HERCEPTIN.RTM.; U.S. Pat. No. 5,821,337) is clinically active in
patients with HER2-overexpressing metastatic breast cancers that
have received extensive prior anti-cancer therapy (Baselga et al.,
J. Clin. Oncol. 14:737-744 (1996)).
[0007] Trastuzumab received marketing approval from the Food and
Drug Administration Sep. 25, 1998 for the treatment of patients
with metastatic breast cancer whose tumors overexpress the HER2
protein. At present, trastuzumab is approved for use as a single
agent or in combination with chemotherapy or hormone therapy in the
metastatic setting, and as single agent or in combination with
chemotherapy as adjuvant treatment for patients with early-stage
HER2-positive breast cancer. trastuzumab-based therapy is now the
recommended treatment for patients with HER2-positive early-stage
breast cancer who do not have contraindications for its use
(Herceptin.RTM. prescribing information; NCCN Guidelines, version
2.2011). Trastuzumab plus docetaxel (or paclitaxel) is a registered
standard of care in the first-line metastatic breast cancer (MBC)
treatment setting (Slamon et al. N Engl J Med. 2001;
344(11):783-792; Marty et al. J Clin Oncol. 2005;
23(19):4265-4274).
[0008] Patients treated with the HER2 antibody trastuzumab are
selected for therapy based on HER2 expression. See, for example,
WO99/31140 (Paton et al.), US2003/0170234A1 (Hellmann, S.), and
US2003/0147884 (Paton et al.); as well as WO01/89566,
US2002/0064785, and US2003/0134344 (Mass et al.). See, also, U.S.
Pat. Nos. 6,573,043, 6,905,830, and US2003/0152987, Cohen et al.,
concerning immunohistochemistry (IHC) and fluorescence in situ
hybridization (FISH) for detecting HER2 overexpression and
amplification. Thus, the optimal management of metastatic breast
cancer now takes into account not only a patient's general
condition, medical history, and receptor status, but also the HER2
status.
[0009] Pertuzumab (also known as recombinant humanized monoclonal
antibody 2C4 (rhuMAb 2C4); Genentech, Inc, South San Francisco)
represents the first in a new class of agents known as HER
dimerization inhibitors (HDI) and functions to inhibit the ability
of HER2 to form active heterodimers or homodimers with other HER
receptors (such as EGFR/HER1, HER2, HER3 and HER4). See, for
example, Harari and Yarden Oncogene 19:6102-14 (2000); Yarden and
Sliwkowski. Nat Rev Mol Cell Biol 2:127-37 (2001); Sliwkowski Nat
Struct Biol 10:158-9 (2003); Cho et al. Nature 421:756-60 (2003);
and Malik et al. Pro Am Soc Cancer Res 44:176-7 (2003).
[0010] Pertuzumab blockade of the formation of HER2-HER3
heterodimers in tumor cells has been demonstrated to inhibit
critical cell signaling, which results in reduced tumor
proliferation and survival (Agus et al. Cancer Cell 2:127-37
(2002)).
[0011] Pertuzumab has undergone testing as a single agent in the
clinic with a phase Ia trial in patients with advanced cancers and
phase II trials in patients with ovarian cancer and breast cancer
as well as lung and prostate cancer. In a Phase I study, patients
with incurable, locally advanced, recurrent or metastatic solid
tumors that had progressed during or after standard therapy were
treated with pertuzumab given intravenously every 3 weeks.
pertuzumab was generally well tolerated. Tumor regression was
achieved in 3 of 20 patients evaluable for response. Two patients
had confirmed partial responses. Stable disease lasting for more
than 2.5 months was observed in 6 of 21 patients (Agus et al. Pro
Am Soc Clin Oncol 22:192 (2003)). At doses of 2.0-15 mg/kg, the
pharmacokinetics of pertuzumab was linear, and mean clearance
ranged from 2.69 to 3.74 mL/day/kg and the mean terminal
elimination half-life ranged from 15.3 to 27.6 days. Antibodies to
pertuzumab were not detected (Allison et al. Pro Am Soc Clin Oncol
22:197 (2003)).
[0012] US 2006/0034842 describes methods for treating
ErbB-expressing cancer with anti-ErbB2 antibody combinations. US
2008/0102069 describes the use of trastuzumab and pertuzumab in the
treatment of HER2-positive metastatic cancer, such as breast
cancer. Baselga et al., J Clin Oncol, 2007 ASCO Annual Meeting
Proceedings Part I, Col. 25, No. 18S (June 20 Supplement),
2007:1004 report the treatment of patients with pre-treated
HER2-positive breast cancer, which has progressed during treatment
with trastuzumab, with a combination of trastuzumab and pertuzumab.
Portera et al., J Clin Oncol, 2007 ASCO Annual Meeting Proceedings
Part I. Vol. 25, No. 18S (June 20 Supplement), 2007:1028 evaluated
the efficacy and safety of trastuzumab+pertuzumab combination
therapy in HER2-positive breast cancer patients, who had
progressive disease on trastuzumab-based therapy. The authors
concluded that further evaluation of the efficacy of combination
treatment was required to define the overall risk and benefit of
this treatment regimen.
[0013] Pertuzumab has been evaluated in Phase II studies in
combination with trastuzumab in patients with HER2-positive
metastatic breast cancer who have previously received trastuzumab
for metastatic disease. One study, conducted by the National cancer
Institute (NCI), enrolled 11 patients with previously treated
HER2-positive metastatic breast cancer. Two out of the 11 patients
exhibited a partial response (PR) (Baselga et al., J Clin Oncol
2007 ASCO Annual Meeting Proceedings; 25:18S (June 20 Supplement):
1004).
[0014] The results of a Phase II neoadjuvant study evaluating the
effect of a novel combination regimen of pertuzumab and trastuzumab
plus chemotherapy (docetaxel) in women with early-stage
HER2-positive breast cancer, presented at the CTRC-AACR San Antonio
Breast Cancer Symposium (SABCS), Dec. 8-12, 2010, showed that the
two HER2 antibodies plus docetaxel given in the neoadjuvant setting
prior to surgery significantly improved the rate of complete tumor
disappearance (pathological complete response rate, pCR, of 45.8
percent) in the breast by more than half compared to trastuzumab
plus docetaxel (pCR of 29.0 percent), p=0.014.
[0015] The Clinical Evaluation of pertuzumab and trastuzumab
(CLEOPATRA) Phase II clinical study assessed the efficacy and
safety of pertuzumab plus trastuzumab plus docetaxel, as compared
with placebo plus trastuzumab plus docetaxel, as first-line
treatment for patients with locally recurrent, unresectable, or
metastatic HER2-positive breast cancer. The combination of
pertuzumab plus trastuzumab plus docetaxel, as compared with
placebo plus trastuzumab plus docetaxel, when used as first-line
treatment for HER2-positive metastatic breast cancer, significantly
prolonged progression-free survival, with no increase in cardiac
toxic effects. (Baselga et al., N Eng J Med 2012 366:2,
109-119).
[0016] The Phase II clinical study NeoSphere assessed the efficacy
and safety of neoadjuvant administration of pertuzumab and
trastuzumab in treatment-naive women (patients who has not received
any previous cancer therapy) with operable, locally advanced, and
inflammatory breast cancer. Patients give pertuzumab and
trastuzumab plus docetaxel showed a significantly improved
pathological complete response rate compared with those given
trastuzumab plus docetaxel, without substantial differences in
tolerability (Gianni et al., Lancet Oncol 2012 13(1):25-32).
Results of 5-year follow-up are reported by Gianni et al., Lancet
Oncol 2016 17(6):791-800).
[0017] Patent Publications related to HER2 antibodies include: U.S.
Pat. Nos. 5,677,171; 5,720,937; 5,720,954; 5,725,856; 5,770,195;
5,772,997; 6,165,464; 6,387,371; 6,399,063; 6,015,567; 6,333,169;
4,968,603; 5,821,337; 6,054,297; 6,407,213; 6,639,055; 6,719,971;
6,800,738; 5,648,237; 7,018,809; 6,267,958; 6,695,940; 6,821,515;
7,060,268; 7,682,609; 7,371,376; 6,127,526; 6,333,398; 6,797,814;
6,339,142; 6,417,335; 6,489,447; 7,074,404; 7,531,645; 7,846,441;
7,892,549; 6,573,043; 6,905,830; 7,129,840; 7,344,840; 7,468,252;
7,674,589; 6,949,245; 7,485,302; 7,498,030; 7,501,122; 7,537,931;
7,618,631; 7,862,817; 7,041,292; 6,627,196; 7,371,379; 6,632,979;
7,097,840; 7,575,748; 6,984,494; 7,279,287; 7,811,773; 7,993,834;
7,435,797; 7,850,966; 7,485,704; 7,807,799; 7,560,111; 7,879,325;
7,449,184; 7,700,299; and US 2010/0016556; US 2005/0244929; US
2001/0014326; US 2003/0202972; US 2006/0099201; US 2010/0158899; US
2011/0236383; US 2011/0033460; US 2005/0063972; US 2006/018739; US
2009/0220492; US 2003/0147884; US 2004/0037823; US 2005/0002928; US
2007/0292419; US 2008/0187533; US 2003/0152987; US 2005/0100944; US
2006/0183150; US2008/0050748; US 2010/0120053; US 2005/0244417; US
2007/0026001; US 2008/0160026; US 2008/0241146; US 2005/0208043; US
2005/0238640; US 2006/0034842; US 2006/0073143; US 2006/0193854; US
2006/0198843; US 2011/0129464; US 2007/0184055; US 2007/0269429; US
2008/0050373; US 2006/0083739; US 2009/0087432; US 2006/0210561; US
2002/0035736; US 2002/0001587; US 2008/0226659; US 2002/0090662; US
2006/0046270; US 2008/0108096; US 007/0166753; US 2008/0112958; US
2009/0239236; US 2004/008204; US 2009/0187007; US 2004/0106161; US
2011/0117096; US 2004/048525; US 2004/0258685; US 2009/0148401; US
2011/0117097; US 2006/0034840; US 2011/0064737; US 2005/0276812; US
2008/0171040; US 2009/0202536; US 2006/0013819; US 2006/0018899; US
2009/0285837; US 2011/0117097; US 2006/0088523; US 2010/0015157; US
2006/0121044; US 2008/0317753; US2006/0165702; US 2009/0081223; US
2006/0188509; US 2009/0155259; US 2011/0165157; US 2006/0204505; US
2006/0212956; US 2006/0275305; US 2007/0009976; US 2007/0020261; US
2007/0037228; US 2010/0112603; US 2006/0067930; US 2007/0224203; US
2008/0038271; US 2008/0050385; 2010/0285010; US 2008/0102069; US
2010/0008975; US 2011/0027190; US 2010/0298156; US 2009/0098135; US
2009/0148435; US 2009/0202546; US 2009/0226455; US 2009/0317387;
and US 2011/0044977.
[0018] Hyaluronidase Enzymes
[0019] Hyaluronidases are a group of generally neutral- or
acid-active enzymes found throughout the animal kingdom.
Hyaluronidases vary with respect to substrate specificity, and
mechanism of action (WO 2004/078140). There are three general
classes of hyaluronidases: 1. Mammalian-type hyaluronidases, (EC
3.2.1.35) which are endo-.beta.-N-acetylhexosaminidases with
tetrasaccharides and hexasaccharides as the major end products.
They have both hydrolytic and transglycosidase activities, and can
degrade hyaluronan and chondroitin sulfates (CS), generally C4-S
and C6-S. 2. Bacterial hyaluronidases (EC 4.2.99.1) degrade
hyaluronan and, and to various extents, CS and DS. They are
endo-.beta.-N-acetylhexosaminidases that operate by a beta
elimination reaction that yields primarily disaccharide end
products. 3. Hyaluronidases (EC 3.2.1.36) from leeches, other
parasites, and crustaceans are endo-beta-glucuronidases that
generate tetrasaccharide and hexasaccharide end products through
hydrolysis of the .beta.1-3 linkage.
[0020] Mammalian hyaluronidases can be further divided into two
groups: neutral-active and acid-active enzymes. There are six
hyaluronidase-like genes in the human genome, HYAL1, HYAL2, HYAL3,
HYAL4, HYALP1 and PH20/SPAM1. HYALP1 is a pseudogene, and HYAL3 has
not been shown to possess enzyme activity toward any known
substrates. HYAL4 is a chondroitinase and exhibits little activity
towards hyaluronan. HYAL1 is the prototypical acid-active enzyme
and PH20 is the prototypical neutral-active enzyme. Acid-active
hyaluronidases, such as HYAL1 and HYAL2 generally lack catalytic
activity at neutral pH (i.e. pH 7). For example, HYAL1 has little
catalytic activity in vitro over pH 4.5 [Frost I. G. and Stern, R.,
"A microtiter-based assay for hyaluronidase activity not requiring
specialized reagents", Anal. Biochemistry, 1997; 251:263-269].
HYAL2 is an acid-active enzyme with a very low specific activity in
vitro.
[0021] The hyaluronidase-like enzymes can also be characterized by
those which are generally locked to the plasma membrane via a
glycosylphosphatidyl inositol anchor such as human HYAL2 and human
PH20 (Danilkovitch-Miagkova et al., Proc. Natl. Acad. Sci. USA,
2003; 100(8):4580-4585; Phelps et al., Science 1988; 240(4860):
1780-1782), and those which are generally soluble such as human
HYAL1 [Frost, I. G. et al., "Purification, cloning, and expression
of human plasma hyaluronidase", Biochem. Biophys. Res. Commun.
1997; 236(1):10-15]. However, there are variations from species to
species: bovine PH20 for example is very loosely attached to the
plasma membrane and is not anchored via a phospholipase sensitive
anchor [Lalancette et al., Biol. Reprod., 2001; 65(2):628-36]. This
unique feature of bovine hyaluronidase has permitted the use of the
soluble bovine testes hyaluronidase enzyme as an extract for
clinical use (Wydase.TM., Hyalase.TM.). Other PH20 species are
lipid anchored enzymes that are generally not soluble without the
use of detergents or lipases. For example, human PH20 is anchored
to the plasma membrane via a GPI anchor. Attempts to make human
PH20 DNA constructs that would not introduce a lipid anchor into
the polypeptide resulted in either a catalytically inactive enzyme,
or an insoluble enzyme [Arming et al., Eur. J. Biochem., 1997;
247(3):810-4]. Naturally occurring macaque sperm hyaluronidase is
found in both a soluble and membrane bound form. While the 64 kDa
membrane bound form possesses enzyme activity at pH 7.0, the 54 kDa
form is only active at pH 4.0 (Cherr et al., Dev. Biol., 1996; 10;
175(1): 142-53). Thus, soluble forms of PH20 are often lacking
enzyme activity under neutral conditions.
[0022] WO2006/091871 describes that small amounts of soluble
hyaluronidase glycoproteins (sHASEGPs) can be introduced into a
formulation in order to facilitate the administration of
therapeutic drug into the hypodermis. By rapidly depolymerizing HA
in the extracellular space sHASEGP reduces the viscosity of the
interstitium, thereby increasing hydraulic conductance and allowing
for larger volumes to be administered safely and comfortably into
the SC tissue. The increased hydraulic conductance induced by
sHASEGP through reduced interstitial viscosity allows for greater
dispersion, potentially increasing the systemic bioavailability of
subcutaneously (SC) administered therapeutic drug.
[0023] When injected in the hypodermis, the depolymerization of HA
by sHASEGP is localized to the injection site in the SC tissue.
Experimental evidence shows that the sHASEGP is inactivated locally
in the interstitial space with a half-life of 13 to 20 minutes in
mice, without detectable systemic absorption in blood following
single intravenous dose in CD-1 mice. Within the vascular
compartment sHASEGP demonstrates a half-life of 2.3 and 5 minutes
in mice and Cynomolgus monkeys, respectively, with doses up to 0.5
mg/kg. The rapid clearance of sHASEGP, combined with the continual
synthesis of the HA substrate in the SC tissue, results in a
transient and locally-active permeation enhancement for other
co-injected molecules, the effects of which are fully reversible
within 24 to 48 hours post administration (Bywaters G. L., et al.,
"Reconstitution of the dermal barrier to dye spread after
Hyaluronidase injection", Br. Med. J., 1951; 2 (4741):
1178-1183).
[0024] In addition to its effects on local fluid dispersion,
sHASEGP also acts as absorption enhancer. Macromolecules greater
than 16 kilodaltons (kDa) are largely excluded from absorption
through the capillaries via diffusion and are mostly absorbed via
the draining lymph nodes. A subcutaneously administered
macromolecule such as e.g. a therapeutic antibody (molecular weight
approximately 150 kDa) must therefore traverse the interstitial
matrix before reaching the draining lymphatics for subsequent
absorption into the vascular compartment. By increasing local
dispersion, sHASEGP increases the rate (Ka) of absorption of many
macromolecules. This leads to increased peak blood levels
(C.sub.max) and potentially to increased bioavailability relative
to SC administration in the absence of sHASEGP (Bookbinder L. H.,
et al., "A recombinant human enzyme for enhanced interstitial
transport of therapeutics", J. Control. Release 2006; 114:
230-241).
[0025] Hyaluronidase products of animal origin have been used
clinically for over 60 years, primarily to increase the dispersion
and absorption of other co-administered drugs and for
hypodermoclysis (SC injection/infusion of fluid in large volume)
(Frost G. I., "Recombinant human hyaluronidase (rHuPH20): an
enabling platform for subcutaneous drug and fluid administration",
Expert Opinion on Drug Delivery, 2007; 4: 427-440). The details on
the mechanism of action of hyaluronidases have been described in
detail in the following publications: Duran-Reynolds F., "A
spreading factor in certain snake venoms and its relation to their
mode of action", CR Soc Biol Paris, 1938; 69-81; Chain E., "A
mucolytic enzyme in testes extracts", Nature 1939; 977-978;
Weissmann B., "The transglycosylative action of testicular
hyaluronidase", J Biol. Chem., 1955; 216: 783-94; Tammi, R.,
Saamanen, A. M., Maibach, H. I., Tammi M., "Degradation of newly
synthesized high molecular mass hyaluronan in the epidermal and
dermal compartments of human skin in organ culture", J. Invest.
Dermatol. 1991; 97:126-130; Laurent, U. B. G., Dahl, L. B., Reed,
R. K., "Catabolism of hyaluronan in rabbit skin takes place
locally, in lymph nodes and liver", Exp. Physiol. 1991; 76:
695-703; Laurent, T. C. and Fraser, J. R. E., "Degradation of
Bioactive Substances: Physiology and Pathophysiology", Henriksen,
J. H. (Ed) CRC Press, Boca Raton, Fla.; 1991. pp. 249-265; Harris,
E. N., et al., "Endocytic function, glycosaminoglycan specificity,
and antibody sensitivity of the recombinant human 190-kDa
hyaluronan receptor for endocytosis (HARE)", J. Biol. Chem. 2004;
279:36201-36209; Frost, G. I., "Recombinant human hyaluronidase
(rHuPH20): an enabling platform for subcutaneous drug and fluid
administration", Expert Opinion on Drug Delivery, 2007; 4: 427-440.
Hyaluronidase products approved in EU countries include Hylase.RTM.
"Dessau" and Hyalase.RTM.. Hyaluronidase products of animal origin
approved in the US include Vitrase.TM., Hydase.TM., and
Amphadase.TM..
[0026] Stable lyophilized antibody formulations comprising a
lyoprotectant, a buffer and a surfactant have been described by
Andya et al. (WO 97/04801 and U.S. Pat. Nos. 6,267,958, 6,685,940,
6,821,151, 7,060,268). WO 2006/044908 provides antibody
formulations, including monoclonal antibodies formulated in
histidine-acetate buffer, pH 5.5 to 6.5, preferably 5.8 to 6.2.
Anti-HER2 antibody formulations are disclosed in U.S. Pat. Nos.
8,372,396; 9,017,671. Subcutaneous anti-HER2 antibody formulations
and their uses are described in U.S. Pat. No. 9,345,661.
Intravenous fixed dose administration of pertuzumab is disclosed in
U.S. Pat. Nos. 7,449,184 and 8,404,234.
SUMMARY OF THE INVENTION
[0027] In one aspect, the invention concerns an article of
manufacture comprising a single dose vial containing a single fixed
dose of a HER2 antibody comprising the variable light chain and
variable heavy chain amino acid sequences of SEQ ID Nos. 7 and 8,
respectively, wherein the fixed dose is about 600 mg or about 1200
mg. Preferably, the HER2 antibody is pertuzumab.
[0028] In one embodiment, the article of manufacture comprises two
single dose vials, wherein a first vial contains a single fixed
dose of about 1200 mg of pertuzumab, and a second vial contains a
single fixed dose of about 600 mg of pertuzumab.
[0029] In a second embodiment, the article of manufacture comprises
two single dose vials, wherein the first vial contains a single
fixed dose of about 600 mg of pertuzumab and the second vial
contains a single fixed dose of about 600 mg of trastuzumab.
[0030] In a third embodiment, the article of manufacture comprises
two single dose vials, wherein the first vial contains a single
fixed dose of about 1200 mg of pertuzumab and a second vial
comprising a single fixed dose of 600 mg of trastuzumab.
[0031] In all embodiments, at least one of the single dose vials
may contain the fixed dose(s) in a liquid formulation for
subcutaneous administration.
[0032] In all embodiments, the liquid formulation for subcutaneous
administration may further comprise a hyaluronidase enzyme, such as
recombinant human hyaluronidase (rHuPH20). rHuPH20 may be present
in an amount sufficient to result in an increase in the dispersion
of the pertuzumab or trastuzumab contained in the same liquid
formulation during subcutaneous administration.
[0033] rHuPH2 may be present in the trastuzumab-containing liquid
formulation, for example at a concentration of between about 150
U/ml and 16,000 U/ml, or at a concentration of between about 600
U/ml and about 16,000 U/ml, or at a concentration of between about
1,000 U/ml and about 2,000 U/ml, e.g. at a concentration of about
2,000 U/ml or at a concentration of at least about 600 U/mL.
[0034] rHuPH20 may be present in the pertuzumab containing liquid
formulation at a concentration of between about 600 U/ml and about
2,000 U/ml, such as at a concentration of about 600 U/mL, or at a
concentration of about 667 U/ml, or at a concentration of about
1,000 U/mL, or at a concentration of about 2,000 U/mL.
[0035] In another embodiment, the single dose vial present in the
article of manufacture further comprises a single fixed dose of
trastuzumab.
[0036] In one embodiment the single fixed dose of pertuzumab and
the single fixed dose of trastuzumab is contained in a single
liquid formulation for subcutaneous administration, where the
liquid formulation may, for example contain a single fixed dose of
about 600 mg of pertuzumab and a single fixed dose of about 600 mg
of trastuzumab, or a single fixed dose of about 1200 mg of
pertuzumab and a single fixed dose of about 600 mg of
trastuzumab.
[0037] The liquid formulation comprising the fixed dose of
pertuzumab and fixed dose of pertuzumab may further comprise a
hyaluronidase enzyme, such as recombinant human hyaluronidase
(rHuPH20), which may be present in said liquid formulation in an
amount sufficient to result in an increase in the dispersion of the
pertuzumab and trastuzumab contained in the same liquid formulation
during subcutaneous administration, such as at a concentration of
at least about 600 U/mL, or at a concentration of between about 600
U/ml and about 2,000 U/ml, e.g. at a concentration of about 1,000
U/mL.
[0038] In some embodiments, the articles of manufacture herein
further comprise a package insert instructing the user to
administer the fixed dose(s) subcutaneously to a patient with HER2
positive cancer.
[0039] In one embodiment, the package insert instructs the user to
administer the fixed doses of pertuzumab and trastuzumab
subcutaneously to a patient with HER2 positive cancer.
[0040] In another embodiment, the package insert instructs the user
to co-administer the fixed dose pertuzumab and the fixed dose of
trastuzumab subcutaneously as two separate subcutaneous
injections.
[0041] In a further embodiment, the package insert instructs the
user to administer the fixed dose pertuzumab co-mixed with the
fixed-dose trastuzumab, as a single subcutaneous injection.
[0042] In yet another embodiment, the package insert instructs the
user to administer the fixed doses of pertuzumab and trastuzumab
subcutaneously to a patient with HER2 positive cancer.
[0043] The cancer may, for example, be breast cancer, peritoneal
cancer, fallopian tube cancer, lung cancer, colorectal cancer,
biliary cancer or bladder cancer, such as early breast cancer (EBC)
or metastatic breast cancer (MBC).
[0044] In another aspect, the invention concerns an article of
manufacture comprising a 10-mL or 20-mL vial holding a single fixed
dose of a HER2 antibody comprising the variable light and variable
heavy amino acid sequences in SEQ ID Nos. 7 and 8, respectively,
wherein the fixed dose is about 600 mg or about 1200 mg of the HER2
antibody, and a package insert instructing the user to administer
the fixed dose subcutaneously to a patient with HER2 positive
cancer.
[0045] In one embodiment, the HER2 antibody is pertuzumab.
[0046] In another embodiment, the fixed dose of pertuzumab is
contained in a liquid formulation for subcutaneous administration,
wherein the liquid formulation may, for example, comprise the
pertuzumab at a concentration of about 100-150 mg/mL, e.g. at a
concentration of about 120 mg/mL.
[0047] In various embodiments, the liquid formulation present in
the article of manufacture further comprises recombinant human
hyaluronidase (rHuPH20) in amount sufficient to result in an
increase in the dispersion of the pertuzumab during subcutaneous
administration, such as at a concentration of about 2,000 U/mL, or
at a concentration of about 1,000 U/mL.
[0048] The article of manufacture may further comprise one of more
excipients selected from the group consisting of buffering agents,
stabilizers and surfactants.
[0049] In one embodiment, the buffering agent is suitable to adjust
the pH to about 5.0 to 6.0, such as pH 5.5 to 5.7, e.g. 5.5. An
exemplary buffer is a histidine buffer, such as L-histidine
acetate.
[0050] The stabilizer may comprise sucrose and optionally
methionine and/or trehalose
[0051] A preferred surfactant is polysorbate 20.
[0052] In a further aspect, the invention concerns an aqueous
formulation for subcutaneous administration comprising pertuzumab
at a concentration of about 120 mg/mL, rHuPH20 at a concentration
of about 1000-2000 U/mL, an L-histidine buffer to adjust to pH to
about 5.5-5.7, sucrose, methionine and polysorbate 20.
[0053] In one embodiment, the rHuPH20 is present at a concentration
of about 1000 U/mL.
[0054] In another embodiment, the rHuPH20 is present at a
concentration of about 2000 U/mL.
[0055] In a further embodiment, the of the aqueous solution is pH
is 5.7.
[0056] The invention further concerns a liquid subcutaneous
pharmaceutical composition comprising a fixed dose of pertuzumab
and a fixed dose of trastuzumab co-formulated in an aqueous
solution further comprising rHuPH20, a buffering agent suitable to
adjust the pH to about 5.0 to 6.0, a stabilizer and a
surfactant.
[0057] In one embodiment, the buffering agent is a histidine
buffer.
[0058] In another embodiment, the buffering agent is L-histidine
acetate.
[0059] In yet another embodiment, the pH is 5.5-5.7, e.g. 5.5.
[0060] In other embodiments, the liquid pharmaceutical composition
comprises sucrose as a stabilizer, and may further comprise
methionine and/or trehalose as a stabilizer.
[0061] In one specific aspect, the liquid pharmaceutical
composition comprises 600 mg pertuzumab at a concentration of 60
mg/ml, 600 mg trastuzumab at a concentration of 60 mg/ml, 1,000
U/mL rHuPH20, 20 mM His-HCl pH 5.5, 105 mM trehalose, 100 mM
sucrose, 0.04% polysorbate 20, 10 mM methionine, and sterile water
for injection up to a total volume of 10 ml, which, for example, be
contained in a 15-ml vial.
[0062] In another specific aspect, the liquid pharmaceutical
composition comprises 1,200 mg pertuzumab at a concentration of 80
mg/ml, 600 mg trastuzumab at a concentration of 40 mg/ml, 1,000
U/mL rHuPH20, 20 mM His-HCl pH 5.5, 70 mM trehalose, 133 mM
sucrose, 0.04% polysorbate 20, 10 mM methionine, and sterile water
for injection up to a total volume of 15 ml, which may be contained
in a 20-ml vial.
[0063] The above articles of manufacture may further comprise a
package insert with instructions to subcutaneously administer the
liquid pharmaceutical composition contained therein to a human
subject with HER2 positive cancer, such as, for example, breast
cancer, peritoneal cancer, fallopian tube cancer, lung cancer,
colorectal cancer, biliary cancer and bladder cancer, e.g. early
breast cancer (EBC) or metastatic breast cancer (MBC).
[0064] In a further aspect, the invention concerns a method for
treating cancer comprising subcutaneously administering to a human
subject with a HER2 positive cancer one or more fixed dose(s) of a
HER2 antibody comprising the variable light and variable heavy
amino acid sequences in SEQ ID Nos. 7 and 8, respectively, in an
amount effective to treat the cancer, wherein the fixed dose is
about 600 mg and/or about 1200 mg.
[0065] The HER2 antibody preferably is pertuzumab.
[0066] In one embodiment, the method comprises administering to the
human subject pertuzumab at a fixed loading dose of about 1200 mg
followed by at least one maintenance dose of about 600 mg.
[0067] In a second embodiment, the administration of the loading
dose is followed by administration of multiple maintenance
doses.
[0068] In a third embodiment, the first maintenance dose of
pertuzumab is administered to the human subject approximately two
weeks or approximately three weeks after administration of the
loading dose of pertuzumab.
[0069] In further embodiments, the fixed doses of pertuzumab are
administered to the human subject approximately every 2 weeks or
approximately every 3 weeks.
[0070] The cancer may be HER2 positive cancer, such as breast
cancer, peritoneal cancer, fallopian tube cancer, lung cancer,
colorectal cancer, biliary cancer and bladder cancer, e.g. early
breast cancer (EBC) or metastatic breast cancer (MBC).
[0071] Optionally, the method may further comprise administering a
second therapeutic agent to the patient, such as a different HER2
antibody, e.g. trastuzumab, or a chemotherapeutic agent.
[0072] In one embodiment, the fixed dose pertuzumab is administered
subcutaneously in combination with subcutaneously administered
trastuzumab.
[0073] In another embodiment, the fixed dose pertuzumab and the
trastuzumab are co-administered subcutaneously as two separate
subcutaneous injections.
[0074] In yet another embodiment, the fixed dose pertuzumab is
co-mixed with fixed dose trastuzumab, and administered as a single
subcutaneous injection.
[0075] In a further embodiment, the fixed dose pertuzumab and fixed
dose trastuzumab are administered as a single co-formulation for
subcutaneous administration, such as any of the co-formulations
described hereinabove and throughout the disclosure.
[0076] The chemotherapeutic agent, if administered, may, for
example, be a taxane and/or an anthracycline, such as paclitaxel,
docetaxel, daunorubicin, doxorubicin, and/or epirubicin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0077] FIG. 1 provides a schematic of the HER2 protein structure,
and amino acid sequences for Domains I-IV (SEQ ID Nos.1-4,
respectively) of the extracellular domain thereof.
[0078] FIGS. 2A and 2B depict alignments of the amino acid
sequences of the variable light (V.sub.L) (FIG. 2A) and variable
heavy (V.sub.H) (FIG. 2B) domains of murine monoclonal antibody 2C4
(SEQ ID Nos. 5 and 6, respectively); V.sub.L and V.sub.H domains of
variant 574/pertuzumab (SEQ ID NOs. 7 and 8, respectively), and
human V.sub.L and V.sub.H consensus frameworks (hum id, light kappa
subgroup I; humIII, heavy subgroup III) (SEQ ID Nos. 9 and 10,
respectively). Asterisks identify differences between variable
domains of pertuzumab and murine monoclonal antibody 2C4 or between
variable domains of pertuzumab and the human framework.
Complementarity Determining Regions (CDRs) are in brackets.
[0079] FIGS. 3A and 3B show the amino acid sequences of pertuzumab
light chain (FIG. 3A; SEQ ID NO. 11) and heavy chain (FIG. 3B; SEQ
ID No. 12). CDRs are shown in bold. Calculated molecular mass of
the light chain and heavy chain are 23,526.22 Da and 49,216.56 Da
(cysteines in reduced form). The carbohydrate moiety is attached to
Asn 299 of the heavy chain.
[0080] FIGS. 4A and 4B show the amino acid sequences of trastuzumab
light chain (FIG. 4A; SEQ ID NO. 13) and heavy chain (FIG. 4B; SEQ
ID NO. 14), respectively. Boundaries of the variable light and
variable heavy domains are indicated by arrows.
[0081] FIGS. 5A and 5B depict a variant pertuzumab light chain
sequence (FIG. 5A; SEQ ID NO. 15) and a variant pertuzumab heavy
chain sequence (FIG. 5B; SEQ ID NO. 16), respectively.
[0082] FIG. 6 shows the study schema of the dose finding study for
subcutaneous administration of pertuzumab alone and in combination
with trastuzumab.
[0083] FIG. 7 Decision Diagram.
[0084] FIG. 8 Study Overview.
[0085] FIG. 9 shows dose normalized concentrations (.mu.g/mL) of
subcutaneously administered pertuzumab, with and without
trastuzumab, as a function of time (days).
[0086] FIG. 10 shows dose-normalized concentrations (.mu.g/mL) of
pertuzumab as a function of time (days) with different
concentrations of rHuPH20.
[0087] FIG. 11 shows the parameter estimations using the pertuzumab
and the historical population PK (popPK) W models in
comparison.
[0088] FIG. 12 Demographics and Age Distribution.
[0089] FIG. 13 Overview of Adverse Events Part 1.
[0090] FIG. 14 Overview of Adverse Events Pat 1, No. of
subjects.
[0091] FIG. 15 Most Common Adverse Events (all grades)--incidence
.gtoreq.5% overall in study, No. of subjects
[0092] FIG. 16 EGFR related toxicity
[0093] FIG. 17 Injection Related Reactions and Injection Site
Reactions
[0094] FIG. 18 LVEF--ECHO Assessments
[0095] FIG. 19 Compositions of the pertuzumab, trastuzumab and
rHuPH20 Subcutaneous Drug Substances
[0096] (SC DS) used in the preparation of the fixed-dose
pertuzumab-trastuzumab Co-Formulations.
[0097] FIG. 20 shows the amount (%) of high molecular weight
species (HMWS) in various subcutaneous pertuzumab and trastuzumab
formulations, and pertuzumab/trastuzumab co-formulations at
5.degree. C. and 25.degree. C., respectively.
[0098] FIG. 21 Mean Serum Pertuzumab Concentration-Time Profile by
Cohort
[0099] FIG. 22 Geometric Mean Dose-Normalized Serum Pertuzumab
Concentration-Time Profile, With and Without Concomitant
Herceptin
[0100] FIG. 23 Geometric Mean Serum Pertuzumab Concentration-Time
Profile With 667 U/mL or 2,000 U/mL rHuPH20 (HMV)
[0101] FIG. 24 Geometric Mean Serum Trastuzumab Concentration-Time
Profile With 667 U/mL or 2,000 U/mL rHuPH20 (HMV).
[0102] FIG. 25 Geometric Mean Serum Pertuzumab Concentration-Time
Profile following Perjeta 600 mg SC and Perjeta 420 mg IV
Doses.
[0103] FIG. 26 Geometric Mean Serum Pertuzumab Concentration-Time
Profile in HMV or EBC Patients.
[0104] FIG. 27 Geometric Mean Dose-Normalized Serum Pertuzumab
Concentration-Time Profile, With 667 U/mL, 1,000 U/mL or 2,000 U/mL
rHuPH20.
[0105] FIG. 28 Geometric Mean Serum Trastuzumab Concentration-Time
Profile With 667 U/mL, or 1,000 U/mL, or 2,000 U/mL rHuPH20.
[0106] FIG. 29 Pertuzumab Drug Substance Stability Scratch &
Sprinkle Test: SEC Data
[0107] FIG. 30 FDC Formulation Differences--Turbidity
[0108] FIG. 31 FDC Formulation Differences--SEC/HMWS
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0109] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of the active ingredient to be effective, and which
contains no additional components which are unacceptably toxic to a
subject to which the formulation would be administered. Such
formulations are sterile.
[0110] A "sterile" formulation is aseptic or free from all living
microorganisms and their spores.
[0111] A "stable" formulation is one in which the protein therein
essentially retains its physical stability and/or chemical
stability and/or biological activity upon storage. Preferably, the
formulation essentially retains its physical and chemical
stability, as well as its biological activity upon storage. The
storage period is generally selected based on the intended
shelf-life of the formulation. Various analytical techniques for
measuring protein stability are available in the art and are
reviewed in Peptide and Protein. Drug Delivery, 247-301, Vincent
Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and
Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example.
Stability can be measured at a selected temperature for a selected
time period. Preferably, the formulation is stable at about
40.degree. C. for at least about 2-4 weeks, and/or stable at about
5.0 and/or 15.degree. C. for at least 3 months, and/or stable at
about -20.degree. C. for at least 3 months or at least 1 year.
Furthermore, the formulation is preferably stable following
freezing (to, e.g., -70.degree. C.) and thawing of the formulation,
for example following 1, 2 or 3 cycles of freezing and thawing.
Stability can be evaluated qualitatively and/or quantitatively in a
variety of different ways, including evaluation of aggregate
formation (for example using size exclusion chromatography, by
measuring turbidity, and/or by visual inspection); by assessing
charge heterogeneity using cation exchange chromatography or
capillary zone electrophoresis; amino-terminal or carboxy-terminal
sequence analysis; mass spectrometric analysis; SDS-PAGE analysis
to compare reduced and intact antibody; peptide map (for example
tryptic or LYS-C) analysis; evaluating biological activity or
antigen binding function of the antibody; etc. Instability may
involve any one or more of: aggregation, deamidation (e.g. Asn
deamidation), oxidation (e.g. Met oxidation), isomerization (e.g.
Asp isomeriation), clipping/hydrolysis/fragmentation (e.g. hinge
region fragmentation), succinimide formation, unpaired cysteine(s),
N-terminal extension, C-terminal processing, glycosylation
differences, etc.
[0112] An antibody which is "susceptible to deamidation" is one
comprising one or more residue which has been found to be prone to
deamidate.
[0113] An antibody which is "susceptible to aggregation" is one
which has been found to aggregate with other antibody molecule(s),
especially upon freezing and/or agitation.
[0114] An antibody which is "susceptible to fragmentation" is one
which has been found to be cleaved into two or more fragments, for
example at a hinge region thereof.
[0115] By "reducing deamidation, aggregation, or fragmentation" is
intended preventing or decreasing the amount of deamidation,
aggregation, or fragmentation relative to the monoclonal antibody
formulated at a different pH or in a different buffer.
[0116] Herein, "biological activity" of a monoclonal antibody
refers to the ability of the antibody to bind to antigen and result
in a measurable biological response which can be measured in vitro
or in vivo. In the case of pertuzumab, in one embodiment, the
biological activity refers to the ability of the formulated
antibody to inhibit proliferation of the human breast cancer cell
line MDA-MB-175-VII.
[0117] By "isotonic" is meant that the formulation of interest has
essentially the same osmotic pressure as human blood. Isotonic
formulations will generally have an osmotic pressure from about 250
to 350 mOsm. Isotonicity can be measured using a vapor pressure or
ice-freezing type osmometer, for example.
[0118] As used herein, "buffer" refers to a buffered solution that
resists changes in pH by the action of its acid-base conjugate
components. The buffer of this invention preferably has a pH in the
range from about 5.0 to about 7.0, preferably from about 5.5 to
about 6.5, for example from about 5.5 to about 6.2, such as, for
example, 5.5 or 5.7. Examples of buffers that will control the pH
in this range include acetate, succinate, succinate, gluconate,
histidine, citrate, glycylglycine and other organic acid buffers.
The preferred buffer herein is a histidine buffer.
[0119] A "histidine buffer" is a buffer comprising histidine ions.
Examples of histidine buffers include histidine chloride, histidine
acetate, histidine phosphate, histidine sulfate. The preferred
histidine buffer identified in the examples herein was found to be
histidine acetate. In the preferred embodiment, the histidine
acetate buffer is prepared by titrating L-histidine (free base,
solid) with acetic acid (liquid). Preferably, the histidine buffer
or histidine-acetate buffer is at pH 5.5 to 6.5, or at pH 5.7 to
6.2, e.g. pH 5.7.
[0120] A "saccharide" herein comprises the general composition
(CH2O)n and derivatives thereof, including monosaccharides,
disaccharides, trisaccharides, polysaccharides, sugar alcohols,
reducing sugars, nonreducing sugars, etc. Examples of saccharides
herein include glucose, sucrose, trehalose, lactose, fructose,
maltose, dextran, glycerin, dextran, erythritol, glycerol,
arabitol, sylitol, sorbitol, mannitol, mellibiose, melezitose,
raffinose, mannotriose, stachyose, maltose, lactulose, maltulose,
glucitol, maltitol, lactitol, iso-maltulose, etc. The preferred
saccharide herein is a nonreducing disaccharide, such as trehalose
or sucrose.
[0121] Herein, a "surfactant" refers to a surface-active agent,
preferably a nonionic surfactant. Examples of surfactants herein
include polysorbate (for example, polysorbate 20 and, polysorbate
80); poloxamer (e.g. poloxamer 188); Triton; sodium dodecyl sulfate
(SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-,
myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-,
linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or
cetyl-betaine; lauroamidopropyl-, cocamidopropyl-,
linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-betaine (e.g. lauroamidopropyl);
myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.); polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.
Pluronics, PF68 etc), etc. The preferred surfactant herein is
polysorbate 20.
[0122] A "HER receptor" is a receptor protein tyrosine kinase which
belongs to the HER receptor family and includes EGFR, HER2, HER3
and HER4 receptors. The HER receptor will generally comprise an
extracellular domain, which may bind an HER ligand and/or dimerize
with another HER receptor molecule; a lipophilic transmembrane
domain; a conserved intracellular tyrosine kinase domain; and a
carboxyl-terminal signaling domain harboring several tyrosine
residues which can be phosphorylated. The HER receptor may be a
"native sequence" HER receptor or an "amino acid sequence variant"
thereof. Preferably the HER receptor is native sequence human HER
receptor.
[0123] The expressions "ErbB2" and "HER2" are used interchangeably
herein and refer to human HER2 protein described, for example, in
Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al.
Nature 319:230-234 (1986) (Genebank accession number X03363). The
term "erbB2" refers to the gene encoding human ErbB2 and "neu"
refers to the gene encoding rat p185.sup.new. Preferred HER2 is
native sequence human HER2.
[0124] Herein, "HER2 extracellular domain" or "HER2 ECD" refers to
a domain of HER2 that is outside of a cell, either anchored to a
cell membrane, or in circulation, including fragments thereof. The
amino acid sequence of HER2 is shown in FIG. 1. In one embodiment,
the extracellular domain of HER2 may comprise four domains: "Domain
I" (amino acid residues from about 1-195; SEQ ID NO:1), "Domain II"
(amino acid residues from about 196-319; SEQ ID NO:2), "Domain III"
(amino acid residues from about 320-488: SEQ ID NO:3), and "Domain
IV" (amino acid residues from about 489-630; SEQ ID NO:4) (residue
numbering without signal peptide). See Garrett et al. Mol. Cell.
11: 495-505 (2003), Cho et al. Nature 421: 756-760 (2003), Franklin
et al. Cancer Cell 5:317-328 (2004), and Plowman et al. Proc. Natl.
Acad. Sci. 90:1746-1750 (1993), as well as FIG. 1 herein.
[0125] "HER3" or "ErbB3" herein refer to the receptor as disclosed,
for example, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as
Kraus et al. PNAS (USA) 86:9193-9197 (1989).
[0126] A "low HER3" cancer is one which expresses HER3 at a level
less than the median level for HER3 expression in the cancer type.
In one embodiment, the low HER3 cancer is epithelial ovarian,
peritoneal, or fallopian tube cancer. HER3 DNA, protein, and/or
mRNA level in the cancer can be evaluated to determine whether the
cancer is a low HER3 cancer. See, for example, U.S. Pat. No.
7,981,418 for additional information about low HER3 cancer.
Optionally, a HER3 mRNA expression assay is performed in order to
determine that the cancer is a low HER3 cancer. In one embodiment,
HER3 mRNA level in the cancer is evaluated, e.g. using polymerase
chain reaction (PCR), such as quantitative reverse transcription
PCR (qRT-PCR). Optionally, the cancer expresses HER3 at a
concentration ratio equal or lower than about 2.81 as assessed
qRT-PCR, e.g. using a COBAS z480.RTM. instrument.
[0127] A "HER dimer" herein is a noncovalently associated dimer
comprising at least two HER receptors. Such complexes may form when
a cell expressing two or more HER receptors is exposed to an HER
ligand and can be isolated by immunoprecipitation and analyzed by
SDS-PAGE as described in Sliwkowski et al., J. Biol. Chem.,
269(20):14661-14665 (1994), for example. Other proteins, such as a
cytokine receptor subunit (e.g. gp130) may be associated with the
dimer. Preferably, the HER dimer comprises HER2.
[0128] A "HER heterodimer" herein is a noncovalently associated
heterodimer comprising at least two different HER receptors, such
as EGFR-HER2, HER2-HER3 or HER2-HER4 heterodimers.
[0129] A "HER antibody" is an antibody that binds to a HER
receptor. Optionally, the HER antibody further interferes with HER
activation or function. Preferably, the HER antibody binds to the
HER2 receptor. HER2 antibodies of interest herein are pertuzumab
and trastuzumab.
[0130] "HER activation" refers to activation, or phosphorylation,
of any one or more HER receptors. Generally, HER activation results
in signal transduction (e.g. that caused by an intracellular kinase
domain of a HER receptor phosphorylating tyrosine residues in the
HER receptor or a substrate polypeptide). HER activation may be
mediated by HER ligand binding to a HER dimer comprising the HER
receptor of interest. HER ligand binding to a HER dimer may
activate a kinase domain of one or more of the HER receptors in the
dimer and thereby results in phosphorylation of tyrosine residues
in one or more of the HER receptors and/or phosphorylation of
tyrosine residues in additional substrate polypeptides(s), such as
Akt or MAPK intracellular kinases.
[0131] "Phosphorylation" refers to the addition of one or more
phosphate group(s) to a protein, such as a HER receptor, or
substrate thereof.
[0132] An antibody which "inhibits HER dimerization" is an antibody
which inhibits, or interferes with, formation of a HER dimer.
Preferably, such an antibody binds to HER2 at the heterodimeric
binding site thereof. The most preferred dimerization inhibiting
antibody herein is pertuzumab or MAb 2C4. Other examples of
antibodies which inhibit HER dimerization include antibodies which
bind to EGFR and inhibit dimerization thereof with one or more
other HER receptors (for example EGFR monoclonal antibody 806, MAb
806, which binds to activated or "untethered" EGFR; see Johns et
al., J. Biol. Chem. 279(29):30375-30384 (2004)); antibodies which
bind to HER3 and inhibit dimerization thereof with one or more
other HER receptors; and antibodies which bind to HER4 and inhibit
dimerization thereof with one or more other HER receptors.
[0133] A "HER2 dimerization inhibitor" is an agent that inhibits
formation of a dimer or heterodimer comprising HER2.
[0134] A "heterodimeric binding site" on HER2, refers to a region
in the extracellular domain of HER2 that contacts, or interfaces
with, a region in the extracellular domain of EGFR, HER3 or HER4
upon formation of a dimer therewith. The region is found in Domain
II of HER2 (SEQ ID NO: 15). Franklin et al. Cancer Cell 5:317-328
(2004).
[0135] A HER2 antibody that "binds to a heterodimeric binding site"
of HER2, binds to residues in Domain II (SEQ ID NO: 2) and
optionally also binds to residues in other of the domains of the
HER2 extracellular domain, such as domains I and III, SEQ ID NOs: 1
and 3), and can sterically hinder, at least to some extent,
formation of a HER2-EGFR, HER2-HER3, or HER2-HER4 heterodimer.
Franklin et al. Cancer Cell 5:317-328 (2004) characterize the
HER2-pertuzumab crystal structure, deposited with the RCSB Protein
Data Bank (ID Code IS78), illustrating an exemplary antibody that
binds to the heterodimeric binding site of HER2.
[0136] An antibody that "binds to domain II" of HER2 binds to
residues in domain II (SEQ ID NO: 2) and optionally residues in
other domain(s) of HER2, such as domains I and III (SEQ ID NOs: 1
and 3, respectively). Preferably the antibody that binds to domain
II binds to the junction between domains I, II and III of HER2.
[0137] For the purposes herein, "pertuzumab" and "rhuMAb 2C4",
which are used interchangeably, refer to an antibody comprising the
variable light and variable heavy amino acid sequences in SEQ ID
NOs: 7 and 8, respectively. Where pertuzumab is an intact antibody,
it preferably comprises an IgG1 antibody; in one embodiment
comprising the light chain amino acid sequence in SEQ ID NO: 11 or
15, and heavy chain amino acid sequence in SEQ ID NO: 12 or 16. The
antibody is optionally produced by recombinant Chinese Hamster
Ovary (CHO) cells. The terms "pertuzumab" and "rhuMAb 2C4" herein
cover biosimilar versions of the drug with the United States
Adopted Name (USAN) or International Nonproprietary Name (INN):
pertuzumab.
[0138] For the purposes herein, "trastuzumab" and rhuMAb4D5'',
which are used interchangeably, refer to an antibody comprising the
variable light and variable heavy amino acid sequences from within
SEQ ID Nos: 13 and 14, respectively. Where trastuzumab is an intact
antibody, it preferably comprises an IgG1 antibody; in one
embodiment comprising the light chain amino acid sequence of SEQ ID
NO: 13 and the heavy chain amino acid sequence of SEQ ID NO: 14.
The antibody is optionally produced by Chinese Hamster Ovary (CHO)
cells. The terms "trastuzumab" and "rhuMAb4D5" herein cover
biosimilar versions of the drug with the United States Adopted Name
(USAN) or International Nonproprietary Name (INN): trastuzumab.
[0139] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (e.g. bispecific antibodies), and antibody
fragments, so long as they exhibit the desired biological
activity.
[0140] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Humanized
HER2 antibodies specifically include trastuzumab (HERCEPTIN.RTM.)
as described in Table 3 of U.S. Pat. No. 5,821,337 expressly
incorporated herein by reference and as defined herein; and
humanized 2C4 antibodies such as pertuzumab as described and
defined herein.
[0141] An "intact antibody" herein is one which comprises two
antigen binding regions, and an Fc region. Preferably, the intact
antibody has a functional Fc region.
[0142] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen binding region thereof.
Examples of antibody fragments include Fab, Fab', F(ab).sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragment(s).
[0143] "Native antibodies" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end.
The constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light-chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light chain and heavy chain variable domains.
[0144] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in
the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (e.g. residues 26-32 (L1),
50-52 (L2) and 91-96 (L3) in the light chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable
domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
"Framework Region" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0145] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain, including native sequence
Fc regions and variant Fc regions. Although the boundaries of the
Fc region of an immunoglobulin heavy chain might vary, the human
IgG heavy chain Fc region is usually defined to stretch from an
amino acid residue at position Cys226, or from Pro230, to the
carboxyl-terminus thereof. The C-terminal lysine (residue 447
according to the EU numbering system) of the Fc region may be
removed, for example, during production or purification of the
antibody, or by recombinantly engineering the nucleic acid encoding
a heavy chain of the antibody. Accordingly, a composition of intact
antibodies may comprise antibody populations with all K447 residues
removed, antibody populations with no K447 residues removed, and
antibody populations having a mixture of antibodies with and
without the K447 residue.
[0146] Unless indicated otherwise, herein the numbering of the
residues in an immunoglobulin heavy chain is that of the EU index
as in Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991), expressly incorporated herein by
reference. The "EU index as in Kabat" refers to the residue
numbering of the human IgG1 EU antibody.
[0147] A "functional Fc region" possesses an "effector function" of
a native sequence Fc region. Exemplary "effector functions" include
C1q binding; complement dependent cytotoxicity; Fc receptor
binding; antibody-dependent cell-mediated cytotoxicity (ADCC);
phagocytosis; down regulation of cell surface receptors (e.g. B
cell receptor; BCR), etc. Such effector functions generally require
the Fc region to be combined with a binding domain (e.g. an
antibody variable domain) and can be assessed using various assays
as herein disclosed, for example.
[0148] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. Native sequence human Fc regions include a native
sequence human IgG1 Fc region (non-A and A allotypes); native
sequence human IgG2 Fc region; native sequence human IgG3 Fc
region; and native sequence human IgG4 Fc region as well as
naturally occurring variants thereof.
[0149] A "variant Fc region" comprises an amino acid sequence which
differs from that of a native sequence Fc region by virtue of at
least one amino acid modification, preferably one or more amino
acid substitution(s). Preferably, the variant Fc region has at
least one amino acid substitution compared to a native sequence Fc
region or to the Fc region of a parent polypeptide, e.g. from about
one to about ten amino acid substitutions, and preferably from
about one to about five amino acid substitutions in a native
sequence Fc region or in the Fc region of the parent polypeptide.
The variant Fc region herein will preferably possess at least about
80% homology with a native sequence Fc region and/or with an Fc
region of a parent polypeptide, and most preferably at least about
90% homology therewith, more preferably at least about 95% homology
therewith.
[0150] Depending on the amino acid sequence of the constant domain
of their heavy chains, intact antibodies can be assigned to
different "classes". There are five major classes of intact
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2,
IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that
correspond to the different classes of antibodies are called
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0151] A "naked antibody" is an antibody that is not conjugated to
a heterologous molecule, such as a cytotoxic moiety or
radiolabel.
[0152] An "affinity matured" antibody is one with one or more
alterations in one or more hypervariable regions thereof which
result an improvement in the affinity of the antibody for antigen,
compared to a parent antibody which does not possess those
alteration(s). Preferred affinity matured antibodies will have
nanomolar or even picomolar affinities for the target antigen.
Affinity matured antibodies are produced by procedures known in the
art. Marks et al. Bio/Technology 10:779-783 (1992) describes
affinity maturation by VH and VL domain shuffling. Random
mutagenesis of CDR and/or framework residues is described by:
Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier
et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.
155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9
(1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).
[0153] A "deamidated" antibody is one in which one or more
asparagine residues thereof has been derivitized, e.g. to an
aspartic acid, a succinimide, or an iso-aspartic acid.
[0154] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth.
[0155] An "advanced" cancer is one which has spread outside the
site or organ of origin, either by local invasion ("locally
advanced") or metastasis ("metastatic"). Accordingly, the term
"advanced" cancer includes both locally advanced and metastatic
disease.
[0156] "Metastatic" cancer refers to cancer which has spread from
one part of the body (e.g. the breast) to another part of the
body.
[0157] A "refractory" cancer is one which progresses even though an
anti-tumor agent, such as a chemotherapy or biologic therapy, such
as immunotherapy, is being administered to the cancer patient. An
example of a refractory cancer is one which is platinum
refractory.
[0158] A "recurrent" cancer is one which has regrown, either at the
initial site or at a distant site, after a response to initial
therapy, such as surgery.
[0159] A "locally recurrent" cancer is cancer that returns after
treatment in the same place as a previously treated cancer.
[0160] A "non-resectable" or "unresectable" cancer is not able to
be removed (resected) by surgery.
[0161] "Early-stage breast cancer" herein refers to breast cancer
that has not spread beyond the breast or the axillary lymph nodes.
Such cancer is generally treated with neoadjuvant or adjuvant
therapy.
[0162] "Neoadjuvant therapy" or "neoadjuvant treatment" or
"neoadjuvant administration" refers to systemic therapy given prior
to surgery.
[0163] "Adjuvant therapy" or "adjuvant treatment" or "adjuvant
administration" refers to systemic therapy given after surgery.
[0164] Herein, a "patient" or "subject" is a human patient. The
patient may be a "cancer patient," i.e. one who is suffering or at
risk for suffering from one or more symptoms of cancer, in
particular breast cancer.
[0165] A "patient population" refers to a group of cancer patients.
Such populations can be used to demonstrate statistically
significant efficacy and/or safety of a drug, such as pertuzumab
and/or trastuzumab.
[0166] A "relapsed" patient is one who has signs or symptoms of
cancer after remission. Optionally, the patient has relapsed after
adjuvant or neoadjuvant therapy.
[0167] A cancer or biological sample which "displays HER
expression, amplification, or activation" is one which, in a
diagnostic test, expresses (including overexpresses) a HER
receptor, has amplified HER gene, and/or otherwise demonstrates
activation or phosphorylation of a HER receptor.
[0168] A cancer or biological sample which "displays HER
activation" is one which, in a diagnostic test, demonstrates
activation or phosphorylation of a HER receptor. Such activation
can be determined directly (e.g. by measuring HER phosphorylation
by ELISA) or indirectly (e.g. by gene expression profiling or by
detecting HER heterodimers, as described herein).
[0169] A cancer cell with "HER receptor overexpression or
amplification" is one which has significantly higher levels of a
HER receptor protein or gene compared to a noncancerous cell of the
same tissue type. Such overexpression may be caused by gene
amplification or by increased transcription or translation. HER
receptor overexpression or amplification may be determined in a
diagnostic or prognostic assay by evaluating increased levels of
the HER protein present on the surface of a cell (e.g. via an
immunohistochemistry assay; IHC). Alternatively, or additionally,
one may measure levels of HER-encoding nucleic acid in the cell,
e.g. via in situ hybridization (ISH), including fluorescent in situ
hybridization (FISH; see WO98/45479 published October, 1998) and
chromogenic in situ hybridization (CISH; see, e.g. Tanner et al.,
Am. J. Pathol. 157(5): 1467-1472 (2000); Bella et al., J. Clin.
Oncol. 26: (May 20 suppl; abstr 22147) (2008)), southern blotting,
or polymerase chain reaction (PCR) techniques, such as quantitative
real time PCR (qRT-PCR). One may also study HER receptor
overexpression or amplification by measuring shed antigen (e.g.,
HER extracellular domain) in a biological fluid such as serum (see,
e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264
published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28,
1995; and Sias et al. J. Immunol. Methods 132: 73-80 (1990)). Aside
from the above assays, various in vivo assays are available to the
skilled practitioner. For example, one may expose cells within the
body of the patient to an antibody which is optionally labeled with
a detectable label, e.g. a radioactive in situfor radioactivity or
by analyzing a biopsy taken from a patient previously exposed to
the antibody.
[0170] A "HER2-positive" cancer comprises cancer cells which have
higher than normal levels of HER2. Optionally, HER2-positive cancer
has an immunohistochemistry (IHC) score of 2+ or 3+ and/or is in
situ hybridization (ISH), fluorescent in situ hybridization (FISH)
or chromogenic in situ hybridization (CISH) positive, e.g. has an
ISH/FISH/CISH amplification ratio of .gtoreq.2.0.
[0171] A "HER2-mutated" cancer comprises cancer cells with a
HER2-activating mutation, including kinase domain mutations, which
can, for example, be identified by next generation sequencing (NGS)
or real-time polymerase chain reaction (RT-PCR). "HER2-mutated"
cancer specifically includes cancer characterized by insertions in
exon 20 of HER2, deletions around amino acid residues 755-759 of
HER2, any of the mutations G309A, G309E, S310F, D769H, D769Y,
V777L, P780-Y781insGSP, V842I, R896C (Bose et al., Cancer Discov
2013; 3:1-14), as well as previously reported identical
non-synonymous putative activating mutations (or indels) in COSMIC
database found in two or more unique specimens. For further details
see, e.g. Stephens et al., Nature 2004; 431:525-6; Shigematsu et
al., Cancer Res 2005; 65:1642-6; Buttitta et al., Int J Cancer
2006; 119:2586-91; Li et al., Oncogene 2008; 27:4702-11; Sequist et
al., J Clin Oncol 2010; 28:3076-83; Arcila et al., Clin Cancer Res
2012; 18:4910-8; Greulich et al., Proc Natl Acad Sci USA 2012;
109:14476-81; and Herter-Sprie et al., Front Oncol 2013;
3:1-10.
[0172] Herein, an "anti-tumor agent" refers to a drug used to treat
cancer. Non-limiting examples of anti-tumor agents herein include
chemotherapy agents, HER dimerization inhibitors, HER antibodies,
antibodies directed against tumor associated antigens,
anti-hormonal compounds, cytokines, EGFR-targeted drugs,
anti-angiogenic agents, tyrosine kinase inhibitors, growth
inhibitory agents and antibodies, cytotoxic agents, antibodies that
induce apoptosis, COX inhibitors, farnesyl transferase inhibitors,
antibodies that binds oncofetal protein CA 125, HER2 vaccines, Raf
or ras inhibitors, liposomal doxorubicin, topotecan, taxane, dual
tyrosine kinase inhibitors, TLK286, EMD-7200, pertuzumab,
trastuzumab, erlotinib, and bevacizumab.
[0173] The "epitope 2C4" is the region in the extracellular domain
of HER2 to which the antibody 2C4 binds. In order to screen for
antibodies which bind essentially to the 2C4 epitope, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Preferably the antibody blocks
2C4's binding to HER2 by about 50% or more. Alternatively, epitope
mapping can be performed to assess whether the antibody binds
essentially to the 2C4 epitope of HER2. Epitope 2C4 comprises
residues from Domain II (SEQ ID NO: 2) in the extracellular domain
of HER2. 2C4 and pertuzumab binds to the extracellular domain of
HER2 at the junction of domains I, II and III (SEQ ID NOs: 1, 2,
and 3, respectively). Franklin et al. Cancer Cell 5:317-328
(2004).
[0174] The "epitope 4D5" is the region in the extracellular domain
of HER2 to which the antibody 4D5 (ATCC CRL 10463) and trastuzumab
bind. This epitope is close to the transmembrane domain of HER2,
and within Domain W of HER2 (SEQ ID NO: 4). To screen for
antibodies which bind essentially to the 4D5 epitope, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Alternatively, epitope mapping
can be performed to assess whether the antibody binds essentially
to the 4D5 epitope of HER2 (e.g. any one or more residues in the
region from about residue 529 to about residue 625, inclusive of
the HER2 ECD, residue numbering including signal peptide).
[0175] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with cancer as well as those in which cancer
is to be prevented. Hence, the patient to be treated herein may
have been diagnosed as having cancer or may be predisposed or
susceptible to cancer.
[0176] The term "effective amount" refers to an amount of a drug
effective to treat cancer in the patient. The effective amount of
the drug may reduce the number of cancer cells; reduce the tumor
size; inhibit (i.e., slow to some extent and preferably stop)
cancer cell infiltration into peripheral organs; inhibit (i.e.,
slow to some extent and preferably stop) tumor metastasis; inhibit,
to some extent, tumor growth; and/or relieve to some extent one or
more of the symptoms associated with the cancer. To the extent the
drug may prevent growth and/or kill existing cancer cells, it may
be cytostatic and/or cytotoxic. The effective amount may extend
progression free survival (e.g. as measured by Response Evaluation
Criteria for Solid Tumors, RECIST, or CA-125 changes), result in an
objective response (including a partial response, PR, or complete
response, CR), increase overall survival time, and/or improve one
or more symptoms of cancer (e.g. as assessed by FOSI).
[0177] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof.
[0178] A "chemotherapy" is use of a chemical compound useful in the
treatment of cancer. Examples of chemotherapeutic agents, used in
chemotherapy, include alkylating agents such as thiotepa and
CYTOXAN.RTM. cyclosphosphamide; alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; TLK 286 (TELCYTA.TM.); acetogenins
(especially bullatacin and bullatacinone);
delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.);
beta-lapachone; lapachol; colchicines; betulinic acid; a
camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; bisphosphonates, such as
clodronate; antibiotics such as the enediyne antibiotics (e. g.,
calicheamicin, especially calicheamicin gamma1I and calicheamicin
omegaI1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186
(1994)) and anthracyclines such as annamycin, AD 32, alcarubicin,
daunorubicin, doxorubicin, dexrazoxane, DX-52-1, epirubicin,
GPX-100, idarubicin, valrubicin, KRN5500, menogaril, dynemicin,
including dynemicin A, an esperamicin, neocarzinostatin chromophore
and related chromoprotein enediyne antiobiotic chromophores,
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, carabicin, carminomycin, carzinophilin,
chromomycinis, dactinomycin, detorubicin,
6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM. doxorubicin (including
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin, liposomal doxorubicin, and
deoxydoxorubicin), esorubicin, marcellomycin, mitomycins such as
mitomycin C, mycophenolic acid, nogalamycin, olivomycins,
peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin,
streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and
zorubicin; folic acid analogues such as denopterin, pteropterin,
and trimetrexate; purine analogs such as fludarabine,
6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs
such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine, and
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, and testolactone;
anti-adrenals such as aminoglutethimide, mitotane, and trilostane;
folic acid replenisher such as folinic acid (leucovorin);
aceglatone; anti-folate anti-neoplastic agents such as ALIMTA.RTM.,
LY231514 pemetrexed, dihydrofolate reductase inhibitors such as
methotrexate, anti-metabolites such as 5-fluorouracil (5-FU) and
its prodrugs such as UFT, S-1 and capecitabine, and thymidylate
synthase inhibitors and glycinamide ribonucleotide
formyltransferase inhibitors such as raltitrexed (TOMUDEX.RTM.,
TDX); inhibitors of dihydropyrimidine dehydrogenase such as
eniluracil; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elfornithine; elliptinium acetate; an
epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;
lonidainine; maytansinoids such as maytansine and ansamitocins;
mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin;
phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK7 polysaccharide complex (JHS Natural Products,
Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine;
trichothecenes (especially T-2 toxin, verracurin A, roridin A and
anguidine); urethan; vindesine (ELDISINE.RTM., FILDESIN.RTM.);
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxanes; chloranbucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine;
mercaptopurine; platinum; platinum analogs or platinum-based
analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine
(VELBAN.RTM.); etoposide (VP-16); ifosfamide; mitoxantrone;
vincristine (ONCOVIN.RTM.); vinca alkaloid; vinorelbine
(NAVELBINE.RTM.); novantrone; edatrexate; daunomycin; aminopterin;
xeloda; ibandronate; topoisomerase inhibitor RFS 2000;
difluorometlhylornithine (DMFO); retinoids such as retinoic acid;
pharmaceutically acceptable salts, acids or derivatives of any of
the above; as well as combinations of two or more of the above such
as CHOP, an abbreviation for a combined therapy of
cyclophosphamide, doxorubicin, vincristine, and prednisolone, and
FOLFOX, an abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovorin.
[0179] Also included in this definition are anti-hormonal agents
that act to regulate or inhibit hormone action on tumors such as
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and FARESTON.RTM.
toremifene; aromatase inhibitors; and anti-androgens such as
flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as
well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense oligonucleotides, particularly those that inhibit
expression of genes in signaling pathways implicated in abherant
cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras,
and epidermal growth factor receptor (EGF-R); vaccines such as gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; PROLEUKIN.RTM.
rIL-2; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; and pharmaceutically acceptable salts, acids or derivatives
of any of the above.
[0180] A "taxane" is a chemotherapy which inhibits mitosis and
interferes with microtubules. Examples of taxanes include
Paclitaxel (TAXOL.RTM.; Bristol-Myers Squibb Oncology, Princeton,
N.J.); cremophor-free, albumin-engineered nanoparticle formulation
of paclitaxel or nab-paclitaxel (ABRAXANE.TM.; American
Pharmaceutical Partners, Schaumberg, Ill.); and Docetaxel
(TAXOTERE.RTM.; Rhone-Poulenc Rorer, Antony, France).
[0181] An "anthacycline" is a type of antibiotic that comes from
the fungus Streptococcus peucetius, examples include: Daunorubicin,
Doxorubicin, Epirubicin, and any other anthracycline
chemotherapeutic agents, including those listed before.
[0182] "Anthracycline-based chemotherapy" refers to a chemotherapy
regimen that consists of or includes one or more anthracycline.
Examples include, without limitation, 5-FU, epirubicin, and
cyclophosphamide (FEC); 5-FU, doxorubicin, and cyclophosphamide
(FAC); doxorubicin and cyclophosphamide (AC); epirubicin and
cyclophosphamide (EC); dose-dense doxorubicin and cyclophosphamide
(ddAC), and the like.
[0183] For the purposes herein, "carboplatin-based chemotherapy"
refers to a chemotherapy regimen that consists of or includes one
or more Carboplatins. An example is TCH (Docetaxel/TAXOL.RTM.,
Carboplatin, and trastuzumab/HERCEPTIN.RTM.).
[0184] An "aromatase inhibitor" inhibits the enzyme aromatase,
which regulates estrogen production in the adrenal glands. Examples
of aromatase inhibitors include: 4(5)-imidazoles,
aminoglutethimide, MEGASE.RTM. megestrol acetate, AROMASIN.RTM.
exemestane, formestanie, fadrozole, RIVISOR.RTM. vorozole,
FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole. In one
embodiment, the aromatase inhibitor herein is letrozole or
anastrozole.
[0185] An "antimetabolite chemotherapy" is use of an agent which is
structurally similar to a metabolite, but cannot be used by the
body in a productive manner. Many antimetabolite chemotherapy
interferes with the production of the nucleic acids, RNA and DNA.
Examples of antimetabolite chemotherapeutic agents include
gemcitabine (GEMZAR.RTM.), 5-fluorouracil (5-FU), capecitabine
(XELODA.TM.), 6-mercaptopurine, methotrexate, 6-thioguanine,
pemetrexed, raltitrexed, arabinosylcytosine ARA-C cytarabine
(CYTOSAR-U.RTM.), dacarbazine (DTIC-DOME.RTM.), azocytosine,
deoxycytosine, pyridmidene, fludarabine (FLUDARA.RTM.), cladrabine,
2-deoxy-D-glucose etc.
[0186] By "chemotherapy-resistant" cancer is meant that the cancer
patient has progressed while receiving a chemotherapy regimen (i.e.
the patient is "chemotherapy refractory"), or the patient has
progressed within 12 months (for instance, within 6 months) after
completing a chemotherapy regimen.
[0187] The term "platin" is used herein to refer to platinum based
chemotherapy, including, without limitation, cisplatin,
carboplatin, and oxaliplatin.
[0188] The term "fluoropyrimidine" is used herein to refer to an
antimetabolite chemotherapy, including, without limitation,
capecitabine, floxuridine, and fluorouracil (5-FU).
[0189] A "fixed" or "flat" dose of a therapeutic agent herein
refers to a dose that is administered to a human patient without
regard for the weight (WT) or body surface area (BSA) of the
patient. The fixed or flat dose is therefore not provided as a
mg/kg dose or a mg/m.sup.2 dose, but rather as an absolute amount
of the therapeutic agent.
[0190] A "loading" dose herein generally comprises an initial dose
of a therapeutic agent administered to a patient, and is followed
by one or more maintenance dose(s) thereof. Generally, a single
loading dose is administered, but multiple loading doses are
contemplated herein. Usually, the amount of loading dose(s)
administered exceeds the amount of the maintenance dose(s)
administered and/or the loading dose(s) are administered more
frequently than the maintenance dose(s), so as to achieve the
desired steady-state concentration of the therapeutic agent earlier
than can be achieved with the maintenance dose(s).
[0191] A "maintenance" dose herein refers to one or more doses of a
therapeutic agent administered to the patient over a treatment
period. Usually, the maintenance doses are administered at spaced
treatment intervals, such as approximately every week,
approximately every 2 weeks, approximately every 3 weeks, or
approximately every 4 weeks, preferably every 3 weeks.
[0192] A "vial" is a container suitable for holding a liquid or
lyophilized preparation. In one embodiment, the vial is a
single-use vial, e.g. a 10 ml or a 20 ml single-use vial with a
stopper, such as a 10 ml single use glass vial with a 20 mm
stopper.
[0193] A "package insert" is a leaflet that, by order of the Food
and Drug Administration (FDA) or other Regulatory Authority, must
be placed inside the package of every prescription drug. The
leaflet generally includes the trademark for the drug, its generic
name, and its mechanism of action; states its indications,
contraindications, warnings, precautions, adverse effects, and
dosage forms; and includes instructions for the recommended dose,
time, and route of administration.
[0194] The expression "safety data" concerns the data obtained in a
controlled clinical trial showing the prevalence and severity of
adverse events to guide the user regarding the safety of the drug,
including guidance on how to monitor and prevent adverse reactions
to the drug.
[0195] "Efficacy data" refers to the data obtained in controlled
clinical trial showing that a drug effectively treats a disease,
such as cancer.
[0196] By "stable mixture" when referring to a mixture of two or
more drugs, such as pertuzumab and trastuzumab, means that each of
the drugs in the mixture essentially retains its physical and
chemical stability in the mixture as evaluated by one or more
analytical assays. Exemplary analytical assays for this purpose
include: color, appearance and clarity (CAC), concentration and
turbidity analysis, particulate analysis, size exclusion
chromatography (SEC), ion-exchange chromatography (IEC), capillary
zone electrophoresis (CZE), image capillary isoelectric focusing
(iCIEF), and potency assay. In one embodiment, mixture has been
shown to be stable for up to 24 hours at 5.degree. C. or 30.degree.
C.
[0197] Administration "in combination" encompasses combined
administration and separate administration, in which case,
administration of one therapeutic agent can occur prior to,
simultaneously, and/or following, administration of another
therapeutic agents. Thus, administration of pertuzumab and
trastuzumab in combination (or administration of a combination of
pertuzumab and trastuzumab) encompasses combined administration and
separate administration in either order.
[0198] A drug that is administered "concurrently" with one or more
other drugs is administered during the same treatment cycle, on the
same day of treatment as the one or more other drugs, and,
optionally, at the same time as the one or more other drugs. For
instance, for cancer therapies given every 3-weeks, the
concurrently administered drugs are each administered on day-1 of a
3-week cycle.
[0199] The term "co-administration" is used herein to refer to
separate administration, including, for example, administration of
pertuzumab and trastuzumab as two separate subcutaneous (SC)
injections.
[0200] The term "co-mixed" is used herein to refer to simultaneous
administration as a single injection, including, for example,
administration of pertuzumab and trastuzumab as a single
subcutaneous (SC) injection, prepared by the health professional on
site, immediately prior to SC administration by mixing separate
pertuzumab and trastuzumab formulations.
[0201] The term "co-formulation" is used herein to refer to a
single ready-to-use pharmaceutical formulation comprising two or
more active ingredients, including, for example, a single
ready-to-use pharmaceutical formulation comprising pertuzumab and
trastuzumab formulated together for subcutaneous (SC)
administration.
II. Antibody and Chemotherapy Compositions
[0202] (i) HER2 Antibodies
[0203] The HER2 antigen to be used for production of antibodies may
be, e.g., a soluble form of the extracellular domain of a HER2
receptor or a portion thereof, containing the desired epitope.
Alternatively, cells expressing HER2 at their cell surface (e.g.
NIH-3T3 cells transformed to overexpress HER2; or a carcinoma cell
line such as SK-BR-3 cells, see Stancovski et al. PNAS (USA)
88:8691-8695 (1991)) can be used to generate antibodies. Other
forms of HER2 receptor useful for generating antibodies will be
apparent to those skilled in the art.
[0204] Various methods for making monoclonal antibodies herein are
available in the art. For example, the monoclonal antibodies may be
made using the hybridoma method first described by Kohler et al.,
Nature, 256:495 (1975), by recombinant DNA methods (U.S. Pat. No.
4,816,567).
[0205] The anti-HER2 antibodies used in accordance with the present
invention, pertuzumab and trastuzumab, are commercially
available.
[0206] U.S. Pat. No. 6,949,245 describes production of exemplary
humanized HER2 antibodies which bind HER2 and block ligand
activation of a HER receptor.
[0207] Humanized HER2 antibodies specifically include trastuzumab
as described in Table 3 of U.S. Pat. No. 5,821,337 expressly
incorporated herein by reference and as defined herein; and
humanized 2C4 antibodies such as pertuzumab as described and
defined herein.
[0208] The humanized antibodies herein may, for example, comprise
nonhuman hypervariable region residues incorporated into a human
variable heavy domain and may further comprise a framework region
(FR) substitution at a position selected from the group consisting
of 69H, 71H and 73H utilizing the variable domain numbering system
set forth in Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991). In one embodiment, the humanized
antibody comprises FR substitutions at two or all of positions 69H,
71H and 73H.
[0209] An exemplary humanized antibody of interest herein comprises
variable heavy domain complementarity determining residues
GFTFTDYTMX (SEQ ID NO: 17), where X is preferably D or S;
DVNPNSGGSIYNQRFKG (SEQ ID NO:18); and/or NLGPSFYFDY (SEQ ID NO:19),
optionally comprising amino acid modifications of those CDR
residues, e.g. where the modifications essentially maintain or
improve affinity of the antibody. For example, an antibody variant
for use in the methods of the present invention may have from about
one to about seven or about five amino acid substitutions in the
above variable heavy CDR sequences. Such antibody variants may be
prepared by affinity maturation, e.g., as described below.
[0210] The humanized antibody may comprise variable light domain
complementarity determining residues KASQDVSIGVA (SEQ ID NO:20);
SASYX.sup.1X.sup.2X.sup.3, where XI is preferably R or L, X.sup.2
is preferably Y or E, and X.sup.3 is preferably T or S (SEQ ID
NO:21); and/or QQYYIYPYT (SEQ ID NO:22), e.g. in addition to those
variable heavy domain CDR residues in the preceding paragraph. Such
humanized antibodies optionally comprise amino acid modifications
of the above CDR residues, e.g. where the modifications essentially
maintain or improve affinity of the antibody. For example, the
antibody variant of interest may have from about one to about seven
or about five amino acid substitutions in the above variable light
CDR sequences. Such antibody variants may be prepared by affinity
maturation, e.g., as described below.
[0211] The present application also contemplates affinity matured
antibodies which bind HER2. The parent antibody may be a human
antibody or a humanized antibody, e.g., one comprising the variable
light and/or variable heavy sequences of SEQ ID Nos. 7 and 8,
respectively (i.e. comprising the VL and/or VH of pertuzumab). An
affinity matured variant of pertuzumab preferably binds to HER2
receptor with an affinity superior to that of murine 2C4 or
pertuzumab (e.g. from about two or about four fold, to about 100
fold or about 1000 fold improved affinity, e.g. as assessed using a
HER2-extracellular domain (ECD) ELISA). Exemplary variable heavy
CDR residues for substitution include H28, H30, H34, H35, H64, H96,
H99, or combinations of two or more (e.g. two, three, four, five,
six, or seven of these residues). Examples of variable light CDR
residues for alteration include L28, L50, L53, L56, L91, L92, L93,
L94, L96, L97 or combinations of two or more (e.g. two to three,
four, five or up to about ten of these residues).
[0212] Humanization of murine 4D5 antibody to generate humanized
variants thereof, including trastuzumab, is described in U.S. Pat.
Nos. 5,821,337, 6,054,297, 6,407,213, 6,639,055, 6,719,971, and
6,800,738, as well as Carter et al. PNAS (USA), 89:4285-4289
(1992). HuMAb4D5-8 (trastuzumab) bound HER2 antigen 3-fold more
tightly than the mouse 4D5 antibody, and had secondary immune
function (ADCC) which allowed for directed cytotoxic activity of
the humanized antibody in the presence of human effector cells.
HuMAb4D5-8 comprised variable light (V.sub.L) CDR residues
incorporated in a V.sub.L .kappa. subgroup I consensus framework,
and variable heavy (V.sub.H) CDR residues incorporated into a
V.sub.H subgroup III consensus framework. The antibody further
comprised framework region (FR) substitutions as positions: 71, 73,
78, and 93 of the V.sub.H (Kabat numbering of FR residues; and a FR
substitution at position 66 of the V.sub.L (Kabat numbering of FR
residues). trastuzumab comprises non-A allotype human .gamma. 1 Fc
region.
[0213] Various forms of the humanized antibody or affinity matured
antibody are contemplated. For example, the humanized antibody or
affinity matured antibody may be an antibody fragment.
Alternatively, the humanized antibody or affinity matured antibody
may be an intact antibody, such as an intact IgG1 antibody.
[0214] (ii) Pertuzumab Compositions
[0215] In one embodiment of a HER2 antibody composition, the
composition comprises a mixture of a main species pertuzumab
antibody and one or more variants thereof. The preferred embodiment
herein of a pertuzumab main species antibody is one comprising the
variable light and variable heavy amino acid sequences in SEQ ID
Nos. 7 and 8, and most preferably comprising a light chain amino
acid sequence of SEQ ID No. 11, and a heavy chain amino acid
sequence of SEQ ID No. 12 (including deamidated and/or oxidized
variants of those sequences). In one embodiment, the composition
comprises a mixture of the main species pertuzumab antibody and an
amino acid sequence variant thereof comprising an amino-terminal
leader extension. Preferably, the amino-terminal leader extension
is on a light chain of the antibody variant (e.g. on one or two
light chains of the antibody variant). The main species HER2
antibody or the antibody variant may be an full length antibody or
antibody fragment (e.g. Fab of F(ab=)2 fragments), but preferably
both are full length antibodies. The antibody variant herein may
comprise an amino-terminal leader extension on any one or more of
the heavy or light chains thereof. Preferably, the amino-terminal
leader extension is on one or two light chains of the antibody. The
amino-terminal leader extension preferably comprises or consists of
VHS-. Presence of the amino-terminal leader extension in the
composition can be detected by various analytical techniques
including, but not limited to, N-terminal sequence analysis, assay
for charge heterogeneity (for instance, cation exchange
chromatography or capillary zone electrophoresis), mass
spectrometry, etc. The amount of the antibody variant in the
composition generally ranges from an amount that constitutes the
detection limit of any assay (preferably N-terminal sequence
analysis) used to detect the variant to an amount less than the
amount of the main species antibody. Generally, about 20% or less
(e.g. from about 1% to about 15%, for instance from 5% to about
15%) of the antibody molecules in the composition comprise an
amino-terminal leader extension. Such percentage amounts are
preferably determined using quantitative N-terminal sequence
analysis or cation exchange analysis (preferably using a
high-resolution, weak cation-exchange column, such as a PROPAC
WCX-10.TM. cation exchange column). Aside from the amino-terminal
leader extension variant, further amino acid sequence alterations
of the main species antibody and/or variant are contemplated,
including but not limited to an antibody comprising a C-terminal
lysine residue on one or both heavy chains thereof, a deamidated
antibody variant, etc.
[0216] Moreover, the main species antibody or variant may further
comprise glycosylation variations, non-limiting examples of which
include antibody comprising a G1 or G2 oligosaccharide structure
attached to the Fc region thereof, antibody comprising a
carbohydrate moiety attached to a light chain thereof (e.g. one or
two carbohydrate moieties, such as glucose or galactose, attached
to one or two light chains of the antibody, for instance attached
to one or more lysine residues), antibody comprising one or two
non-glycosylated heavy chains, or antibody comprising a sialidated
oligosaccharide attached to one or two heavy chains thereof
etc.
[0217] The composition may be recovered from a genetically
engineered cell line, e.g. a Chinese Hamster Ovary (CHO) cell line
expressing the HER2 antibody, or may be prepared by peptide
synthesis.
[0218] For more information regarding exemplary pertuzumab
compositions, see U.S. Pat. Nos. 7,560,111 and 7,879,325 as well as
US 2009/0202546A1.
[0219] (iii) Trastuzumab Compositions
[0220] The trastuzumab composition generally comprises a mixture of
a main species antibody (comprising light and heavy chain sequences
of SEQ ID NOS: 13 and 14, respectively), and variant forms thereof,
in particular acidic variants (including deamidated variants).
Preferably, the amount of such acidic variants in the composition
is less than about 25%, or less than about 20%, or less than about
15%. See, U.S. Pat. No. 6,339,142. See, also, Harris et al., J.
Chromatography, B 752:233-245 (2001) concerning forms of
trastuzumab resolvable by cation-exchange chromatography, including
Peak A (Asn30 deamidated to Asp in both light chains); Peak B
(Asn55 deamidated to isoAsp in one heavy chain); Peak 1 (Asn30
deamidated to Asp in one light chain); Peak 2 (Asn30 deamidated to
Asp in one light chain, and Asp102 isomerized to isoAsp in one
heavy chain); Peak 3 (main peak form, or main species antibody);
Peak 4 (Asp102 isomerized to isoAsp in one heavy chain); and Peak C
(Asp102 succinimide (Asu) in one heavy chain). Such variant forms
and compositions are included in the invention herein.
[0221] (iv) Subcutaneous Formulations Comprising a Hyaluronidase
Enzyme
[0222] Hyaluronidase enzyme acts primarily as a permeation enhancer
to increase the dispersion and absorption of other co-administered
drugs. Hyaluronidase transiently hydrolyses hyaluronan, component
of the SC matrix, leading to reduced viscosity of the extracellular
matrix of the hypodermis and, thus, to an improved delivery of
subcutaneously administered drugs to the systemic circulation.
[0223] Soluble Hyaloronidase glycoproteins (sHASEGP), a process for
preparing the same and their use in pharmaceutical compositions
have been described in WO 2004/078140. The use of soluble
Hyaluronidase glycoproteins in combination with a variety of
exemplary antibodies, such as e.g. trastuzumab, has been mentioned
in WO 2006/091871.
[0224] The hyaluronidase enzyme in the formulations of the present
invention enhances the delivery of the anti-HER2 antibody or
antibodies (e.g. pertuzumab and/or trastuzumab) to the systemic
circulation, e.g. by increasing the absorption of the active
substance (it acts as a permeation enhancer). The hyaluronidase
enzyme also increases the delivery of the therapeutic HER2 antibody
or antibodies (e.g. pertuzumab and/or trastuzumab) into the
systemic circulation via the subcutaneous application route by the
reversible hydrolyzation of hyaluronan, an extracellular component
of the SC interstitial tissue. The hydrolysis of hyaluronan in the
hypodermis temporarily opens channels in the interstitial space of
the SC tissue and thereby improves the delivery of the therapeutic
anti-HER2 antibody into the systemic circulation. In addition, the
administration shows reduced pain in humans and less volume-derived
swelling of the SC tissue.
[0225] Hyaluronidase, when administered locally has its entire
effect locally. In other word hyaluronidase is inactivated and
metabolized locally in minutes and has not been noted to have
systemic or long term effects. The rapid inactivation of
hyaluronidase within minutes when it enters the blood stream
precludes a realistic ability to perform comparable biodistribution
studies between different hyaluronidase products. This property
also minimizes any potential systemic safety concerns because the
hyaluronidase product cannot act at distant sites.
[0226] The unifying feature of all hyaluronidase enzymes is their
ability to depolymerize hyaluronan, regardless of differences in
chemical structure, in species source, in tissue sources, or in the
batches of drug product sourced from the same species and tissue.
They are unusual in that their activity is the same (except for
potency) in spite of having different structures.
[0227] The hyaluronidase enzyme excipient in accordance with the
formulation of the present invention is characterized by having no
adverse effect on the molecular integrity of the HER2 antibody or
antibodies in the stable pharmaceutical formulations described
herein. Furthermore, the hyaluronidase enzyme merely modifies the
delivery of the HER2 antibody or HER2 antibodies to the systemic
circulation but does not possess any properties that could provide
or contribute to the therapeutic effects of systemically absorbed
HER2 antibody or antibodies. The hyaluronidase enzyme is not
systemically bioavailable and does not adversely affect the
molecular integrity of the HER2 antibody or antibodies at the
recommended storage conditions of the stable pharmaceutical
formulation in accordance with the invention.
[0228] A number of suitable hyaluronidase enzymes in accordance
with the present invention are known from the prior art. The
preferred enzyme is a human hyaluronidase enzyme, most preferably
the recombinant human hyaluronidase enzyme known as rHuPH20.
rHuPH20 is a member of the family of neutral and acid-active
.beta.-1,4 glycosyl hydrolases that depolymerize hyaluronan by the
hydrolysis of the .beta.-1,4 linkage between the C.sub.1 position
of N-acetyl glucosamine and the C.sub.4 position of glucuronic
acid. Hyaluronan is a polysaccharide found in the intracellular
ground substance of connective tissue, such as the subcutaneous
interstitial tissue, and of certain specialized tissues, such as
the umbilical cord and vitreous humor. The hydrolysis of hyaluronan
temporarily decreases the viscosity of the interstitial tissue and
promotes the dispersion of injected fluids or of localized
transudates or exudates, thus facilitating their absorption. The
effects of hyaluronidase are local and reversible with complete
reconstitution of the tissue hyaluronan occurring within 24 to 48
hours (Frost, G. I., "Recombinant human hyaluronidase (rHuPH20): an
enabling platform for subcutaneous drug and fluid administration",
Expert Opinion on Drug Delivery, 2007; 4:427-440). The increase in
the permeability of connective tissue through the hydrolysis of
hyaluronan correlates with the efficacy of hyaluronidase for their
capability to increase the dispersion and absorption of
co-administered molecules.
[0229] The human genome contains several hyaluronidase genes. Only
the PH20 gene product possesses effective hyaluronidase activity
under physiologic extracellular conditions and acts as a spreading
agent, whereas acid-active hyaluronidases do not have this
property.
[0230] rHuPH20 is the first and only recombinant human
hyaluronidase enzyme currently available for therapeutic use.
Naturally occurring human PH20 protein has a lipid anchor attached
to the carboxy terminal amino acid that anchors it to the plasma
membrane. The rHuPH20 enzyme developed by Halozyme is a truncated
deletion variant that lacks such amino acids in the carboxy
terminus responsible for the lipid attachment. This gives rise to a
soluble, neutral pH-active enzyme similar to the protein found in
bovine testes preparations. The rHuPH20 protein is synthesized with
a 35 amino acid signal peptide that is removed from the N-terminus
during the process of secretion. The mature rHuPH20 protein
contains an authentic N-terminal amino acid sequence orthologous to
that found in some bovine hyaluronidase preparations.
[0231] The PH20 hyaluronidases, including the animal derived PH20
and recombinant human rHuPH20, depolymerize hyaluronan by the
hydrolysis of the .beta.-1,4 linkage between the C.sub.1 position
of N-acetyl glucosamine and the C.sub.4 position of glucuronic
acid. The tetrasaccharide is the smallest digestion product
(Weissmann, B., "The transglycosylative action of testicular
hyaluronidase", J. Biol. Chem., 1955; 216: 783-94). This N-acetyl
glucosamine/glucuronic acid structure is not found in N-linked
glycans of recombinant biological products and therefore rHuPH20
will not affect the glycosylation of antibodies it is formulated
with, such as e.g. pertuzumab or pertuzumab and trastuzumab. The
rHuPH20 enzyme itself possesses six N-linked glycans per molecule
with core structures similar to that found in monoclonal
antibodies. As anticipated, these N-linked structures do not change
over time, confirming the lack of enzymatic activity of rHuPH20 on
these N-linked glycan structures. The short half-life of rHuPH20
and the constant synthesis of hyaluronan lead to a short and local
action of the enzyme on tissues.
[0232] The hyaluronidase enzyme present in the subcutaneous
formulation in accordance with the present invention can be
prepared by using recombinant DNA technology. In this way it is
ensured that the same protein (identical amino acid sequence) is
obtained all the time and that an allergic reaction, e.g. caused by
contaminating proteins co-purified during extraction from a tissue,
is avoided. The hyaluronidase enzyme used in the formulation as
exemplified herein is a human enzyme, viz. rHuPH20.
[0233] The amino acid sequence of rHuPH20 (HYLENEX.TM.) is well
known and available under CAS Registry No. 75971-58-7. The
approximate molecular weight is 61 kDa.
[0234] While the safety and efficacy of hyaluronidase products has
been established, there are only two monoclonal antibodies
(Herceptin.RTM. and MabThera.RTM.) that have been approved for
subcutaneous delivery, using hyaluronidase containing formulations.
There is no known hyaluronidase containing subcutaneous formulation
comprising two antibodies in the same formulation (co-formulation
of two antibodies).
[0235] The concentration of the hyaluronidase enzyme depends on the
actual hyaluronidase enzyme used in the preparation of the
formulation in accordance with the invention. An effective amount
of the hyaluronidase enzyme can be readily determined by the person
skilled in the art based on the disclosure further below.
[0236] The hyaluronidase enzyme should be provided in sufficient
amount to result in an increase in the dispersion and absorption of
the co-administered anti-HER2 antibody or antibodies, such as
pertuzumab and/or trastuzumab. The minimal amount of the
hyaluronidase enzyme is at least about 150 U/ml. More particularly
the effective amount of the hyaluronidase enzyme is about 150 U/ml
to about 16,000 U/ml, or about 600 U/ml to about 16,000 ml, or
about 1,000 to 16,000 U/ml, where the latter corresponds to about
0.01 mg to 0.16 mg protein based on an assumed specific activity of
100,000 U/mg. Alternatively the concentration of the hyaluronidase
enzyme is about 1,500 to 12,000 U/ml, or more particularly about
2,000 U/ml or about 12,000 U/ml. The amounts specified correspond
to the amount of hyaluronidase enzyme initially added to the
formulation. The hyaluronidase enzyme concentrations measured in
the final formulation may vary within a certain range. The ratio
(w/w) of the hyaluronidase enzyme to the anti-HER2 antibody or
antibodies is generally in the range of 1:1,000 to 1:8,000, or in
the range of 1:4,000 to 1:5,000 or about 1:6,000.
[0237] The hyaluronidase enzyme may be derived from animals, human
samples or manufactured based on the recombinant DNA technology as
described further below.
[0238] In some embodiments, the subcutaneous HER2 antibody
formulations herein comprise recombinant human hyaluronidase
(rHuPH20) at a concentration of about 600 U/mL to about 16,000
U/mL, or about 1,000 U/mL to about 16,000 U/mL, or about 1,000 to
about 2,000 U/ml, or at a concentration of about 600 U/ml, or about
667 U/mL, or about 1,000 U/mL, or about 2,000 U/mL, preferable
about 1,000 U/mL.
[0239] In some embodiments the highly concentrated, stable
pertuzumab formulations of the present invention comprise a fixed
dose of 600 mg or 1200 mg of pertuzumab and recombinant human
hyaluronidase (rHuPH20) at a concentration of 1,000 U/mL.
[0240] As noted above the soluble hyaluronidase glycoprotein may be
considered to be a further excipient in the anti-HER2 formulation.
The soluble hyaluronidase glycoprotein may be added to the
anti-HER2 formulation at the time of manufacturing the anti-HER2
formulation or may be added shortly before the injection.
Alternatively the soluble hyaluronidase glycoprotein may be
provided as a separate injection. In the latter case the soluble
hyaluronidase glycoprotein may be provided in a separate vial
either in lyophilized form which must be reconstituted with
suitable diluents before the subcutaneous injection takes place, or
may be provided as a liquid formulation by the manufacturer. The
anti-HER2 formulation and the soluble hyaluronidase glycoprotein
may be procured as separate entities or may also be provided as
kits comprising both injection components and suitable instructions
for their subcutaneous administration. Suitable instructions for
the reconstitution and/or administration of one or both of the
formulations may also be provided.
[0241] In addition to the hyaluronidase enzyme, such as rHuPH20,
the subcutaneous formulations of the present invention comprise one
or more additional excipients, such as one or more buffering
agents, one or more stabilizers, and/or one or more
surfactants.
[0242] The buffer used in the formulations in accordance with the
present invention has a pH in the range from about 5.0 to about
7.0, or from about 5.0 to about 6.0, or from about 5.3 to about
5.8, or from about 5.5 to about 5.7.
[0243] For the subcutaneous (SC) pertuzumab formulations the pH of
about 5.7 has been found most suitable. A preferred pH of a
subcutaneous (SC) trastuzumab formulation is about 5.5.
[0244] Examples of buffering agents that will control the pH in
this range include acetate, succinate, gluconate, histidine,
citrate, glycylglycine and other organic acid buffers. The most
suitable buffer in accordance with the present invention is a
histidine buffer, such as, for example, histidine chloride,
histidine acetate, histidine phosphate, histidine sulfate,
preferably a histidine chloride buffer. A histidine chloride buffer
can be prepared by titrating L-histidine (free base, solid) with
diluted hydrochloric acid. In particular the histidine buffer or
histidine chloride buffer is an L-histidine buffer at pH of
5.5.+-.0.6, more particularly at a pH from about 5.3 to about 5.8,
and most particularly has a pH of 5.5 or 5.7.
[0245] The stabilizer may, for example, be a saccharide or a
combination of saccharides, including monosaccharides,
disaccharides, trisaccharides, polysaccharides, sugar alcohols,
reducing sugars, nonreducing sugars, etc. Examples of saccharides
herein include glucose, sucrose, trehalose, lactose, fructose,
maltose, dextran, glycerin, dextran, erythritol, glycerol,
arabitol, sylitol, sorbitol, mannitol, mellibiose, melezitose,
raffinose, mannotriose, stachyose, maltose, lactulose, maltulose,
glucitol, maltitol, lactitol, and iso-maltulose. A particularly
suitable saccharide for use in the trastuzumab SC formulations is
trehalose, and a particularly suitable saccharide for use in the
pertuzumab SC formulations is sucrose.
[0246] The surfactant preferably is a nonionic surfactant. Examples
of surfactants herein include polysorbate; poloxamer (e.g.
poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel
sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or
stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or
stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;
lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,
myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine
(e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQU AT.TM. series (Mona
Industries, Inc., Paterson, N.J.); polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.
Pluronics, PF68 etc); etc. Polysorbate 20 (PS20) and Polysorbate 80
(PS80), respectively are particularly suitable for use in the
formulations described herein.
III. Selecting Patients for Therapy
[0247] Detection of HER2 expression or amplification can be used to
select patients for treatment in accordance with the present
invention. Several FDA-approved commercial assays are available to
identify HER2-positive, HER2-expressing, HER2-overexpressing or
HER2-amplified cancer patients. These methods include
HERCEPTEST.RTM. (Dako) and PATHWAY'HER2 (immunohistochemistry (IHC)
assays) and PathVysion.RTM. and HER2 FISH pharmDx.TM. (FISH
assays). Users should refer to the package inserts of specific
assay kits for information on the validation and performance of
each assay.
[0248] For example, HER2 expression or overexpression may be
analyzed by IHC, e.g. using the HERCEPTEST.RTM. (Dako). Paraffin
embedded tissue sections from a tumor biopsy may be subjected to
the IHC assay and accorded a HER2 protein staining intensity
criteria as follows:
[0249] Score 0 no staining is observed or membrane staining is
observed in less than 10% of tumor cells.
[0250] Score 1+a faint/barely perceptible membrane staining is
detected in more than 10% of the tumor cells. The cells are only
stained in part of their membrane.
[0251] Score 2+a weak to moderate complete membrane staining is
observed in more than 10% of the tumor cells.
[0252] Score 3+a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0253] Those tumors with 0 or 1+ scores for HER2 overexpression
assessment may be characterized as HER2-negative, whereas those
tumors with 2+ or 3+ scores may be characterized as
HER2-positive.
[0254] Tumors overexpressing HER2 may be rated by
immunohistochemical scores corresponding to the number of copies of
HER2 molecules expressed per cell, and can been determined
biochemically:
[0255] 0=0-10,000 copies/cell,
[0256] 1+=at least about 200,000 copies/cell,
[0257] 2+=at least about 500,000 copies/cell,
[0258] 3+=at least about 2,000,000 copies/cell.
[0259] Overexpression of HER2 at the 3+ level, which leads to
ligand-independent activation of the tyrosine kinase (Hudziak et
al., Proc. Natl. Acad. Sci. USA, 84:7159-7163 (1987)), occurs in
approximately 30% of breast cancers, and in these patients,
relapse-free survival and overall survival are diminished (Slamon
et al., Science, 244:707-712 (1989); Slamon et al., Science,
235:177-182 (1987)).
[0260] The presence of HER2 protein overexpression and gene
amplification are highly correlated, therefore, alternatively, or
additionally, the use of in situ hybridization (ISH), e.g.
fluorescent in situ hybridization (FISH), assays to detect gene
amplification may also be employed for selection of patients
appropriate for treatment in accordance with the present invention.
FISH assays such as the INFORM.TM. (sold by Ventana, Arizona) or
PathVysion.RTM. (Vysis, Illinois) may be carried out on
formalin-fixed, paraffin-embedded tumor tissue to determine the
extent (if any) of HER2 amplification in the tumor.
[0261] Most commonly, HER2-positive status is confirmed using
archival paraffin-embedded tumor tissue, using any of the foregoing
methods.
[0262] Preferably, HER2-positive patients having a 2+ or 3+ IHC
score and/or who are FISH or ISH positive are selected for
treatment in accordance with the present invention. Patients with
3+ IHC score and FISH/ISH positivity are particularly suitable for
treatment in accordance with the present invention.
[0263] HER2 mutations associated with responsiveness to
HER2-directed therapy have also been identified. Such mutations
include, without limitation, insertions in exon 20 of HER2,
deletions around amino acid residues 755-759 of HER2, any of the
mutations G309A, G309E, S310F, D769H, D769Y, V777L,
P780-Y781insGSP, V842I, R896C (Bose et al., Cancer Discov 2013;
3:1-14), as well as previously reported identical non-synonymous
putative activating mutations (or indels) in COSMIC database found
in two or more unique specimens.
[0264] See also U.S. Pat. No. 7,981,418 for alternative assays for
screening patients for therapy with pertuzumab, and the
Examples.
IV. Pharmaceutical Formulations
[0265] Therapeutic formulations of the HER2 antibodies used in
accordance with the present invention are prepared for storage by
mixing an antibody having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), generally in the form of lyophilized
formulations or aqueous solutions. Antibody crystals are also
contemplated (see US Pat Appln 2002/0136719). Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG). Lyophilized
antibody formulations are described in WO 97/04801, expressly
incorporated herein by reference.
[0266] Lyophilized antibody formulations are described in U.S. Pat.
Nos. 6,267,958, 6,685,940 and 6,821,515, expressly incorporated
herein by reference. The preferred HERCEPTIN.RTM. (trastuzumab)
formulation is a sterile, white to pale yellow preservative-free
lyophilized powder for intravenous (W) administration, comprising
440 mg trastuzumab, 400 mg .alpha.alpha.,.alpha.-trehalose
dehydrate, 9.9 mg L-histidine-HCl, 6.4 mg L-histidine, and 1.8 mg
polysorbate 20, USP. Reconstitution of 20 mL of bacteriostatic
water for injection (BWFI), containing 1.1% benzyl alcohol as a
preservative, yields a multi-dose solution containing 21 mg/mL
trastuzumab, at pH of approximately 6.0. For further details, see
the trastuzumab prescribing information.
[0267] The preferred pertuzumab formulation for therapeutic use
comprises 30 mg/mL pertuzumab in 20 mM histidine acetate, 120 mM
sucrose, 0.02% polysorbate 20, at pH 6.0. An alternate pertuzumab
formulation comprises 25 mg/mL pertuzumab, 10 mM histidine-HCl
buffer, 240 mM sucrose, 0.02% polysorbate 20, pH 6.0.
[0268] The formulation of the placebo used in the clinical trials
described in the Examples is equivalent to pertuzumab, without the
active agent.
[0269] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Various drugs which can be combined
with the HER dimerization inhibitor are described in the Method
Section below. Such molecules are suitably present in combination
in amounts that are effective for the purpose intended.
[0270] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0271] Exemplary specific formulations suitable for use in the
methods of the present invention are as follows:
[0272] Pertuzumab W: pertuzumab 420 mg/14 ml concentrate for i.v.
infusion is a sterile, clear to slightly opalescent, colorless to
pale brown liquid supplied in 20 ml single-use glass vials with 20
mm stoppers. Each single-use vial contains 420 mg of pertuzumab at
a concentration of 30 mg/mL in 20 mM L-histidine acetate (pH 6.0),
120 mM sucrose and 0.02% polysorbate 20.
[0273] Pertuzumab SC with rHuPH20: pertuzumab 600 mg/5 ml with
rHuPH20 solution for s.c. injection is a sterile,
preservative-free, colorless to slightly brownish liquid supplied
in 10 ml single-use vials with 20 mm stoppers. Vials are filled to
enable delivery and transfer of 5.0 ml of the study drug filled
with about 5.4 ml drug product). Each vial is composed of a
formulations containing 120 mg/mL R04368451 in L-histidine acetate
buffer containing excipients sucrose, polysorbate 20, methionine,
and rHuPH20 (2000 U/mL) at pH 5.7.
[0274] A specific pertuzumab SC formulation with rHuPH20 has the
following ingredients:
[0275] 120 mg/mL pertuzumab
[0276] 240 mM Sucrose
[0277] 0.02% Polysorbate 20
[0278] 10 mM Methionine
[0279] 2000 U/mL rhuPH20
[0280] 20 mM Histidine/Acetate
[0281] pH 5.7
[0282] Pertuzumab SC without rHuPH20: pertuzumab 500 mg/5 ml
solution for s.c. injection is a sterile, preservative-free,
colorless to slightly brownish liquid supplied in 10 ml single-use
glass vials with 20 mm stoppers. Vials are filled to enable
delivery and transfer of 5.0 ml of study drug. Each vial is
composed of a formulation containing 120 mg/mL pertuzumab in
L-histidine acetate buffer containing excipients sucrose,
polysorbate 20, and methionine at pH 5.7.
[0283] Trastuzumab SC: trastuzumab for subcutaneous administration
typically contains the following ingredients: recombinant human
hyaluronidase (rHuPH20); L-histidine; L-histidine hydrochloride
monohydrate; .alpha.,.alpha.-trehalose dehydrate; L-methionine;
Polysorbate 20; Water for injections, trastuzumab 600 mg/5 ml. The
trastuzumab solution for s.c. injection is a sterile,
preservative-free, colorless to slightly brownish liquid supplied
in 6 ml single-use glass vials with 20 mm stoppers, Each vial is
composed of a formulation containing 120 mg/mL of trastuzumab in
L-Histidine-HCl buffer containing excipients trehalose, polysorbate
20, methionine, and rHuPH20 (2000 U/mL) at pH 5.5.
[0284] A specific trastuzumab SC formulation has the following
ingredients:
[0285] 120 mg/mL trastuzumab
[0286] 210 mM Trehalose
[0287] 0.04% Polysorbate 20
[0288] 10 mM Methionine
[0289] 2,000 U/mL rHuPH20
[0290] 20 mM Histidine-HCl
[0291] pH 5.5
[0292] A specific pertuzumab-trastuzumab SC fixed-dose combination
(FDC) Loading dosage form has the following composition:
trastuzumab 600 mg and pertuzumab 1,200 mg in 15 ml solution for
s.c. injection is a sterile, preservative-free, colorless to
slightly brownish liquid supplied in 20 ml single-use glass vials
with 20 mm stoppers, Each vial is composed of a formulation
containing 40 mg/mL of trastuzumab and 80 mg/ml of pertuzumab in
L-Histidine-HCl buffer containing excipients trehalose, sucrose,
polysorbate 20, methionine, and rHuPH20 (1,000 U/mL) at pH 5.5.
[0293] A specific pertuzumab-trastuzumab SC fixed-dose combination
(FDC) Maintenance dosage form has the following composition:
trastuzumab 600 mg and pertuzumab 600 mg in 10 ml solution for s.c.
injection is a sterile, preservative-free, colorless to slightly
brownish liquid supplied in 15 ml single-use glass vials with 20 mm
stoppers, Each vial is composed of a formulation containing 60
mg/mL of trastuzumab and 60 mg/ml of pertuzumab in L-Histidine-HCl
buffer containing excipients trehalose, sucrose, polysorbate 20,
methionine, and rHuPH20 (1,000 U/mL) at pH 5.5.
V. Treatment Methods
[0294] For intravenous administration, pertuzumab and trastuzumab
are administered according to applicable prescribing
information.
[0295] Pertuzumab is typically administered every three weeks by
intravenous infusion, starting with a first 840 mg infusion
administered over 60 minutes, followed by a second and any
subsequent intravenous infusions of 420 mg administered over 30 to
60 minutes. Further details of suitable administration schedules
are given in the trastuzumab Prescribing Information.
[0296] Trastuzumab is typically administered every three weeks by
intravenous infusion starting with a first 8 mg/kg loading dose
over 90 minutes, followed by a second and any subsequent
intravenous infusions 6 mg/kg maintenance doses administered over
30 to 60 minutes. Further details of suitable administration
schedules are given in the trastuzumab Prescribing Information.
[0297] Pertuzumab and trastuzumab can be administered during the
same visit, in either order.
[0298] According to the present invention, pertuzumab or
pertuzumab+trastuzumab are administered subcutaneously.
[0299] Pertuzumab SC is typically administered every three weeks as
subcutaneous injection, starting with a fixed loading dose of about
1200 mg, followed by a second and any subsequent fixed maintenance
doses of about 600 mg as hereinabove disclosed and as described in
the Examples. The injection site should be alternated between the
left and the right thigh. New injections should be given at least
2.5 cm from the old site on healthy skin and not into areas where
the skin is red, bruised, tender or hard.
[0300] Trastuzumab SC is typically administered as subcutaneous
injections at a 600 mg dose over 2-5 minutes every three weeks. The
injection site should be alternated between the left and the right
thigh. New injections should be given at least 2.5 cm from the old
site on healthy skin and not into areas where the skin is red,
bruised, tender or hard.
[0301] Pertuzumab/trastuzumab SC co-formulations are administered
in a similar manner.
[0302] For the co-mix subcutaneous administration of pertuzumab and
trastuzumab, it is necessary to compound the final mixture in
syringes using a syringe connector. The subcutaneous injection is
finally administered using a disposable plastic syringe and
stainless steel needle.
VI. Articles of Manufacture
[0303] In another embodiment of the invention, an article of
manufacture containing materials useful for the treatment of
cancer. The article of manufacture comprises a vial with a fixed
dose of the pertuzumab for subcutaneous administration, wherein the
fixed dose is approximately 600 mg or approximately 1200 of
pertuzumab. The article of manufacture preferably further comprises
a package insert. The package insert may provide instructions to
administer the fixed dose to a patient with HER2-expressing, e.g.
HER2-positive, HER2-amplified, or HER2-mutated cancer
subcutaneously, alone or in combination with subcutaneous
administration of trastuzumab, where administration in combination
includes, without limitation, co-administration, co-mixed
administration and administration of a co-formulation, as
hereinabove defined and described and as described in the Examples.
In certain embodiments, the cancer is breast cancer, ovarian
cancer, peritoneal cancer, fallopian tube cancer, lung cancer,
prostate cancer, colorectal cancer, biliary cancer and bladder
cancer. In other embodiments, the cancer is breast cancer,
peritoneal cancer, fallopian tube cancer, lung cancer, colorectal
cancer, biliary cancer and bladder cancer. In a particular
embodiment, the cancer is breast cancer, such as early breast
cancer or metastatic breast cancer.
[0304] In one embodiment, the article of manufacture is a
single-use glass vial equipped with a stopper, which contains the
formulation to be administered.
[0305] Another form of an article of manufacture is a syringe,
containing the formulation to be administered, which may be
attached to a stainless streel hypodermic needle for subcutaneous
administration.
[0306] In one embodiment, the article of manufacture comprises two
vials, wherein a first vial contains a fixed dose of approximately
1200 mg of pertuzumab, and a second vial contains a fixed dose of
approximately 600 mg of pertuzumab.
[0307] In another embodiment, the article of manufacture of
comprises two vials, wherein a first vial contains a fixed dose of
approximately 600 mg of pertuzumab, and a second vial contains a
fixed dose of approximately 600 mg of trastuzumab.
[0308] In another embodiment, the article of manufacture comprises
a single-dose vial containing about 600 mg of pertuzumab.
IV. Deposit of Biological Materials
[0309] The following hybridoma cell lines have been deposited with
the American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. 20110-2209, USA (ATCC):
TABLE-US-00001 Antibody Designation ATCC No. Deposit Date 4D5 ATCC
CRL 10463 May 24, 1990 2C4 ATCC HB-12697 Apr. 8, 1999
TABLE-US-00002 TABLE 1 TABLE OF SEQUENCES Description SEQ ID NO
FIG. HER2 domain I 1 1 HER2 domain II 2 1 HER2 domain III 3 1 HER2
domain IV 4 1 2C4 variable light 5 2A 2C4 variable heavy 6 2B
574/pertuzumab variable light 7 2A 574/pertuzumab variable heavy 8
2B human V.sub.L consensus framework 9 2A Human V.sub.H consensus
framework 10 2B pertuzumab light chain 11 3A pertuzumab heavy chain
12 3B trastuzumab light chain 13 4A trastuzumab heavy chain 14 4B
Variant pertuzumab light chain 15 5A Variant pertuzumab heavy chain
16 5B GFTFTDYTMX 17 DVNPNSGGSIYNQRFKG 18 NLGPSFYFDY 19 KASQDVSIGVA
20 SASYX.sup.1X.sup.2X.sup.3 21 QQYYIYPYT 22
[0310] Further details of the invention are illustrated by the
following non-limiting Examples. The disclosures of all citations
in the specification are expressly incorporated herein by
reference.
TABLE-US-00003 TABLE 2 LIST OF ABBREVIATIONS AND DEFINITIONs OF
TERMS Abbreviation Definition ALP alkaline phosphatase ALT alanine
aminotransferase ARDS acute respiratory distress syndrome ARR
administration-related reaction AST aspartate aminotransferase ATA
anti-therapeutic antibody AUC area under the serum
concentration-time curve AUC.sub.0-21 day area under the serum
concentration-time curve AUC from start of study treatment to Day
21 AUC.sub.0-inf area under the concentration-time curve from start
of study treatment 0 to infinity BMI body mass index BP blood
pressure CHF congestive heart failure CL/F mean apparent clearance
C.sub.max maximum serum concentration C.sub.trough trough
concentration CTCAE Common Terminology Criteria for Adverse Events
EBC early breast cancer EC Ethics Committee ECG electrocardiogram
ECHO echocardiography eCRF electronic Case Report Form EDC
electronic data capture EGFR epidermal growth factor receptor FDC
fixed-dose combination FEC fluorouracil, epirubicin, and
cyclophosphamide GLP Good Laboratory Practice HBcAb total hepatitis
B core antibody HBsAg hepatitis B surface antigen HCG human
chorionic gonadotropin HCV hepatitis C virus HER2 human epidermal
growth factor receptor 2 HIV human immunodeficiency virus HMV
healthy male volunteer HR heart rate IB Investigator's Brochure ICH
International Conference on Harmonisation Ig immunoglobulin IMP
investigational medicinal product IND Investigational New Drug
Application INN International Non-Proprietary Name IRB
Institutional Review Board IRR infusion-related reaction IV
intravenous IUD intrauterine device LPLV last patient, last visit
LVEF left ventricular ejection fraction mAb monoclonal antibody MBC
metastatic breast cancer MedDRA Medical Dictionary for Regulatory
Activities MUGA multi-gated acquisition (scan) NCI National Cancer
Institute NYHA New York Heart Association ORR overall response rate
PK pharmacokinetic(s) popPK population pharmacokinetics Q3W every 3
weeks QTc QT interval corrected for rate QTcF QT interval corrected
using Fridericia's formula RBC red blood cell rHuPH20 recombinant
human hyaluronidase SC subcutaneous SD standard deviation SI
International System of Units SID single-use injection device SYR
syringe TBD to be determined t.sub.max time of maximum serum
concentration t.sub.1/2 elimination half-life TK Toxicokinetic ULN
upper limit of normal WBC white blood cell
Example 1
Phase I Pertuzumab Subcutaneous Dose-Finding Study in Combination
with Trastuzumab
[0311] This is a Phase I, open-label, two-part multicenter clinical
pertuzumab subcutaneous dose-finding study in combination with
trastuzumab in healthy male volunteers and female patients with
early breast cancer.
[0312] The study is designed the safety and PK of pertuzumab SC for
Q3W treatment by applying a PK-based approach to compare an SC
formulation to the approved W formulation. In this dose-finding
study, it is intended to identify the SC dose that is comparable to
IV with respect to serum concentrations. pertuzumab Q3W SC serum
C.sub.trough concentrations are unknown.
[0313] Different types of pertuzumab SC injections will be assessed
in the study: [0314] Separate administration of pertuzumab SC with
or without trastuzumab SC as separate injections
(co-administration) [0315] Simultaneous administration of
pertuzumab SC and trastuzumab SC as single injection (co-mixed)
[0316] Administration of pertuzumab and trastuzumab SC as a single
injection (co-mixed)
[0317] Initially, in Part 1, Healthy Mail Volunteers (HMVs) will
receive a single dose of IV or SC pertuzumab (with or without
trastuzumab SC) to select the SC dose(s) of pertuzumab expected to
result in serum concentrations comparable to IV pertuzumab, both
when given as co-administration or co-mixed injection. The
pertuzumab SC dose(s) will then be confirmed in patients with
EBC.
[0318] Based on PK data in Part 1 (healthy volunteer cohorts), the
pertuzumab popPK model will be used to identify the target dose(s)
for Part 2. (See, Garg et al., Cancer Chemother Pharmacol (2014)
74:819-829.)
[0319] Upon selection of the target dose and on the basis of
information about the feasibility of an FDC, patients with early
breast cancer (EBC) who have completed their standard treatment
will be enrolled in Part 2 to receive pertuzumab SC at the dose(s)
identifies in Part 1. This identified dose of pertuzumab will be
either co-administered with trastuzumab SC, co-mixed with
trastuzumab SC, or co-formulated with trastuzumab SC in a
fixed-dose combination (FDC). Part 2 will include pertuzumab SC
dose confirmation as well as a comparison of PK from co-mixed and
FDC.
[0320] In this study in which two monoclonal antibodies (mAbs) will
be administered at a volume of up to approximately 15 mL, the
concentration of rHuPH20 will also be evaluated. Previously, 2000
U/mL of the absorption enhancer has been used with trastuzumab SC
and Rituximab SC, however, these are single antibodies and at
volumes less than the pertuzumab/trastuzumab combination studied
here.
[0321] In order to determine if less rHuPH20 would lead to adequate
mAb absorption, the study has been designed to test an enzyme
concentration of 2000 U/mL when both antibodies are given (15 mL
volume) and an enzyme concentration of 667 U/mL (using pertuzumab
that does not contain rHuPH20) when both antibodies are given (15
mL volume). If the PK parameters are approximately equivalent, the
reduced amount of rHuPH20 may be potentially used in the
development of the FDC co-formulated product.
[0322] Objectives and Endpoints
[0323] Primary Objectives
[0324] Part 1(Dose Finding)
[0325] The primary objectives for Part 1 of this study are as
follows: [0326] To select the subcutaneous (SC) loading and
maintenance dose of pertuzumab that results in comparable exposure
to intravenous (W) pertuzumab when pertuzumab SC is given as a
single-agent injections (for eventual use in co-administration with
trastuzumab SC). [0327] To select the SC loading and maintenance
dose of pertuzumab that results in comparable exposure to IV
pertuzumab when pertuzumab SC is given mixed with trastuzumab SC as
a single injection (co-mixed). [0328] To assess whether additional
rHuPH20 is needed when pertuzumab SC and trastuzumab SC are
co-mixed SC.
[0329] Part 2 (Dose Confirmation)
[0330] The primary objectives for Part 2 of this study are as
follows: [0331] To confirm the maintenance dose of pertuzumab SC
when given as a single-agent injection as part of co-administration
with trastuzumab SC
[0332] or [0333] To confirm the maintenance dose of pertuzumab SC
when given mixed with trastuzumab SC in a single injection
(co-mixed) or co-formulated with trastuzumab SC in a ready-to-use
single injection (fixed-dose combination (FDC)).
[0334] Secondary Objectives
[0335] The secondary objectives of this study are as follows:
[0336] To assess the safety and tolerability of pertuzumab SC give
alone or in combination with trastuzumab SC (co-mixed or FDC) in
healthy male volunteers (HMV) and female patients with early breast
cancer (EBC) who have completed standard breast cancer therapy, on
the basis of the following endpoints: [0337] Incidence, nature and
severity of adverse events graded according to the National [0338]
Cancer Institute Common Terminology Criteria for Adverse Events
(NCI CTCAE) v4.03; [0339] Changes in vital sign, left ventricular
ejection fraction (LVEF), and electrocardiogram (ECG) parameters;
[0340] Changes in clinical laboratory results; [0341] Incidence of
anti-therapeutic antibody (ATA) response.
[0342] Study Design
[0343] Description of the Study
[0344] Overview of Study Design
[0345] This is an open-label, two-part, multi-center study of
pertuzumab SC.
[0346] Part 1 of the study is dose finding, in which the loading
and maintenance dose of pertuzumab
[0347] SC will be determined in HMVs. Two types of pertuzumab SC
injections will be assessed: pertuzumab given as a single-agent
injection (for eventual use in co-administration with trastuzumab
SC single-agent injection) and pertuzumab SC co-mixed with
trastuzumab SC in a single injection.
[0348] Part 2 of the study will confirm the pertuzumab SC dose(s)
in patients with EBC who have completed standard breast cancer
therapy. The dose of pertuzumab SC in Part 2 will be
co-administered with trastuzumab SC, co-mixed with trastuzumab SC,
or co-formulated with trastuzumab SC as an FDC. Part 2 will include
pertuzumab SC dose confirmation as well as a comparison of PK from
co-mixed and FDC before a Phase III study.
[0349] See FIG. 6 for the study schema. Safety will be monitored
and blood samples for PK assessment will be drawn according to the
schedule of assessments.
[0350] Part 1 (Dose Finding)
[0351] HMVs were enrolled into Cohorts 1 to 8 (6 subjects per
cohort). Each subject received a single injection. Cohorts 2-4
assessed different pertuzumab SC doses. Cohorts 5 to 8 assessed
doses of pertuzumab+trastuzumab co-mixed. The doses evaluated
within each cohort are as follows: [0352] Cohort 1: 420 mg
pertuzumab W (control) [0353] Cohort 2: 400 mg pertuzumab SC [0354]
Cohort 3: 600 mg pertuzumab SC [0355] Cohort 4: 1200 mg pertuzumab
SC [0356] Cohort 5: 600 mg trastuzumab SC (control) [0357] Cohort
6: 400 mg pertuzumab SC plus 600 mg trastuzumab SC (co-mixed)
[0358] Cohort 7: 1200 mg pertuzumab SC plus 600 mg trastuzumab SC
(co-mixed) [0359] Cohort 8: 1200 mg pertuzumab SC (without rHuPH20)
plus 600 mg trastuzumab SC (co-mixed)
[0360] The different pertuzumab doses were administered by
adjusting the dosing volume. The concentration of pertuzumab and
trastuzumab was 120 mg/mL and rHuPH20 was 2,000 U/mL in the SC
dosing solutions.
[0361] Cohorts 6 and 7 received pertuzumab SC and trastuzumab SC
both containing rhuPH20 in a concentration of 2,000 U/mL, while
HMVs in Cohort 8 were given pertuzumab SC containing no rHuPH20,
co-mixed with trastuzumab SC containing rhuPH20 in a concentration
of 2,000 U/mL, therefore the overall rhuPH20 concentration received
by Cohort 8 was approximately 667 U/mL. Cohort 8 was planned to
assess the impact of a lower concentration of rHuPH20 on the PK of
pertuzumab and trastuzumab when administered in a co-mixed
injection.
[0362] Time to Observe
[0363] To ensure safety during the trial, the first healthy
volunteer in Cohort 2 was closely monitored for safety and
tolerability after treatment with pertuzumab SC and until the end
of Day 3. The 3-day monitoring was completed prior to expanding
Cohort 2 and prior to commencing dosing in Cohort 3 or 4.
[0364] If the pertuzumab SC dose was deemed safe and tolerated in
the first healthy volunteer in Cohort 2, subsequent healthy
volunteers were treated in parallel in Cohorts 2, 3, and 4 without
the addition of the 3-day time period to observe.
[0365] Similarly, 3 healthy volunteers in Cohort 6 were treated
with pertuzumab and trastuzumab SC and closely monitored for safety
and tolerability for 3 days post-dose, prior to expanding Cohort 6
and prior to commencing dosing in Cohort 7 or 8.
[0366] If the pertuzumab and trastuzumab SC dose was deemed safe
and tolerated in the first 3 healthy volunteers in Cohort 6,
subsequent healthy volunteers will be treated in parallel in Cohort
6 without the addition of the 3-day time period to observe.
[0367] Cohorts 7 and 8 were opened at the same time. Three HMVs
were treated with pertuzumab and trastuzumab SC, and closely
monitored for safety and tolerability for 3 days post-dose, prior
to expanding the respective cohorts.
[0368] If the pertuzumab and trastuzumab SC dose was deemed safe
and tolerated in the first 3 healthy volunteers in Cohorts 7 and 8,
subsequent healthy volunteers will be treated in parallel in those
cohorts without the addition of the 3-day time period to
observe.
[0369] Healthy volunteers in Cohorts 1 and 5 can be dosed in
parallel and may be enrolled prior to the first healthy volunteer
in Cohort 2.
[0370] Dose Selection for Part 2
[0371] The selection of the pertuzumab SC doses in Part 1 (Cohorts
2, 3, and 4) is based on the pertuzumab IV population
pharmacokinetics (popPK) model with the values of the trastuzumab
SC PK parameters incorporated. Once the sufficient amount of data
in Part 1 allows the estimation of fixed PK parameters (i.e.,
C.sub.trough, AUC0-inf, maximum serum concentration [C.], time of
maximum serum concentration [T.sub.max]), the pertuzumab SC
(maintenance) dose(s) will be selected for Part 2. This SC dose
will be calculated to deliver a similar pertuzumab exposure to that
of W pertuzumab at 420 mg. Equally, based on PK parameters, one
pertuzumab SC (loading) dose will be calculated to deliver a
similar pertuzumab exposure to that of IV pertuzumab at 840 mg. The
pertuzumab W popPK model will be updated with pertuzumab SC
parameters using the Part 1 data and will be used to correctly
identify the SC maintenance and loading doses.
[0372] The trastuzumab SC 600-mg dose was determined in the Phase
Ib dose--finding study BP22023 and confirmed in the Phase III
HannaH study.
[0373] Additional dose-finding cohorts may be opened if doses from
planned cohorts result in pertuzumab exposure different from the
target concentration or if the variability in pharmacokinetics is
too high to determine a dose for Part 2 of the study.
[0374] Using Part 1 data, a pertuzumab SC dose was calculated to
deliver a similar pertuzumab exposure to that of pertuzumab IV at
420 mg (maintenance dose) and 840 mg (loading doses). The selected
rHuPH20 concentration for the co-mixed administration of pertuzumab
and trastuzumab in
[0375] Cohort B was based on comparbility of safety and pertuzumab
exposure in Cohorts 7 and 8.
[0376] Part 2 (Dose Confirmation)
[0377] Women with EBC who have completed standard (neo) adjuvant
breast cancer therapy were enrolled in Part 2.
[0378] If the feasibility of an FDC product is confirmed, Cohorts B
and C only (not Cohort A; co-administration) may be enrolled in
Part 2. This would allow confirmation of the Part 1 dose and show
comparability between a co-mixed injection and an FDC product. If
there is a PK interaction between pertuzumab and trastuzumab when
co-mixed or if the development of the FDC is not feasible, only
Cohort A may be enrolled in Part 2, which would allow confirmation
of the Part 1 dose. This study design will allow for the selection
of a pertuzumab SC dose and formulation option for the Phase III
study while enrolling the fewest number of patients. The overall
schema for Part 2 shall therefore be Cohort A only or Cohort B and
Cohort C (see 185H FIG. 2). Each cohort will enroll 20 patients and
each patient will receive one dose of pertuzumab and
trastuzumab.
[0379] Each Cohort enrolled 20 patients and each patient received
one dose of pertuzumab and trastuzumab. [0380] Cohort A: pertuzumab
SC (dose determined in Part 1) with 600 mg trastuzumab SC; each
agent administered separately (co-administration)
[0381] or [0382] Cohort B: pertuzumab SC (dose determined in Part
1) with 600 mg trastuzumab SC; both agents administered in one
injection (co-mixed)
[0383] and [0384] Cohort C: pertuzumab SC (dose determined in Part
1) with 600 mg trastuzumab SC; both agents formulated together and
administered in one injection (FDC)
[0385] The highest planned pertuzumab SC dose administered in Part
1 and Part 2 will not exceed 1200 mg.
[0386] Note: The pertuzumab and trastuzumab doses will be modified
by adjusting the dosing volume. The concentration of pertuzumab and
trastuzumab is 120 mg/mL and of rHuPH20 (when present) is 2000 U/mL
in the SC dosing solutions.
[0387] The decision diagram is shown in FIG. 7.
[0388] If there was a PK interaction between pertuzumab and
trastuzumab wen administered in a co-mixed injection, or if the
development of the FDC was not feasible, only Cohort A
(co-administration) was to be enrolled in Part 2. This study design
allowed for the selection of a pertuzumab SC dose and formulation
option for further evaluation in the Phase III study while
enrolling the fewest number of patients. The overall schema for
Part 2 was therefore Cohort A only or Cohort B and Cohort C.
[0389] Criteria for Continuing or Stopping Dosing
[0390] Safety, tolerability, and PK data will be assessed
continuously and prior to expanding cohorts or (if needed) adding
cohorts. The starting doses will be pertuzumab 420 mg IV and 400,
600, and 1200 mg SC.
[0391] To allow informed decisions regarding dosing healthy
volunteers in SC cohorts, the relevant safety and tolerability data
of certain subjects will be reviewed after 3 days before dosing the
next subject in the cohort or opening other cohorts.
[0392] The decision to continue dosing will be made jointly by the
investigator and the Roche Medical Monitor and any other person
that the investigator or Medical Monitor considers necessary to
assist with this decision.
[0393] The dose will not be further administered in any other
healthy volunteer or EBC if the tolerability or safety in a
preceding healthy volunteer or EBC is not acceptable as judged by
the Investigator and the Medical Monitor. Dosing should not be
further administered in any other healthy volunteer or EBC if any
of the events listed below occurs, unless it is obvious that the
occurrence is not related to the administration of the treatment:
[0394] Severe drug-related adverse event [0395] Hypersensitivity
reactions according to the NCI CTCAE (Grades 3 to 5) [0396] An LVEF
drop of >10% points or to <50% (for HMV) [0397] An LVEF drop
of >10% points and to <50% (for EBC) [0398] A repeat
assessment must be carried out within 3 weeks of the first
documented drop and the case must be reviewed by a Cardiologist.
New York Heart Association (NYHA) class II congestive heart failure
(CHF) or greater must be confirmed by a cardiologist.
[0399] It should be made clear that these are guidelines only and
the Investigator together with the Medical Monitor can make an
exception. However, when such an exception is made, the reasons for
it should be clearly documented on the electronic Case Report Form
(eCRF).
[0400] End and Length of the Study
[0401] The end of this study is defined as the date when the last
patient, last visit (LPLV) occurs. LPLV is expected to occur 7
months after the last patient is enrolled. The total length of the
study, from screening of the first patient to the end of the study,
is expected to be approximately 16 to 24 months. There will be a
maximum of 34 weeks for healthy volunteers/patients from screening
to follow-up (up to 4 weeks for screening period and 30 weeks for
the study conduct and follow-up). For each study participant (HMV
and EBC patients), the screening period was up to 4 weeks and the
follow-up was performed approximately 7 months after the study drug
administration.
[0402] Materials and Methods
[0403] Study Population
[0404] Part 1 Inclusion Criteria
[0405] HMVs must meet the following criteria for study entry:
[0406] Signed Informed Consent Form [0407] Healthy male subjects,
ages 18 to 45 years inclusive [0408] Able to comply with the study
protocol, in the investigator's judgment [0409] LVEF .gtoreq.55%
measured by echocardiography (ECHO) or multi-gated acquisition
(MUGA) scan [0410] A body mass index (BMI) between 18 and 32
kg/m.sup.2 inclusive [0411] Agreement to remain abstinent (refrain
from heterosexual intercourse) or use contraceptive measures and
agreement to refrain from donating sperm, as defined below: [0412]
With female partners of childbearing potential, men must remain
abstinent or use a condom plus an additional contraceptive method
that together result in a failure rate of <1% per year during
the treatment period and for at least 7 months after the
administration of pertuzumab and/or trastuzumab. The reliability of
sexual abstinence should be evaluated in relation to the duration
of the clinical trial and the preferred and usual lifestyle of the
patient. Periodic abstinence (e.g., calendar, ovulation,
symptothermal, or postovulation methods) and withdrawal are not
acceptable methods of contraception. [0413] Men must refrain from
donating sperm during this same period. [0414] With pregnant female
partners, men must remain abstinent or use a condom during the
treatment period and for at least 7 months after the administration
of pertuzumab and/or trastuzumab to avoid exposing the embryo.
[0415] No contraindications from detailed medical and surgical
history and physical [0416] Intact normal skin without potentially
obscuring tattoos, pigmentation, or lesions in the area for
intended injection in the thighs.
[0417] Part 1 Exclusion Criteria [0418] HMVs who meet any of the
following criteria will be excluded from study entry: [0419]
Positive urine test for drugs of abuse as per local standard [0420]
Positive result on hepatitis B virus (HBV), hepatitis C virus
(HCV), or human immunodeficiency virus (HW) 1 or 2 test [0421]
History of exposure to HBV, HCV, or HIV [0422] Active viral
hepatitis infection (hepatitis B or C) or HIV infection [0423]
Systolic blood pressure (BP) .gtoreq.140 mmHg or <90 mmHg, or
diastolic BP >0 mmHg or <50 mmHg [0424] Use of prohibited
medications or herbal remedies within 10 days or 5 times the
elimination half-life (whichever is longer) prior to study drug
administration [0425] Clinically significant abnormalities in
laboratory test results (including hepatic and renal panels,
complete blood count, chemistry panel, and urinalysis) [0426]
Clinically relevant ECG abnormalities on screening or baseline ECG,
including but not limited to the following: [0427] QTc interval
(QTcB >450 msec) [0428] Notable resting tachycardia (HR >100
bpm) [0429] Difference between highest and lowest of any baseline
QTc at a specific timepoint >30 msec [0430] Measurement of QT
interval imprecise (e.g., flat T waves, arrhythmias, etc.) [0431]
Evidence of atrial fibrillation, atrial flutter, right or left
bundle branch block, Wolf-Parkinson-White syndrome, or cardiac
pacemaker [0432] Any other significant abnormality [0433] History
of any cardiac condition or LVEF <55% [0434] Participation in an
investigational drug or device study within 90 days prior to
screening [0435] Donation of blood >500 mL within 3 months prior
to screening [0436] Known allergy to hyaluronidase, bee, or vespid
venom, or any other ingredient in the formulation of rHuPH20
(Hylenex.RTM. recombinant [hyaluronidase human injection]) [0437]
Known hypersensitivity to any of the study treatments or to
excipients of recombinant human or humanized antibodies [0438]
History of hypersensitivity or significant allergic reactions,
spontaneous or following any prior drug administration [0439]
Apparent clinically relevant family history of hypersensitivity,
allergy, or severe cardiac diseases [0440] Lower extremity edema or
pathology (e.g., cellulitis, lymphatic disorder or prior surgery,
preexisting pain syndrome, previous lymph node dissection, etc.)
that could interfere with any protocol-specified outcome assessment
[0441] Any clinically relevant history of systemic disease (e.g.,
malignancy, diabetes mellitus, gastrointestinal, renal, hepatic,
cardiovascular, rheumatological, or pulmonary disease) [0442]
History of breast cancer, treatment for breast cancer, or treatment
with anthracyclines or other cardiotoxic drugs [0443] Current
disease or condition that could interfere with, or for which the
treatment of might interfere with, the conduct of the study, or
that would, in the opinion of the investigator, pose an
unacceptable risk to the subject in this study [0444] Current
chronic daily treatment (continuous for >3 months) with
corticosteroids (dose .gtoreq.10 mg/day methylprednisolone),
excluding inhaled corticosteroids [0445] Receipt of W antibiotics
for infection within 7 days prior to enrollment into the study.
[0446] Study Entry Criteria: Part 2 (Female Patients with Early
Breast Cancer)
[0447] Part 2 Inclusion Criteria [0448] Patients must meet the
following criteria for study entry: [0449] Signed Informed Consent
Form [0450] Females age .gtoreq.18 years [0451] Able to comply with
the study protocol, in the investigator's judgment [0452] Eastern
Cooperative Oncology Group performance status of 0 [0453] Current
non-metastatic adenocarcinoma of the breast that meets the
following criteria: [0454] a) Treated with adequate surgical
procedure [0455] b) Completed standard anticancer (neo)adjuvant
treatment (chemotherapy/biological) >7 months prior to study
drug administration [0456] c) Treated with radiotherapy if
applicable [0457] Baseline LVEF .gtoreq.55% measured by ECHO or
MUGA scan [0458] Negative pregnancy test in women of childbearing
potential who are premenopausal or less than 12 months of
amenorrhea post-menopause, and have not undergone surgical
sterilization. [0459] For women of childbearing potential:
agreement to remain abstinent (refrain from heterosexual
intercourse) or use non-hormonal contraceptive methods that result
in a failure rate of <1% per year during the treatment period
and for at least 7 months after the administration of pertuzumab
and trastuzumab [0460] A woman is considered to be of childbearing
potential if she is postmenarcheal, has not reached a
postmenopausal state (.gtoreq.12 continuous months of amenorrhea
with no identified cause other than menopause), and has not
undergone surgical sterilization (removal of ovaries and/or
uterus). [0461] Examples of contraceptive methods with a failure
rate of <1% per year include bilateral tubal ligation, male
sterilization and copper intrauterine devices (IUDs). [0462] The
reliability of sexual abstinence should be evaluated in relation to
the duration of the clinical trial and the preferred and usual
lifestyle of the patient. Periodic abstinence (e.g., calendar,
ovulation, symptothermal, or postovulation methods) and withdrawal
are not acceptable methods.
[0463] Part 2 Exclusion Criteria [0464] Patients who meet any of
the following criteria will be excluded from study entry: [0465]
Concurrent other malignancy requiring therapy of any modality that
may interfere with PK investigations or result in unexpected
toxicity [0466] Maximum cumulative dose of doxorubicin >360
mg/m.sup.2 or maximum cumulative dose of epirubicin >720
mg/m.sup.2 or any prior anthracyclines unrelated to the present
breast cancer [0467] Serious, uncontrolled concomitant disease that
would contraindicate the use of any of the investigational drugs
used in this study or that would put the patient at high risk for
treatment-related complications. [0468] History of other malignancy
within 5 years prior to screening, except for appropriately treated
carcinoma in situ of the cervix, non-melanoma skin carcinoma, or
Stage I uterine cancer [0469] Patients currently participating in
other studies of investigational agents unless agreed by the
investigator and Sponsor [0470] Serious cardiac illness or medical
conditions [0471] Any previous or concurrent condition suggesting
susceptibility to hypersensitivity or allergic reactions. Patients
with mild or seasonal allergies may be included after discussion
between the investigator and Sponsor. [0472] Severe
infusion-related reactions (IRRs) experienced during any previous
therapy with pertuzumab or trastuzumab [0473] Known allergy to
hyaluronidase, bee, or vespid venom, or any other ingredient in the
formulation of Hylenex.RTM. [0474] Any of the following abnormal
laboratory tests on Day -1 prior to trastuzumab treatment: Serum
total bilirubin >1.25.times. upper limit of normal (ULN; with
the exception of Gilbert's syndrome)
[0475] Alanine aminotransferase (ALT) or aspartate aminotransferase
(AST) >1.25.times.ULN;
[0476] Albumin <25 g/L
[0477] Alkaline phosphatase (ALP) >2.5.times.ULN
[0478] Serum creatinine >1.5.times.ULN
[0479] Total white blood cell (WBC) count <2500 cells/mm3
[0480] Absolute neutrophil count <1500 cells/mm3
[0481] Platelets <100,000 cells/mm3 [0482] Pregnant or lactating
women, or women intending to become pregnant during the study
[0483] Women of childbearing potential or less than 1 year after
menopause (unless surgically sterile) who are unable or unwilling
to use adequate contraceptive measures during study treatment and
for 7 months after study drug administration [0484] Residual
toxicity resulting from previous therapy (e.g., hematologic,
cardiovascular or neurologic that is Grade .gtoreq.2). Alopecia is
permitted. [0485] Uncontrolled hypertension (systolic BP >150
mmHg and/or diastolic BP >100 mmHg) [0486] Clinically
significant (i.e., active) cardiovascular disease, including but
not limited to cerebrovascular accident/stroke or myocardial
infarction within 6 months prior to first study treatment; unstable
angina; CHF of NYHA Grade II or higher; serious cardiac arrhythmia
requiring medication; or other cardiovascular problem that is
uncontrolled or is currently controlled with medication [0487]
Positive result on HBV, HCV, or HW 1 or 2 test [0488] History of
exposure to HBV, HCV, or HIV [0489] Active viral hepatitis
infection (hepatitis B or C) or HIV infection [0490] Receipt of W
antibiotics for infection within 7 days prior to enrollment into
the study [0491] Current chronic daily treatment (continuous for
>3 months) with corticosteroids (dose equivalent to or greater
than 10 mg/day methylprednisolone), excluding inhaled steroids
[0492] Known hypersensitivity to any of the study treatments or to
excipients of recombinant human or humanized antibodies.
[0493] Method of Treatment Assignment
[0494] Healthy volunteers and patients will be identified for
potential recruitment using pre-screening enrollment logs,
Institutional Review Board (IRB)/Ethics Committee
(EC).quadrature.approved newspaper/radio advertisements, and
mailing lists prior to consenting to participate in the study.
[0495] Part 1 (Healthy Volunteers)
[0496] Approximately 48 healthy volunteers will be recruited
initially for Part 1. Patient numbers will be allocated
sequentially in the order in which they are enrolled. Additional
dose-finding cohorts may be opened if necessary.
[0497] Part 2 (Patients with EBC)
[0498] Approximately 40 patients with EBC will be recruited for
Part 2. Patient numbers will be allocated sequentially in the order
in which they are enrolled.
[0499] Study Treatment
[0500] The investigational medicinal products (IMPs) for this study
are pertuzumab and trastuzumab.
[0501] Formulation, Packaging and Handling
[0502] Study drug packaging will be overseen by the Roche clinical
trial supplies department and bear a label with the identification
required by local law, the protocol number, and drug identification
and dosage. The packaging and labeling of the study drug will be in
accordance with Roche standard and local regulations. Upon arrival
of IMPs at the site, site personnel should check them for damage
and verify proper identity, quantity, integrity of seals and
temperature conditions and report any deviations or product
complaints to the monitor upon discovery. The qualified individual
responsible for dispensing the study drug will prepare the correct
dose according to the schedule. This individual will write the date
dispensed, date administered, and patient number and initials, as
appropriate, on the label of the study drug vial and/or on the Drug
Accountability Record. This individual will also record the
study-drug batch or lot number received by each patient during the
study.
[0503] Pertuzumab
[0504] Three formulations of pertuzumab were used:
[0505] Pertuzumab Formulation I is a sterile, colorless to slightly
brownish concentrate for solution for infusion provided as
single-use W formulation containing 30 mg/mL pertuzumab in
L-Histidine acetate buffer containing excipients sucrose and
polysorbate 20. Each 20-mL vial contains 420 mg of pertuzumab (14.0
mL/vial).
[0506] Pertuzumab Formulation 2 is a sterile, colorless to slightly
brownish solution for injection provided as single-use SC
formulation containing 120 mg/mL pertuzumab in L-histidine acetate
buffer containing excipients sucrose, polysorbate 20, methionine,
and rHuPh20 (2000 U/mL). Each 10-mL vial contains 600 mg of
pertuzumab (5.0 mL/vial).
[0507] Pertuzumab Formulation 3 is a sterile, colorless to slightly
brownish solution for injection provided as single-use SC
formulation containing 120 mg/mL pertuzumab in L-Histidine acetate
buffer containing excipients sucrose, polysorbate 20, and
methionine. Each 10-mL vial contains 600 mg of pertuzumab (5.0
mL/vial).
[0508] No preservative is used with pertuzumab since the vials are
intended for single use only. The recommended storage conditions
for the drug product are between 2.degree. C. and 8.degree. C.,
protected from light. The drug product must not be frozen.
[0509] Trastuzumab
[0510] Trastuzumab formulation is a sterile, colorless to slightly
brownish concentrate solution for injection containing 120 mg/mL of
trastuzumab in L-Histidine/Histidine-HCl buffer containing
excipients trehalose, polysorbate 20, methionine, and rHuPh20 (2000
U/mL). Each 5-mL vial contains 600 mg of R00452317 (5.0
mL/vial).
[0511] No preservative is used with trastuzumab since the vials are
intended for single use only. The recommended storage conditions
for the drug product are between 2.degree. C. and 8.degree. C.,
protected from light. The drug product must not be frozen.
[0512] Dosage, Administration and Compliance
[0513] Pertuzumab and Trastuzumab SC
[0514] The qualified individual responsible for dispensing the
study drug will prepare the correct dose. This individual will
write the date dispensed and subject number and initials on the
study drug vial label and on the Drug Accountability Record. This
individual will also record the study drug batch or lot number
received by each subject during the study.
[0515] HMVs will receive a single dose of pertuzumab IV, pertuzumab
SC, trastuzumab SC, or pertuzumab SC and trastuzumab SC mixed
together (co-mixed). Patients will receive a single dose of
pertuzumab and trastuzumab as two single-agent injections
(co-administration) or one injection of pertuzumab and trastuzumab
mixed together (co-mixed) or pertuzumab co-formulated with
trastuzumab as one FDC injection.
[0516] Healthy volunteers and patients may also be administered a
pre-medication (e.g., acetaminophen [paracetamol] and/or
promethazine), prior to the administration of pertuzumab and/or
trastuzumab SC, at the discretion of the investigator to reduce to
risk of infusion- or injection-related reactions.
[0517] Any overdose or incorrect administration of study drug
should be noted on the Study Drug Administration eCRF. Adverse
events associated with an overdose or incorrect administration of
study drug should be recorded on the Adverse Event eCRF.
[0518] Administered (Pertuzumab IV)
[0519] Healthy volunteers receiving pertuzumab IV (Cohort
1--control) were given a dose of 420 mg.
[0520] The dose of pertuzumab was administered over 60 (.+-.10)
minutes, and healthy volunteers were observed for a further 60
minutes. The infusion should be slowed or interrupted if the
patient experiences infusion-related symptoms.
[0521] Administered Doses (Pertuzumab SC and Trastuzumab SC)
[0522] Healthy volunteers and patients receiving pertuzumab SC
(Cohorts 2-8, A and B) were given doses between 400 and 1200 mg.
Healthy volunteers and patients receiving trastuzumab SC (Cohorts
5-8, A and B) were given a dose of 600 mg (see Table 3).
TABLE-US-00004 TABLE 3 Doses and Cohorts Injection Dose Volume
Cohort Agent (mg) (mL) HMV 1 pertuzumab IV 420 2 pertuzumab SC 400
3.3 3 pertuzumab SC 600 5 4 pertuzumab SC 1200 10 5 trastuzumab SC
600 5 6 pertuzumab SC + 400 + 600 8.3 trastuzumab SC (co-mixed) 7
pertuzumab SC + 1200 + 600 15 trastuzumab SC (co-mixed) 8
pertuzumab SC.sup.a + 1200 + 600 15 trastuzumab SC (co-mixed)
9.sup.b pertuzumab IV/SC +/- X +/- 600 X trastuzumab SC Patients A
pertuzumab SC + trastuzumab TBD + 600 TBD SC (co-admin) B
pertuzumab SC + trastuzumab TBD + 600 TBD SC (co-mixed) C
pertuzumab SC + trastuzumab TBD + 600 TBD SC (FDC) FDC = fixed-dose
combination; HMV = healthy male volunteer; IV = intravenous;
rHuPH20 = recombinant human hyaluronidase; SC = subcutaneous; TBD =
to be determined. .sup.arHuPH20 concentration = 667 U/mL only
.sup.bIf additional cohort necessary
[0523] SC injections were administered into the anterior thigh
region. Patients in Cohort A will receive the two co-administration
injections in opposite thighs, with the second injection
administered immediately after the first.
[0524] The appropriate amount of solution should be withdrawn from
the vials. Refer to the pharmacy manual for instruction.
[0525] The 27-gauge injection needle is inserted using sterile
technique in the SC tissue of the thigh. The needle should be fully
inserted, being careful that the tip of the needle is deeper than
the dermis but not as deep as the underlying muscle. The goal of
the placement angle and needle depth is to achieve uniform
placement into every patient's SC tissue. Study drug should not be
injected into moles, scars, or bruises. The skin should be pinched
and needle inserted before the skin is released and the pressure on
the syringe can be applied.
[0526] The injection should be manually pushed at a flow rate of no
more than 2 mL/min, therefore administration should take
approximately 2-8 minutes depending on the dose being administered.
If there is a request by the subject to interrupt the injection,
the pressure on the syringe should initially be eased to alleviate
the pain. If the pain is not alleviated, the injection should be
stopped and the subject should be asked when they are comfortable
to resume the injection.
[0527] Timing in Relation to Meals, Physical Activities, and
Procedures
[0528] Meals were similar in composition and time of administration
across all cohorts. The consumption of foods and beverages
containing caffeine (e.g., tea, coffee, chocolate, and soft drinks)
or alcohol will not be permitted from Day -1 to Day 2. The use of
tobacco is not permitted during the in-clinic portion of the
study.
[0529] Light ambulatory activities will be permitted, with the
level of activities kept as similar as possible on all days in the
clinical research unit.
[0530] Concomitant Therapy, Prohibited Food, and Additional
Restrictions
[0531] Concomitant therapy includes any medication (e.g.,
prescription drugs, over-the-counter drugs, vaccines, herbal or
homeopathic remedies, nutritional supplements) used by a healthy
volunteer/patient within 30 days of study screening. All such
medications should be reported to the investigator and recorded on
the Concomitant Medications eCRF.
[0532] Permitted Therapy
[0533] For the healthy volunteers, no concomitant medication will
be permitted, with the exception of medications to treat adverse
events, unless the rationale for exception is discussed between the
investigator and Medical Monitor and clearly documented.
[0534] For patients with EBC, the following treatments are
permitted during the study:
[0535] Acceptable methods of contraception must be used when the
female patient or male partner is not surgically sterilized or does
not meet the study definition of postmenopausal (.gtoreq.12 months
of amenorrhea)
[0536] H.sub.1 and H.sub.2 antagonists (e.g., diphenhydramine,
cimetidine)
[0537] Cardiovascular medications: angiotensin-converting enzyme
(ACE) inhibitors, angiotensin receptor blockers, .beta. blockers,
calcium-channel blockers and diuretics (for treatment of arterial
hypertension with a goal to reduce blood pressure to <140/90
mmHg), .beta. blockers, calcium-channel blockers, and digoxin (for
heart rate control), and thrombocyte aggregation inhibitors
[0538] Analgesics/anti-inflammatories (e.g.,
paracetamol/acetaminophen, meperidine, opioids)
[0539] Short-term use of corticosteroids to treat or prevent
allergic or infusion reactions
[0540] Anti-emetics (approved prophylactic serotonin antagonists,
benzodiazepines, dopamine antagonists, etc.)
[0541] Medication to treat diarrhea (e.g., loperamide)
[0542] Estrogen-receptor antagonists (e.g., tamoxifen), aromatase
inhibitors (e.g., anastrazole, exemestane), and gonadotrophin
hormone-releasing hormone agonists (e.g., buserelin, triptorelin)
after surgery, as per local practice and guidelines
[0543] Ovarian suppression (luteinizing hormone-releasing hormone
[LHRH] analog)
[0544] Bisphosphonates (to be used in accordance with the approved
labeled indication and/or nationally recognized treatment
guidelines)
[0545] At the discretion of the investigator, healthy volunteers
and patients may also be administered a pre-medication (e.g.,
acetaminophen [paracetamol] and/or promethazine) prior to the
administration of pertuzumab and/or trastuzumab SC to reduce the
risk of IRRs or injection-related reactions.
[0546] Prohibited Therapy
[0547] Use of the following therapies is prohibited during the
study and for at least 10 days prior to initiation of study
treatment: [0548] Anti-cancer therapies other than those
administered in this study or listed in permitted therapies above,
including cytotoxic chemotherapy, radiotherapy, immunotherapy, and
biological anti-cancer therapy [0549] Any targeted therapy, other
than those used in this study [0550] Any investigational agent,
except for those used for this study [0551] Initiation of herbal
remedies: Herbal remedies initiated prior to study entry and
continuing during the study are not permitted and must be reported
on the appropriate eCRF. [0552] Any systemically active, oral,
injected, or implanted hormonal method of contraception, except for
progesterone-coated IUDs that had been previously implanted [0553]
Estrogen-replacement therapy (hormone-replacement therapy) [0554]
No prescription medicines, over-the-counter medicines, or herbal
remedies are allowed for at least 10 days before study drug dose,
through the end of the study unless either agreed by study
doctor.
[0555] Prohibited Food
[0556] The consumption of foods and beverages containing caffeine
(e.g., tea, coffee, chocolate, and soft drinks) or alcohol will not
be permitted from Day -1 to Day 2.
[0557] Additional Restrictions
[0558] Meals will be similar in composition and time of
administration across all cohorts. The use of tobacco is not
permitted during the in-clinic portion of the study. Light
ambulatory activities will be permitted, with the level of
activities kept as similar as possible on all days in the clinical
research unit.
[0559] Study Assessments
[0560] Part 1 (Male Healthy Volunteers)
[0561] Healthy volunteers will report to the unit on Day -1 for
pre-dose assessments and will stay overnight (for 3 nights) at the
unit. Healthy volunteers may be discharged on the morning of Day 2
at the discretion of the investigator and return to the clinic on
Day 3.
[0562] On Day 1, healthy volunteers will be given pertuzumab by IV
infusion or a SC injection of pertuzumab, trastuzumab SC, or
pertuzumab and trastuzumab SC (co-mixed) into the anterior thigh
region. Injection sites will be digitally photographed after a SC
injection if a severe adverse reaction is observed at the injection
site.
[0563] Safety and pharmacokinetic assessments will be performed at
regular intervals during the study according to the schedule of
assessments. Healthy volunteers will remain in the unit until the
48-hour pharmacokinetic assessment is complete. They will return
for PK and safety assessments on specified days afterwards.
[0564] A follow-up visit will be performed 7 months after study
drug administration. Healthy volunteers will be discharged from the
study by a responsible physician upon completion of the follow-up
visit.
[0565] Part 2 (Female Patients with EBC)
[0566] Patients will report to the unit on Day -1 for pre-dose
assessments. Patients will return to the unit on Day 1 and will be
given pertuzumab and trastuzumab as an SC injection into the
anterior thigh region. Patients in Cohort A will receive the 2
injections in opposite thighs, with the second injection
administered immediately after the first. Injection sites will be
digitally photographed after a SC injection if a severe adverse
reaction at the injection site is observed.
[0567] Safety and PK assessments will be performed at regular
intervals during the study as per the schedule of assessments.
Patients will remain in the unit until 12 hours post-dose. They
will return for pharmacokinetic and safety assessments on specified
days afterwards.
[0568] A follow-up visit will be performed 7 months after study
drug administration. Patients will be discharged from the study by
a responsible physician upon completion of the follow-up visit.
[0569] Follow-Up Visit
[0570] For HMVs or EBC patients with ongoing cardiac adverse events
(regardless of cause) or study treatment--related adverse events,
serious adverse events, or events of special interest on Day 85 or
adverse events, serious adverse events, or events of special
interest occurring between the Day 85 and the follow-up visit, all
assessments in the follow-up visit will be performed and PK/ATA
samples taken.
[0571] For HMVs with no cardiac adverse events (regardless of
cause) or study treatment-related adverse events, serious adverse
events, or adverse events of special interest ongoing on Day 85 and
none occurring between the Day 85 and the follow-up visit, only the
pregnancy follow-up of female partners is required. This visit may
be performed by phone call.
[0572] For EBC patients with no cardiac adverse events (regardless
of cause) or study treatment--related adverse events, serious
adverse events, and adverse events of special interest ongoing on
Day 85 and none occurring between the Day 85 and the follow-up
visit, only the pregnancy test (for patients of childbearing
potential) is required at this visit.
[0573] For postmenopausal EBC patients (.gtoreq.12 months of
amenorrhea) with no cardiac adverse events (regardless of cause) or
study treatment--related adverse events, serious adverse events,
and adverse events of special interest ongoing on Day 85 and none
occurring between the Day 85 and the follow-up visit, the follow up
visit may be performed by phone call.
[0574] Safety Parameters and Adverse Events
[0575] Safety assessments will consist of monitoring and recording
adverse events, including serious adverse events and adverse events
of special interest, performing protocol-specified safety
laboratory assessments, measuring protocol-specified vital signs,
and conducting other protocol-specified tests that are deemed
critical to the safety evaluation of the study.
[0576] Adverse Events
[0577] According to the Guidelines for Good Clinical Practice, an
adverse event is any untoward medical occurrence in a clinical
investigation subject administered a pharmaceutical product,
regardless of causal attribution. An adverse event can therefore be
any of the following: [0578] Any unfavorable and unintended sign
(including an abnormal laboratory finding), symptom, or disease
temporally associated with the use of a medicinal product, whether
or not considered related to the medicinal product. [0579] Any new
disease or exacerbation of an existing disease (a worsening in the
character, frequency, or severity of a known condition). [0580]
Recurrence of an intermittent medical condition (e.g., headache)
not present at baseline. [0581] Any deterioration in a laboratory
value or other clinical test (e.g., ECG, X-ray) that is associated
with symptoms or leads to a change in study treatment or
concomitant treatment or discontinuation from study drug. [0582]
Adverse events that are related to a protocol-mandated
intervention, including those that occur prior to assignment of
study treatment (e.g., screening invasive procedures such as
biopsies).
[0583] Serious Adverse Events (Immediately Reportable to the
Sponsor)
[0584] A serious adverse event is any adverse event that meets any
of the following criteria: [0585] Is fatal (i.e., the adverse event
actually causes or leads to death) [0586] Is life threatening
(i.e., the adverse event, in the view of the investigator, places
the patient at immediate risk of death). This does not include any
adverse event that had it occurred in a more severe form or was
allowed to continue might have caused death. [0587] Requires or
prolongs inpatient hospitalization. [0588] Results in persistent or
significant disability/incapacity (i.e., the adverse event results
in substantial disruption of the patient's ability to conduct
normal life functions. [0589] Is a congenital anomaly/birth defect
in a neonate/infant born to a mother exposed to study drug. [0590]
Is a significant medical event in the investigator's judgment
(e.g., may jeopardize the patient or may require medical/surgical
intervention to prevent one of the outcomes listed above).
[0591] The terms "severe" and "serious" are not synonymous.
Severity refers to the intensity of an adverse event (e.g., rated
as mild, moderate, or severe, or according to NCI CTCAE v4.03; the
event itself may be of relatively minor medical significance (such
as severe headache without any further findings).
TABLE-US-00005 TABLE 4 Adverse Event Severity Grading Scale for
Events Not Specifically Listed in NCI CTCAE Grade Severity 1 Mild;
asymptomatic or mild symptoms; clinical or diagnostic observations
only; or intervention not indicated 2 Moderate; minimal, local, or
non-invasive intervention indicated; or limiting age-appropriate
instrumental activities of daily living .sup.a 3 Severe or
medically significant, but not immediately life-threatening;
hospitalization or prolongation of hospitalization indicated;
disabling; or limiting self-care activities of daily living .sup.b,
c 4 Life-threatening consequences or urgent intervention indicated
.sup.d 5 Death related to adverse event .sup.d NCI CTCAE = National
Cancer Institute Common Terminology Criteria for Adverse Events.
Note: Based on the most recent version of NCI CTCAE (v4.03) .sup.a
Instrumental activities of daily living refer to preparing meals,
shopping for groceries or clothes, using the telephone, managing
money, etc. .sup.b Examples of self-care activities of daily living
include bathing, dressing and undressing, feeding oneself, using
the toilet, and taking medications, as performed by patients who
are not bedridden. .sup.c If an event is assessed as a "significant
medical event," it must be reported as a serious adverse event, per
the definition of serious adverse event. .sup.d Grade 4 and 5
events must be reported as serious adverse events, per the
definition of serious adverse event.
[0592] Adverse Events of Special Interest (Immediately Reportable
to the Sponsor)
[0593] Adverse events of special interest are required to be
reported by the investigator to the Sponsor immediately (i.e., no
more than 24 hours after learning of the event. Adverse events of
special interest for this study include the following: [0594] Cases
of potential drug-induced liver injury that include an elevated ALT
or AST in combination with either an elevated bilirubin or clinical
jaundice, as defined by Hy's law. [0595] Suspected transmission of
an infectious agent by the study drug, as defined: Any organism,
virus, or infectious particle (e.g., prion protein transmitting
transmissible spongiform encephalopathy), pathogenic or
non-pathogenic, is considered an infectious agent. A transmission
of an infectious agent may be suspected from clinical symptoms or
laboratory findings that indicate an infection in a patient exposed
to a medicinal product. This term applies only when a contamination
of the study drug is suspected. [0596] An asymptomatic decline in
LVEF requiring treatment. Note: In general, asymptomatic declines
in LVEF should not be reported as adverse events since LVEF data
are collected separately on the eCRF. Exceptions to this rule are
as follows: [0597] An asymptomatic decline in LVEF to a value of
10-percentage points below baseline or lower and <50% must be
reported as an adverse event. [0598] An asymptomatic decline in
LVEF that requires treatment or that leads to discontinuation of
study treatment must be reported in an expedited manner using the
Adverse Event eCRF and classifying the event as a non-serious event
of special interest that is immediately reportable.
[0599] Selected Adverse Events
[0600] Heart Failure
[0601] Symptomatic LVSD (referred to as heart failure) should be
reported as a serious adverse event. If the diagnosis is heart
failure, it should be reported as such, and not as individual signs
and symptoms of heart failure. On the eCRF, signs and symptoms
should be recorded. A cardiac consultation is recommended for
patients who develop symptomatic LVSD (heart failure). Heart
failure should be graded according to NCI CTCAE v4.03 (Grade 2, 3,
4, or 5), as well as according to the NYHA classification (Class
II, III, and W). Left ventricular systolic dysfunction should not
be used to describe symptomatic dysfunction, as per NCI CTCAE
v4.03.
[0602] Heart failure occurring during the study and up to 5 years
after the last patient enrolled must be reported irrespective of
causal relationship and followed until one of the following
occurs:
[0603] resolution or improvement to baseline status, no further
improvement can be expected, or death.
[0604] Asymptomatic Declines in Left Ventricular Ejection
Fraction
[0605] Asymptomatic declines in LVEF should not be reported as
adverse events because LVEF data are collected separately on the
eCRF. Exceptions to this rule are as follows: [0606] An
asymptomatic decline in LVEF of .gtoreq.10-percentage points from
baseline to an LVEF <50% must be reported as an adverse event
with the term of ejection fraction decreased, as per NCI CTCAE
v4.03. In addition, a comment in the adverse events comments field
should confirm that the event was asymptomatic. [0607] An
asymptomatic decline in LVEF requiring treatment or leading to
discontinuation of pertuzumab and trastuzumab must also be
reported. This adverse event should also be captured as a
non-serious event of special interest on the serious adverse event
form, and a comment should be added to the adverse events comments
field, confirming that the event was asymptomatic.
[0608] Table 5 shows the New York Heart Association Classification
and Left Ventricular Systolic Dysfunction National Cancer Institute
Common Terminology Criteria for Adverse Events, Version 4.03
grading.
TABLE-US-00006 TABLE 5 Class I Patients with cardiac disease but
without resulting limitations of physical activity. Ordinary
physical activity does not cause undue fatigue, palpitation,
dyspnea or anginal pain. Class II Patients with cardiac disease
resulting in slight limitations of physical activity. They are
comfortable at rest. Ordinary physical activity results in fatigue,
palpitation, dyspnea or anginal pain. Class III Patients with
cardiac disease resulting in marked limitations of physical
activity. They are comfortable at rest. Less than ordinary physical
activity causes fatigue, palpitation, dyspnea or anginal pain.
Class IV Patients with cardiac disease resulting in inability to
carry on any physical activity without discomfort. Symptoms of
cardiac insufficiency or of the angina syndrome may be present even
at rest. If any physical activity is undertaken, discomfort is
increased.
[0609] Weatherall D J, Lendingham J G G, editors. Oxford Rextbook
of Medicine. Third Edition. New York: Oxford University Press,
1996.
[0610] Table 6 summarizes the reporting conventions for LVSD and
Heart Failure:
TABLE-US-00007 TABLE 6 Reporting Conventions for left Ventricular
Systolic Dysfunction/Heart Failure Observation How to Report Term
to be Reported Grading Asymptomatic decline in LVEF of <10% No
additional reporting required; LVEF NA NA points from baseline
results to be reported on eCRF. or to an LVEF of .gtoreq.50%
Asymptomatic decline in LVEF of .gtoreq.10% AE .sup.a Ejection
fraction decreased .sup.a NCI CTCAE for "ejection points from
baseline (eCRF AE eForm) fraction decreased" to an LVEF of <50%
Asymptomatic decline in LVEF requiring AE (eCRF AE eForm) and
report as a Ejection fraction decreased .sup.a NCI CTCAE for
"ejection treatment or leading non-serious AESI fraction decreased"
to discontinuation of (reported on an SAE form) pertuzumab and/or
trastuzumab Heart failure/CHF AE (eCRF AE eForm) and "Heart
failure" NCI CTCAE for "ejection (symptomatic LVSD) .sup.b SAE (SAE
form) fraction decreased" AE = adverse event; AESI = adverse event
of special interest; CHF = congestive heart failure; eCRF =
electronic Case Report Form; LVEF = left ventricular ejection
fraction; LVSD = left ventricular systolic dysfunction; NA = not
applicable; NCI CTCAE = National Cancer Institute CommonTerminology
Criteria for Adverse Events; NYHA = New York Heart Association; SAE
= serious adverse event. Note: Any symptomatic LVSD event must be
reported as heart failure. .sup.a Report the status as asymptomatic
and provide the LVEF value in the comments field as appropriate.
.sup.b Any symptomatic LVSD event must be reported as "heart
failure."
[0611] The adverse event severity grading scale for the NCI CTCAE
(v4.03) will be used for assessing adverse event severity. See
Table 4 above for assessing severity for adverse events that are
not specifically listed in the NCI CTCAE.
[0612] Assessment of Casualty of Adverse Events
[0613] Investigators should use their own knowledge of the patient,
the circumstances surrounding the event, and an evaluation of any
potential alternative causes to determine whether an adverse event
is considered to be related to the study drug, indicating "yes" or
"no" accordingly. The following guidance should be taken into
consideration: [0614] Temporal relationship of event onset to the
initiation of study drug [0615] Course of the event, considering
especially the effects of dose reduction, discontinuation of study
drug, or reintroduction of study drug (as applicable)\ [0616] Known
association of the event with the study drug or with similar
treatments [0617] Known association of the event with the disease
under study [0618] Presence of risk factors in the patient or use
of concomitant medications known to increase the occurrence of the
event [0619] Presence of non-treatment-related factors that are
known to be associated with the occurrence of the event
[0620] For patients receiving combination therapy, causality will
be assessed individually for each protocol-mandated therapy.
[0621] Infusion-Related Reactions, Injection Reactions, and Local
Injection Site Reactions
[0622] Adverse events that occur during or within 24 hours after
study drug administration and are judged to be related to study
drug infusion or injection should be captured as a diagnosis (e.g.,
"infusion-related reaction," "injection reaction," "injection-site
reaction") on the Adverse Event eCRF. If possible, avoid ambiguous
terms such as "systemic reaction."
[0623] Associated signs and symptoms should be recorded on the
dedicated Infusion-Related Reaction eCRF, Injection Reaction eCRF,
or Injection-Site Reaction eCRF. If a patient experiences both a
local and systemic reaction to the same dose of study drug, each
reaction should be recorded separately on the Adverse Event eCRF
with signs and symptoms also recorded separately on the dedicated
Infusion-Related Reaction eCRF, Injection Reaction eCRF, or
Injection-Site Reaction eCRF.
[0624] Adverse Events that are Secondary to Other Events
[0625] In general, adverse events that are secondary to other
events (e.g., cascade events or clinical sequelae) should be
identified by their primary cause, with the exception of severe or
serious secondary events. A medically significant secondary adverse
event that is separated in time from the initiating event should be
recorded as an independent event on the Adverse Event eCRF. For
example: [0626] If vomiting results in mild dehydration with no
additional treatment in a healthy adult, only vomiting should be
reported on the eCRF [0627] If vomiting results in severe
dehydration, both events should be reported separately on the eCRF
[0628] If a severe gastrointestinal hemorrhage leads to renal
failure, both events should be reported separately on the eCRF
[0629] If dizziness leads to a fall and consequent fracture, all
three events should be reported separately on the eCRF. [0630] If
neutropenia is accompanied by an infection, both events should be
reported separately on the eCRF.
[0631] All adverse events should be recorded separately on the
Adverse Event eCRF if it is unclear as to whether the events are
associated.
[0632] Persistent of Recurrent Adverse Events
[0633] A persistent adverse event is one that extends continuously,
without resolution, between patient evaluation timepoints. Such
events should only be recorded once on the Adverse Event eCRF. The
initial severity (intensity or grade) of the event will be recorded
at the time the event is first reported. If a persistent adverse
event becomes more severe, the most extreme severity should also be
recorded on the Adverse Event eCRF. If the event becomes serious,
it should be reported to the Sponsor immediately (i.e., no more
than 24 hours after learning that the event became serious. The
Adverse Event eCRF should be updated by changing the event from
"non-serious" to "serious," providing the date that the event
became serious, and completing all data fields related to serious
adverse events.
[0634] A recurrent adverse event is one that resolves between
patient evaluation timepoints and subsequently recurs. Each
recurrence of an adverse event should be recorded as a separate
event on the Adverse Event eCRF.
[0635] Abnormal Laboratory Values
[0636] Not every laboratory abnormality qualifies as an adverse
event. A laboratory test result must be reported as an adverse
event if it meets any of the following criteria: [0637] Is
accompanied by clinical symptoms [0638] Results in a change in
study treatment (e.g., dosage modification, treatment interruption,
or treatment discontinuation) [0639] Results in a medical
intervention (e.g., potassium supplementation for hypokalemia) or a
change in concomitant therapy [0640] Is clinically significant in
the investigator's judgment. Note: For oncology trials, certain
abnormal values may not qualify as adverse events.
[0641] It is the investigator's responsibility to review all
laboratory findings. Medical and scientific judgment should be
exercised in deciding whether an isolated laboratory abnormality
should be classified as an adverse event.
[0642] If a clinically significant laboratory abnormality is a sign
of a disease or syndrome (e.g., ALP and bilirubin 5.times.ULN
associated with cholestasis), only the diagnosis (i.e.,
cholestasis) should be recorded on the Adverse Event eCRF.
[0643] If a clinically significant laboratory abnormality is not a
sign of a disease or syndrome, the abnormality itself should be
recorded on the Adverse Event eCRF, along with a descriptor
indicating whether the test result is above or below the normal
range (e.g., "elevated potassium," as opposed to "abnormal
potassium"). If the laboratory abnormality can be characterized by
a precise clinical term per standard definitions, the clinical term
should be recorded as the adverse event. For example, an elevated
serum potassium level of 7.0 mEq/L should be recorded as
"hyperkalemia."
[0644] Observations of the same clinically significant laboratory
abnormality from visit to visit should only be recorded once on the
Adverse Event eCRF.
[0645] Abnormal Vital Sign Values
[0646] Not every vital sign abnormality qualifies as an adverse
event. A vital sign result must be reported as an adverse event if
it meets any of the following criteria: [0647] Is accompanied by
clinical symptoms [0648] Results in a change in study treatment
(e.g., dosage modification, treatment interruption, or treatment
discontinuation) [0649] Results in a medical intervention or a
change in concomitant therapy [0650] Is clinically significant in
the investigator's judgment
[0651] It is the investigator's responsibility to review all vital
sign findings. Medical and scientific judgment should be exercised
in deciding whether an isolated vital sign abnormality should be
classified as an adverse event.
[0652] If a clinically significant vital sign abnormality is a sign
of a disease or syndrome (e.g., high BP), only the diagnosis (i.e.,
hypertension) should be recorded on the Adverse Event eCRF.
[0653] Observations of the same clinically significant vital sign
abnormality from visit to visit should only be recorded once on the
Adverse Event eCRF.
[0654] Results of Pk Studies and Dose Selection
[0655] Part 1 SC PK Analyses and Dose Selection
[0656] The selection of the pertuzumab SC doses in Part 1 (Cohorts
2, 3, and 4) was based on the pertuzumab IV popPK model with the
values of the trastuzumab SC PK parameters incorporated.
[0657] For better accuracy, IV popPK from historical model
parameter estimates was also relied on in the SC dose selection
analyses. Following the estimation of fixed PK parameters (i.e.,
C.sub.trough, AUC0-inf, maximum serum concentration [C.sub.max],
time of maximum serum concentration [T.sub.max]), the pertuzumab SC
(maintenance) dose(s) was selected for Part 2. This SC dose was
calculated to deliver a similar pertuzumab exposure to that of IV
pertuzumab at 420 mg. Equally, based on PK parameters, one
pertuzumab SC (loading) dose was calculated to deliver a similar
pertuzumab exposure to that of IV pertuzumab at 840 mg. The
pertuzumab IV popPK model was updated with pertuzumab SC parameters
using the Part 1 data and was used to correctly identify the SC
maintenance and loading doses.
[0658] FIG. 8 shows the study overview, including the antibody
dosages, injections volumes, and rHuPH20 (Halozyme) concentrations
and amounts for cohorts 1-8.
[0659] FIG. 9 shows dose normalized concentrations (.mu.g/mL) of
subcutaneously administered pertuzumab, with and without
trastuzumab, as a function of time (days). The data show that there
is no PK interaction between pertuzumab SC and trastuzumab SC when
administered simultaneously.
[0660] No differences were seen between the PK of trastuzumab SC
administered as monotherapy or with pertuzumab SC.
[0661] FIG. 10 shows dose normalized concentrations (.mu.g/mL) of
pertuzumab as a function of time (days). There were no significant
differences in pertuzumab PK or trastuzumab PK (not shown) when
administered with 2,000 U/mL or 667 U/mL rHuPH20.
[0662] FIG. 11 shows the parameter estimations using the pertuzumab
and the historical population PK (popPK) W models in comparison at
different pertuzumab concentrations administered W or SC, with and
without trastuzumab.
[0663] Characterization of Pertuzumab Pharmacokinetics--Part 1
[0664] Mean pertuzumab concentration-time profiles for Cohorts 1-4
and 6-8 are shown in FIG. 24. The pertuzumab concentrations after
an intravenously administered dose of 420 mg followed a biphasic
pattern with a distinct distribution and elimination phase.
Subcutaneously administered pertuzumab resulted in a time to reach
maximum concentration (T.sub.max) between 4-7 days and dose-related
increases in exposure. Variability within some of the 1200 mg SC
cohorts was observed, likely due to a small sample size.
[0665] Pertuzumab geometric mean dose-normalized concentrations
when administered with and without trastuzumab were compared to
assess the potential impact of trastuzumab on the PK of pertuzumab.
As shown in FIG. 22, there was no apparent impact of trastuzumab on
the PK of pertuzumab when the two antibodies were delivered SC
co-mixed. This is consistent with PK data from previous studied
where the two antibodies were administered sequentially.
[0666] Pertuzumab geometric mean concentrations when administered
with 667 U/mL or 2,000 U/mL rHuPH20 were compared to assess the
potential impact of the absorption enhancing enzyme rhuPH20 on
pertuzumab PK. There was no apparent impact of lowering the rHuPH20
concentration from 2,000 U/mL to 667 U/mL on the PK of pertuzumab
or trastuzumab, as shown in FIGS. 20 and 21, respectively.
[0667] The characterization of pertuzumab PK indicated that
trastuzumab (resulting from simultaneous trastuzumab SC
administration) has no apparent impact on pertuzumab. Pertuzumab PK
appeared mainly unaltered by lowering the rHuPH20 concentration
from 2,000 U/mL to 667 U/mL, however, as only a small number of
subjects were exposed to each concentration (n=6 per cohort), it
was difficult to rule out possible differences particularly in the
terminal phase and C.sub.tough. These observations supported
further analyses to determine the pertuzumab SC dose that was
non-inferior to pertuzumab IV 420 mg.
[0668] Population PK Model Development to Select Pertuzumab SC
Dose
[0669] A pertuzumab SC 1200 loading dose was selected based on
pertuzumab dose-proportionality and linear pharmacokinetics
suggested by the popPK model developed. Model-based simulations
confirmed comparable exposures between pertuzumab 1200 SC and 840
mg W. Observed pertuzumab exposures from the 1200 mg SC cohorts
from Part 1 further confirmed the loading dose selection when
compared with IV exposures observed in historical studies.
[0670] Characterization of Pertuzumab Pharmacokinetics--Part 2
[0671] Given no apparent pharmacokinetic (PK) drug-drug interaction
(DDI) between pertuzumab and trastuzumab when co-mixed in Part 1,
and technical development of the fixed-dose co-formulation (FDC)
was e feasible, Cohort A (co-administration of 600 mg pertuzumab
with 600 mg trastuzumab) was not enrolled in part 2. Cohort B
investigated a co-mixed injection of pertuzumab SC and trastuzumab
SC with 1,000 U/mL rHuPH20 to confirm the pertuzumab dose selected
in Part 1 of the study. The co-mixed material in Cohort B serves as
a surrogate for FDC (described in Example 2), which will be tested
in Cohort C.
[0672] Non-compartmental and population PK analyses of PK data in
Part 2 were conducted: [0673] to confirm the lack of PK drug-drug
interaction (DDI) between pertuzumab and trastuzumab when both were
administered co-mixed SC, [0674] to investigate the impact of 1,000
U/mL rHuPH20 on pertuzumab and trastuzumab PK, and [0675] to
confirm the pertuzumab SC maintenance dose of 600 mg was
non-inferior when compared to pertuzumab IV 420 mg (administered to
HMVs) in terms of steady state C.sub.trough in 20 EBC patients.
[0676] Pertuzumab geometric mean concentrations following a
co-mixed dose of pertuzumab SC 500 mg, trastuzumab SC 600 mg and
rHuPH20 1,000 U/mL to 20 EBC patients in Part 2 Cohort B were
compared to pertuzumab geometric mean concentrations following a
dose of pertuzumab IV 420 mg to 6 HMVs in Part 1 Cohort 1. As shown
in FIG. 22, pertuzumab exposures (C.sub.trough and AUC) are similar
between 600 mg SC (EBC patients) and 420 mg W (HMVs).
[0677] Pertuzumab geometric mean dose-normalized concentrations
following a co-mixed dose of pertuzumab SC 600 mg, trastuzumab SC
600 mg and rHuPH20 1,000 U/mL to 20 EBC patients in Part 2 Cohort B
were also compared to pertuzumab geometric mean concentrations
following a dose of pertuzumab SC 600 mg and rHuPH20 2,000 U/mL to
6 HMVs in Part 1 Cohort 3. As shown in FIG. 23, the pertuzumab PK
profiles are very similar between EBC patients (Part 2 Cohort B)
and HMVs following a partuzumab SC 600 mg dose (Part 1 Cohort
3).
[0678] Comparing pertuzumab and trastuzumab geometric mean
dose-normalized concentrations when administered with 667 U/mL,
1,000 U/mL or 2,000 U/mL rHuPH20 can assess the potential impact of
the absorption enhancing enzyme rHuPH20 on pertuzumab and
trastuzumab PK. As shown in FIG. 24, there was no apparent impact
of lowering the rHuPH20 concentration from 2,000 U/mL to 1,000 U/mL
or 667 U/mL on the PK of pertuzumab. The comparison of pertuzumab
PK data from EBC patients to HNVs further confirms the lack of
interaction between pertuzumab and trastuzumab when pertuzumab and
trastuzumab are administered co-mixed subcutaneously.
[0679] As shown in FIG. 25, there were no apparent differences in
trastuzumab PK when administered with 667 U/mL, 2,000 U/mL or 2,000
U/mL rHuPH20. The characterization of pertuzumab PK in Part 2
Cohort B confirmed the results in Part 1 of the study. Part 2
Cohort B indicated that trastuzumab (resulting from simultaneous
trastuzumab SC administration) had no apparent impact on pertuzumab
PK, and pertuzumab and trastuzumab PK are similar at rhuPH20
concentrations from 2,000 U/mL to 1,000 U/mL.
[0680] Observed PK data, model simulations using the popPK model
refreshed with additional PK data collected in Part 2 Cohort B and
resulting probabilities in Part 2 Cohort B were nearly identical to
the data obtained in Part 1. These data were further supported
after a second popPK model was built using SC data from Parts 1 and
2 of the current Phase I study and W data from the historical
pertuzumab IV popPK model (Garg et al., Cancer Chemother Pharmacol.
2014; 74:819-829). The addition of a robust pertuzumab IV dataset
in patients coupled with the SC data in HMVs confirmed the PK
parameter estimates and simulations and provided agreement with
selection of a pertuzumab SC loading and maintenance dose of 1200
mg and 600 mg, respectively.
[0681] rHuPH20 Pharmacokinetics
[0682] Plasma rHuPH20 concentrations were measured at predose and
at 0.5 and 24 hours postdose for patients in Cohorts 2-8 of Part 1
and Cohort B of Part 2. A validated sandwich immunoassay using an
electroluminescence (ECL) readout, was used to measure the plasma
rHuPH20 concentrations. The minimum quantifiable concentration was
0.061444 ng/mL.
[0683] Plasma rHuPH20 concentrations were below the limit of
quantification for all sampling time points, indicating no
quantifiable systemic exposure to the enzyme at the rHuPH20 doses
used in this study.
[0684] Conclusions
[0685] The PK of subcutaneously administered pertuzumab has been
consistent with the PK of subcutaneously administered trastuzumab.
The tested lowed and higher amounts/concentrations of rHuPH20
showed no impact on the PK of subcutaneously administered
pertuzumab. Thus, both of the tested rHuPH20 concentrations (667
U/mL and 1,000 U/mL) are suitable for use in the methods described
herein.
[0686] Similar pertuzumab and trastuzumab PK were observed when
using 2,000-, 1,000-, or 667-U/mL rHUPH20. The safety profiles were
comparable in co-mixed cohorts receiving 1,000-, 1,000-. Or
667-U/mL rHuPH20. However, since different concentrations of
rHuPH20 were assessed in two groups of populations (HMVs and EBC
patients) each with a small number of subjects, a potential impact
of a lower concentration (e.g., 1,000 U/mL) on pertuzumab PK and/or
safety in these different subject populations cannot be ruled out,
and the recommended concentration of rHuPH20 in the FDC (2,000
U/mL) was determined with the totality of other available clinical
experience.
[0687] Safety Results
[0688] Part 1 Safety Data
[0689] The demographics and age distribution of the study
population is shown in FIG. 12.
[0690] FIG. 13 shows an overview of Adverse Events in Part 1 of the
study. The total number of adverse events was 145, and the % of the
Grade 1, Grade 2 and .gtoreq. Grade 3 adverse events for the
various cohorts is listed in parenthesis. The observed one Grade 3
AE of diarrhea on day 32 was an unrelated concurrent illness, a
possible viral infection assessed as concurrent upper respiratory
tract infection (URTI). There was no Serious Adverse Event (SAE),
Adverse Event of Special Interest (AESI), or Adverse Event (AE)
leading to discontinuation, or AE leading to death. At the time of
evaluation some AEs were ongoing, therefore no final Extreme Grade
can be provided.
[0691] FIG. 14 shows an overview shows a summary of the Adverse
Events in Part 1 of the study, listing the number of subjects for
each cohost and adverse event.
[0692] FIG. 15 is a tabulation of the most common Adverse Events
(all grades) with an overall incidence of .gtoreq.5% in the study
No. of subjects for Cohorts 18.
[0693] FIG. 16 shows the EGF related toxicities, i.e. diarrhea,
mucositis and EGFR associated rash.
[0694] FIG. 17 summarizes the injection related reactions and
injection site reactions, including systemic and local reactions,
for Cohorts 1 to 8. There was one injections site reaction in
Cohort 7 (1200 mg pertuzumab (P) SC+600 mg trastuzumab (H) SC with
rHuPH20. The symptoms were discomfort, pain, tightness and numbness
at the injection site. The systemic injection Related Reactions
included fever, chills, nausea, stiffness, lightheadedness, skin
sensitivity, photophobia, temperature fluctuation, and
headache.
[0695] FIG. 18 shows the results of LVEF--Echo assessments in
cohorts 1-8. In cohort 3 (600 mg pertuzumab (P) SC), one Healthy
Male Volunteer (HMV) has a drop of >10% from 67% at Baseline to
56% at Day 22. Follow up Ejection Fraction (EF) at Day 85 was 60%.
The cardiologist confirmed no evidence of cardiotoxicity. The drop
is believed to be due to variability of the imaging method.
[0696] Common Adverse Events
[0697] In Part 1 (Cohorts 1-8), a total of 148 AEs were reported in
44 out of 48 (91.7%) healthy kale volunteers (HMVs). The majority
of AEs were reported to be of low intensity (Grade 1 or 2). The
most common SOC was infections and infestations, with 22 (45.8%)
HMVs experiencing a total of 33 AEs in this category, of which a
majority of events were considered not related to study drug by the
investigator. The most commonly observed AEs (by PT) across
different cohorts were: upper respiratory tract infection (13 HMVs
[27.1%]), headache (9 HMVs [18.8%]), drug eruption (9 HMVs
[18.8%]), and diarrhea (9 HMVs [18.8%]). Among these common AEs,
study drug related AEs (assessed by the investigator) were reported
in 4 (8.3%), 8 (18.8%), and 7 (14.6%) HMVs, respectively. All AEs
in Part 1 resolved by the end of Part 1.
[0698] In Cohort 1 (control), in which HMVs received a single IV
injection of 420 mg pertuzumab, commonly observed AEs (by patient)
included: diarrhea (3 out of 6 HMVs [50%]), upper respiratory tract
infection (2 HMVs [33.3%]), and angular cheilitis (2 HMVs
[33.3%]).
[0699] In Cohorts 2-4, in which HMVs received a single SC injection
of pertuzumab at 400 mg, 600 mg, and 1200 mg, respectively, the
most commonly observed AEs (by patient) were respiratory tract
infection, rash, and diarrhea, each occurred in 4 out of 18 (22.2%)
HMVs.
[0700] In Cohort 5 (control), in which HMVs received a single SC
injection of 600 mg trastuzumab, the most commonly observed AEs (by
patient) was pain in extremity (at buttocks and upper thighs, not
at injection site) (2 out of 6 HMVs [33.3%]). All other AEs were
reported in 1 HMV only.
[0701] In Cohorts 6-8, in which HMVs received a SC injection of 600
mg trastuzumab co-mixed with pertuzumab at 400 mg, 1200 mg (with
2000 U/mL rHuPH20), and 1200 mg (with 667 U/mL rHuPH20),
respectively, the incidence of AEs was similar to that in HMVs who
received pertuzumab alone (Cohorts 2-4). The most commonly observed
AEs were upper respiratory tract infection and drug eruption, each
occurring in 7 out of 18 (38.9%) HMVs. Other common AEs reported in
at least 20% of HMVs were angular cheilitis (4 HMVs [22.2%]) and
headache (4 HMVs [22.2%]).
[0702] The incidence of overall AEs was similar between HMVs
receiving a concentration of 2.000 U/mL rHuPH20 (Cohort 7) and
those receiving a concentration of 667 U/mL rHuPH20 (Cohort 8) as
part of a co-mixed injection of pertuzumab and trastuzumab.
[0703] Regarding additional safety objectives there were: [0704] No
significant overall changes in blood pressure (BP), heart rate (HR)
or in beat-to-beat intervals (RR) [0705] Four subjects had a rise
in temperature post-dose on Day 1 associated with Injection Related
Reaction [0706] No clinically significant ECG changes were
reported
[0707] Regarding laboratory changes there was: [0708] No
significant AE laboratory abnormality [0709] One subject had Grade
4 Urate increase (Day 22), which was confirmed by site as Not
Clinically Significant and probably exercise related, the result
was normal at Day 85. [0710] Four subjects had Grade 3 Urate
increase, which were all confirmed by site as Not Clinically
Significant and Grade 1.
[0711] Part 2 Safety Data
[0712] In Part 2, all 20 [1005] female EBC patients received a
co-mixed SC injection of 600 mg pertuzumab and 600 mg trastuzumab
(with 1.000 U/mL rHuPH20). All patients experienced at least one
AE, with a total of 102 AEs reported by the time of clinical trial
cut-off. The majority of AEs were reported of low intensity (Grade
1 or 2).
[0713] The SOCs in which the most common AEs (reported in 50%
patients) occurred included the following: [0714] Nervous System
Disorders (14 patients [70%]) [0715] Gastrointestinal Disorders (10
[50%]), study drug related AEs (10 [50%]) [0716] Musculoskeletal
and Connective Tissue Disorders (10 [50%])
[0717] The most commonly observed AEs (by patient) reported in at
least 20% of patients were: headache (13 patients [65%]), myalgia
(7 patients [35%]), diarrhea (6 patients [30%]), injection site
reaction (6 patients [30%]), and nausea (4 patients [20%]). Among
these common AEs, study drug related AEs (assessed by the
investigator) were reported in 9 (45%), 6 (30%), 4 (20%), 6 (30%),
and 1 (5%) patients, respectively.
[0718] Both Cohort B (EBC patients) or Part 2 and Cohorts 6-8
(HMVs) of Part 1 received a co-mixed injection of pertuzumab and
trastuzumab, and the type of AEs that were reported (by patient)
with pertuzumab SC and trastuzumab SC in EBC patients are
consistent with known risks associated with the combination
therapy. Injection site reaction, all of Grade 1 or 2, occurred
with higher frequency in EBC patients compared to HMVs (6 [30%] vs.
1 [5.6%]).
[0719] Conclusions
[0720] In Part 1, all adverse events (AEs) in subcutaneously dose
cohorts were Grade 1 or Grade 2.
[0721] There were no Serious Adverse Events (SAEs), Adverse Events
of Special Interest (AESI), or >G3 AEs leading to
discontinuation, or fatal events.
[0722] No significant cardiac events were observed.
[0723] There were higher numbers of AEs in most P SC and P+H SC
cohorts compared to control cohort (P IV, H SC), however, no
consistent pattern was observed with increasing dose or addition
trastuzumab (H).
[0724] The most common AEs (occurring in >5% of subjects) were
upper respiratory tract infection, diarrhea, headache and drug
eruption. There was no difference between Cohorts 7 and 8 (with
lower rHuPH20 concentration).
[0725] Four Injection Related Reactions (1 in H SC) and one
Injection Site Reaction (Cohort 7) were observed. All reactions
were Grade 1/2 and comparable to the subcutaneously administered
trastuzumab (H SC) profile.
[0726] In conclusion, the safety profile of subcutaneously
administered pertuzumab (P), and the results in general were
consistent with the known safety profile of intravenously
administered pertuzumab and subcutaneously administered
trastuzumab. Accordingly, it was safe to continue the study and
move to Part 2.
[0727] Pertuzumab SC, given as a loading dose of 1200 mg and
maintenance dose of 600 mg provides similar C.sub.tough and AUC as
pertuzumab W 840 mg and 420 mg, respectively, as determined in
HMVs. Pertuzumab SC 600 mg dose in EBC patients provides similar
C.sub.trough and AUC to the 420 mg IV and 600 mg SC cohorts in HMVs
in Part 1 and dose proportionality through PK linearity confirms a
pertuzumab SC 1200 loading dose. Therefore, pertuzumab SC doses
(1200 mg loading, 600 mg maintenance) are confirmed in EBC
patients.
[0728] In general, the safety profile of pertuzumab SC is
consistent with the known safety profile of pertuzumab IV, and is
well tolerated when given in combination with trastuzumab SC. There
were no new safety signals identified. The majority of HMVs in Part
1 and all EBC patients in Part 2 Cohort B experienced at least one
AE. There were 2 Grade 3 AEs in Part 1 and 1 Grade 3 AE in Part 2
during the study. The remainder of the AEs were of low intensity
(Grade 1 or 2). There were no SAEs, deaths, or AEs leading to
withdrawals during the study. Following a co-mixed SC injection of
pertuzumab and trastuzumab, female EBC patients experienced higher
incidences of injection site reactions compared to HMVs.
[0729] In view of the PK and safety findings in the current study
(Parts 1 and 2), the safety, tolerability and PK results of this
Phase I study support the continuation of the study and enrollment
of Cohort C to receive pertuzumab+trastuzumab fixed-dose
co-formulation (FDC).
Example 2
Stable Subcutaneous Fixed-Dose Co-Formulations (SC FDC) of
Pertuzumab and Trastuzumab
[0730] Stable fixed-dose co-formulations (FDC) of pertuzumab and
trastuzumab were developed for subcutaneous (SC)
administration.
[0731] The co-formulation studies used the pertuzumab and
trastuzumab SC Drug Substance (DS) compositions and rHuPH20
composition shown in FIG. 19.
[0732] The amount (%) of high molecular weight species (HMWS) in
various subcutaneous pertuzumab and trastuzumab formulations, and
pertuzumab/trastuzumab co-formulations containing trehalose and/or
sucrose as stabilizer at 5.degree. C. and 25.degree. C. are shown
in FIG. 23.
[0733] The following SC FDC loading and maintenance formulations
were found to be stable and suitable for subcutaneous
administration of a single co-formulation of pertuzumab and
trastuzumab to human patients:
[0734] Loading Dose
[0735] Pertuzumab [0736] Dose: 1,200 mg [0737] Concentration: 80
mg/mL
[0738] Trastuzumab [0739] Dose: 600 mg [0740] Concentration: 40
mg/mL
[0741] rHuPH20 [0742] Concentration: 1,000 U/mL or 2,000 U/mL
[0743] pH: 5.5
[0744] 20 mM L-Histidine/HCl
[0745] Trehalose: 70 mM
[0746] Sucrose: 133 mM
[0747] Polysorbate 20 (PS20): 0.04%; 0.4 mg/mL
[0748] 10 mM Methionine
[0749] Nominal fill volume 15 mL
[0750] Vial: 20 mL/20 mm
[0751] Maintenance Dose:
[0752] Pertuzumab [0753] Dose: 600 mg [0754] Concentration: 60
mg/mL
[0755] Trastuzumab [0756] Dose: 600 mg [0757] Concentration: 60
mg/mL
[0758] rHuPH20 [0759] Concentration: 1,000 U/mL or 2,000 U/mL
[0760] pH: 5.5
[0761] 20 mM L-Histidine/HCl
[0762] Trehalose: 105 mM
[0763] Sucrose: 100 mM
[0764] Polysorbate PS20: 0.04%; 0.4 mg/mL
[0765] 10 mM methionine
[0766] Nominal fill volume: 10 mL
[0767] Vial: 15 mL/20 mm
[0768] Pertuzumab Drug Substance Stability
[0769] Scratch & Sprinkle Test
[0770] Protein aggregation can occur due to excipient (sugar)
crystallization in the frozen state under storage conditions of the
drug substance. In the scratch and sprinkle test, vials of drug
substance are frozen and some sugar (trehalose or sucrose) is added
to top of frozen formulation, then scratched with a metal spatula
to accelerate any potential sugar crystallization in the
formulation while frozen. At predetermined time points the
formulations are thawed and analyzed by Size Exclusion
Chromatography (SEC).
[0771] The SEC data shown in FIG. 29 demonstrate that sucrose is
superior excipient for pertuzumab drug substance stored at
-20.degree. C.
[0772] The effect of formulation differences in the following
pertuzumab-trastuzumab fixed-dose combinations (FDCs) on turbidity
and amount of high molecular weight species (HMWS) was tested.
TABLE-US-00008 Code F1 F2 F3 F4 Fixed Dose Combination Maintenance
Maintenance Loading Loading Dose Dose Dose Dose Buffer (20 mM)
His-HCl His-HCl His-HCl His-HCl pH 5.5 pH 5.5 pH 5.5 pH 5.5
Trastuzumab 60 60 40 40 (mg/mL) Pertuzumab 60 60 80 80 (mg/mL)
rHuPH20 2000 2000 2000 2000 (EU/mL) PS20 0.04 0.04 0.04 0.04 (%)
Met 10 10 10 10 (mM) Sucrose 100 -- 133 -- (mM) Trehalose 105 -- 70
-- (mM) NaCl -- 130 -- 130 (mM)
[0773] The data shown in FIG. 30 demonstrate that NaCl as excipient
results in high turbidity in the pertuzumab-trastuzumab SC
fixed-dose combination (FDC). Similarly, the data shown in FIG. 31
demonstrate that NaCl as an excipient results in higher amounts of
high molecular weight species (HMWs). Accordingly, sucrose and
trehalose are superior excipients for the FDC.
[0774] While certain embodiments of the present invention have been
shown and described herein, it will be understood by those skilled
in the art that such embodiments are provided by way of example
only. Numerous variations, changes, and substitutions will now
occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 1
1
221195PRTHomo sapiens 1Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu
Arg Leu Pro Ala Ser1 5 10 15Pro Glu Thr His Leu Asp Met Leu Arg His
Leu Tyr Gln Gly Cys Gln 20 25 30Val Val Gln Gly Asn Leu Glu Leu Thr
Tyr Leu Pro Thr Asn Ala Ser 35 40 45Leu Ser Phe Leu Gln Asp Ile Gln
Glu Val Gln Gly Tyr Val Leu Ile 50 55 60Ala His Asn Gln Val Arg Gln
Val Pro Leu Gln Arg Leu Arg Ile Val65 70 75 80Arg Gly Thr Gln Leu
Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp 85 90 95Asn Gly Asp Pro
Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro 100 105 110Gly Gly
Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys 115 120
125Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr
130 135 140Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala
Leu Thr145 150 155 160Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His
Pro Cys Ser Pro Met 165 170 175Cys Lys Gly Ser Arg Cys Trp Gly Glu
Ser Ser Glu Asp Cys Gln Ser 180 185 190Leu Thr Arg 1952124PRTHomo
sapiens 2Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu
Pro Thr1 5 10 15Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly
Pro Lys His 20 25 30Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser
Gly Ile Cys Glu 35 40 45Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr
Asp Thr Phe Glu Ser 50 55 60Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe
Gly Ala Ser Cys Val Thr65 70 75 80Ala Cys Pro Tyr Asn Tyr Leu Ser
Thr Asp Val Gly Ser Cys Thr Leu 85 90 95Val Cys Pro Leu His Asn Gln
Glu Val Thr Ala Glu Asp Gly Thr Gln 100 105 110Arg Cys Glu Lys Cys
Ser Lys Pro Cys Ala Arg Val 115 1203169PRTHomo sapiens 3Cys Tyr Gly
Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr1 5 10 15Ser Ala
Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser 20 25 30Leu
Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr 35 40
45Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu
50 55 60Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro
Asp65 70 75 80Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg
Ile Leu His 85 90 95Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly
Ile Ser Trp Leu 100 105 110Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser
Gly Leu Ala Leu Ile His 115 120 125His Asn Thr His Leu Cys Phe Val
His Thr Val Pro Trp Asp Gln Leu 130 135 140Phe Arg Asn Pro His Gln
Ala Leu Leu His Thr Ala Asn Arg Pro Glu145 150 155 160Asp Glu Cys
Val Gly Glu Gly Leu Ala 1654142PRTHomo sapiens 4Cys His Gln Leu Cys
Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr1 5 10 15Gln Cys Val Asn
Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu 20 25 30Glu Cys Arg
Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg 35 40 45His Cys
Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val 50 55 60Thr
Cys Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr65 70 75
80Lys Asp Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro
85 90 95Asp Leu Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly
Ala 100 105 110Cys Gln Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val
Asp Leu Asp 115 120 125Asp Lys Gly Cys Pro Ala Glu Gln Arg Ala Ser
Pro Leu Thr 130 135 1405107PRTMus musculus 5Asp Thr Val Met Thr Gln
Ser His Lys Ile Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser Ile
Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30Val Ala Trp Tyr
Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75
80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
1056119PRTMus musculus 6Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Thr1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
Phe Thr Phe Thr Asp Tyr 20 25 30Thr Met Asp Trp Val Lys Gln Ser His
Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Asp Val Asn Pro Asn Ser Gly
Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60Lys Gly Lys Ala Ser Leu Thr
Val Asp Arg Ser Ser Arg Ile Val Tyr65 70 75 80Met Glu Leu Arg Ser
Leu Thr Phe Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Leu
Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr
Leu Thr Val Ser Ser 1157107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 7Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
1058119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Thr Asp Tyr 20 25 30Thr Met Asp Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asp Val Asn Pro Asn Ser Gly
Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60Lys Gly Arg Phe Thr Leu Ser
Val Asp Arg Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Leu
Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 1159107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 9Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Leu Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10510119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 10Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Gly Asp Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Arg
Val Gly Tyr Ser Leu Tyr Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 11511214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 11Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21012448PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp
Tyr 20 25 30Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn
Gln Arg Phe 50 55 60Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Leu Gly Pro Ser Phe Tyr
Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440 44513214PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 13Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21014449PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
14Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp
Thr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr
Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180
185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410
415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 435 440 445Gly15217PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 15Val His Ser Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala1 5 10 15Ser Val Gly Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val 20 25 30Ser Ile Gly Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg 50 55 60Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser65 70 75
80Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile
85 90 95Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr 100 105 110Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu 115 120 125Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro 130 135 140Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly145 150 155 160Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200
205Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 21516449PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
16Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp
Tyr 20 25 30Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn
Gln Arg Phe 50 55 60Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Leu Gly Pro Ser Phe Tyr
Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440 445Lys1710PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptideMOD_RES(10)..(10)Asp or Ser
17Gly Phe Thr Phe Thr Asp Tyr Thr Met Xaa1 5 101817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Asp
Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys1 5 10
15Gly1910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr1 5
102011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala1 5
10217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(5)..(5)Arg or LeuMOD_RES(6)..(6)Tyr or
GluMOD_RES(7)..(7)Thr or Ser 21Ser Ala Ser Tyr Xaa Xaa Xaa1
5229PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 22Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr1 5
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