AU2007200478A1

AU2007200478A1 - APO2 ligand/trail formulations

Info

Publication number: AU2007200478A1
Application number: AU2007200478A
Authority: AU
Inventors: Heather Flores; Tanya P Lin; Timothy C Matthews; Roger Pai; Zahra Shahrokh
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 2001-11-13
Filing date: 2007-02-05
Publication date: 2007-02-22
Also published as: AU2010201713B2; AU2010201713A1

Description

05/02 2007 16:25 FAX 61 3 0 0 ci ~0

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0 00 0 0 ci 0\ 92438333 GRIFFITH HACK 4IPAUSTRALIA [a005

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant:

I

GENENTEC j, INC.

Invention Tit e: APO2 LIkI/TRAIL FORMULATIONS The followingistatement is a full description of this invention, iniluding the best method for performing it known to us: COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:25 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 00i06 APO-2 LIGAND/TRLLL FORMULATIONS PzFELD OF THE INETIOR The psetinvention relates generally to Apo2L/TRAIL forulaion.1 in particular, such Apo2L/TRML formulations o include lyopljilized and crystal compositions.

AOKGROUND OF THE INVENTION o Variousl molecules, such as tumoer necrosis factor-alpha ("TNFalha),tuI necrosis factor-beta ("TNF-beta" or 'lymphotoxinalpha") lyrphotoxin-beta ("LT-betal) CD30 ligani, CD27 ligand, CD0 ignI OX-40 ligand, 4-lBS ligand, Ape-i ligand (also referred to as Fas ligand or CD95 ligand), Ape-2 ligand (also referred to as Apo2t. or TRAIL), Apo-3 ligand (also referredl to as TWEAK), APRIA, 020 ligand (also referred to as RANK ligad, ODF, or TRANCE), land TA4LL-i (also referred to as BJ-yS, BAFF or THANK) have been ihentified as members of the tumor necrosis factor ("TrJFP) fam:ily of cytokines [See, Grilas and Dower, Blood, 95:3378-3404 (1995); Schmid et a1., ProC. Nati. Acad- Sci-, 83:1881 (1986), Dealtry et Eur. J. Inirunol., 17:689 (1987); Pitti et, alj J3. Biol. Chemt-, 271:12687-12690 (1996); Wiley et al., Immunitij, 3:673-682 (1995)1 Browning et al., Cell, 72:847-856 (1993); Anr~itage et al. 'Nature, 357:80-82 (1992). WO 97/01633 published Jan'uary 16, 1999; we 97/25428 published July 17, 1997; Marsters et L 4 Curr. Biol-, 8:525-528 (1998); Chicheportiche et al., Biol. dhemn-, 272:32401-32410 (19971: Hahne et al., J. Exp.

Med., 189:1lP85-1190 (1998) WO98/28426 published July 2, 1998; W098/46751 pulblished October 22, 1998; TR0/98/18921 published May 7, 1998; Moore et Science, 285z260-263 (1999); Shu et al., J_ Leukocyte Bib-., 6S:680 (1.999); Schneider et al., J_ Exp. Med., 189;l747l175 (1999); Mukhopadhyay et al., J. Biel. Chem., 274:15978-15181 (1999)]. Among these molecules, TNF-alpha, TNFbeta, CD30 jligand, 4-lEE ligand, Ape-i ligand, Apo-2 ligand (Apo2L,/TRAIL and Apo-3 ligand (TWEAK) have been reported to be involved in Apoptotic cell death.

Apo2L/T+AIL was identified several years ago as a member of COMS ID No: SBMI-061 42977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:26 FAX 61 3 924: the TNF fami 3:673-682 (1i (1996)] The amino acid 1 produce a i enzymatic cl [Mariani et SCrystallograr homotrimeric \1 10 related prote Hymowitz et Sunlike other unique struc position 23 coordinate a trimer stabi supra; Bodme 4 It has play a role diseases suce [see, e.g., Johnsen et Exp. Med., 191:1095-110: 152 (1998); Miura et al.

Soluble induce apopt, colon, lung, .tumors, as Wiley FEBS Letters Invest., 104 163 (1999); Mizutani et Leukemia, 13 2389 (2000); 1759 (2000)] suggest tha 38333 GRIFFITH HACK 4 IPAUSTRALIA 007 ly of cytokines. [see, Wiley et al., Immunity, Pitti et al., J. Biol. Chem., 271:12697-12690 full-length human Apo2L/TRAIL polypeptide is a 281 ong, Type II transmembrane protein. Some cells can Latural soluble form of the polypeptide, through eavage of the polypeptide's extracellular region al., J. Cell. Biol., 137:221-229 (1997)].

hic studies of soluble forms of Apo2L/TRAIL reveal a structure similar to the structures of TNF and other tins [Hymowitz et al., Molec. Cell, 4:563-571 (1999); al., Biochemistry, 39:633-644 (2000)]. Apo2L/TRAIL, TNF family members however, was found to have a tural feature in that three cysteine residues (at of each subunit in the homotrimer) together zinc atom, and that the zinc binding is important for lity and biological activity. [Hymowitz et al., et al., J. Biol. Chem., 275:20632-20637 (2000)] been reported in the literature that Apo2L/TRAIL may in immune system modulation, including autoimmune Sas rheumatoid arthritis, and in the treatment of HIV Thomas et al., J. Immunol., 161:2195-2200 (1998); Cytokine, 11:664-672 (1999); Griffith et al., J_ 189:1343-1353 (1999); Song et al., J. Exp. Med., S(2000); Jeremias et al., Eur. J. Inmunol., 28:143- Katsikis et al., J. Exp. Med., 186:1365-1372 (1997); J. Exp. Med., 193:651-660 (2001)].

forms of Apo2L/TRAIL have also been reported to isis in a variety of cancer cells in vitro, including breast, prostate, bladder, kidney, ovarian and brain 11 as melanoma, leukemia, and multiple myeloma [see, et al., supra; Pitti et al., supra; Rieger et al., 427:124-128 (1998); Ashkenazi et al., J. Clin.

:155-162 (1999); Walczak et al., Nature Med., 5:157- Keane et al., Cancer Research, 59:734-741 (1999); al., Clin. Cancer Res., 5:2605-2612 (1999); Gazitt, :1817-1824 (1999); Yu et al., Cancer Res.. 60:2384- Chinnaiyan et al., Proc. Natl. Acad. Sci., 97:1754- S In vivo studies in murine tumor models further t Apo2L/TRAIL, alone or in combination with 2 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:26 FAX 61 3 92438333 r GRIFFITH HACK IPAUSTRALIA 008 o I chemotherapy or radiation therapy, can exert substantial antitumor effect 4 [see, Ashkenazi et al., supra; Walzcak et al., 4 supra; Gliniak et al., Cancer Res., 59:6153-6158 (1999); SChinnaiyan ei al., supra; Roth et al., Biochem. Biophys. Res.

Comm., 265:199 (1999)]. In contrast to many types of cancer cells, most jnormal human cell types appear to be resistant to 00 apoptosis induction by certain recombinant forms of Apo2L/TRAIL [Ashkenazi e al-. supra; Walzcak et al., supra]. Jo et al. has o reported that a polyhistidine-tagged soluble form of Apo2L/TRAIL induced apoptosis in vitro in normal isolated human, but not nonhuman, hepatlcytes [Jo et al., Nature Med-, 6:564-567 (2000); see o also, Nagata Nature Med., 6:502-503 (2000)]. It is believed that certain recombinant Apo2L/TRAIL preparations may vary in terms of biochemical properties and biological activities on diseased versus normal cells, depending, for example, on the presence or absence of d tag molecule, zinc content, and trimer content [See, LawrenCe et al., Nature Med., Letter to the Editor, 7:383- 385 (2001); Qin et al., Nature Med., Letter to the Editor, 7:385- 386 (2001)].

Inductibn of various cellular responses mediated by such TNF family cytokines is believed to be initiated by their binding to specific celt receptors. Previously, two distinct TNF receptors of approximately 55-kDa (TNFRl) and 75-kDa (TNFR2) were identified [Hohman et J. Biol. Chem., 264:14927-14934 (1989); Brockhaus et al., Procl. Natl. Acad. Sci., 87:3127-3131 (1990); EP 417,563, published March 20, 1991; Loetscher et al-, Cell, 61:351 (1990); Schall et 41., Cell, 61:361 (1990); Smith et al., Science, 248:1019-102J (1990); Lewis et al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991); Goodwin et al., Mol. Cell. Biol., 11:3020- 3026 (1991) Those TNFRs were found to share the typical structure o cell surface receptors including extracellular, transmembran 4 and intracellular regions- The extracellular portions of both receptors were found naturally also as soluble TNF-binding proteins [Nophar, Y. et al-, EMBO 9:3269 (1990); and Kohno, T et al., Proc. Natl. Acad- Sci. 87:8331 (1990); Hale et al., J. Cell. Biochem. Supplement 15F, 1991, p.

113 (P424)], The extracellular portion of type 1 and type 2 TNFRs (TNFR1 and TNFR2) ontains a repetitive amino acid sequence pattern of 3 1 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:; 26 FAX 61 3 92438333 GRIFFITH HACK 4IPAUSTRALIA 1@009 four cystein -rich domains (CRDs) designated 1 through 4, starting from the NH- terminus. [Schall et al., supra: Loetscher et al., supra; Smith et al., supra; Nophar et al., supra; Kohno et al., supra; Bainn et al., Cell, 73:431-435 (1993)]. A similar repetitive ttern of CRDs exists in several other cell-surface proteins, i luding the p75 nerve growth factor receptor (NGFR) [Johnson et al., Cell, 47:545 (1986); Radeke et al., Nature, 325:593 (198j)], the B cell antigen CD40 [Stamenkovic et al., EMBO 8:1403 the T cell antigen 0X40 [Mallet et al., EMBO 9:1063 990)] and the Fas antigen [Yonehara et al., supra and Itoh et al., Cell, 66:233-243 (1991)]. CRDs are also found in the soluble TNFR (sTNFR)-like T2 proteins of the Shope and myxoma poxviruses pton et al., Virology, 160:20-29 (1987); Smith et al., Biochem. Biophys. Res. Commun., 176:335 (1991); Upton et al., Virology, 18_:370 (1991)]. Optimal alignment of these sequences indicates that the positions of the cysteine residues are well conserved. These receptors are sometimes collectively referred to as memberi of the TNF/NGF receptor superfamily.

The TNF family ligands identified to date, with the exception of lymphotox n-beta, are typically type II transmembrane proteins, whose c-term nlus is extracellular- In contrast, most receptors in the TNF receptor (TNFR) family identified to date are typically type I tran membrane proteins. In both the TNF ligand and receptor families, however, homology identified between family members has Aeen found mainly in the extracellular domain Several of he TNF family cytokines, including TNF-alpha, Apo-1 ligand and D40 ligand, are cleaved proteolytically at the cell surface; the resulting protein in each case typically forms a homotrimeric molecule that functions as a soluble cytokine- TNF receptor fam ly proteins are also usually cleaved proteolytically to release s luble receptor ECDs that can function as inhibitors of the cognat e cytokines.

Pan et al. have disclosed another TNF receptor family member referred to Is *DR4" (Pan et al., Science, 276:111-113 (1997); see also W098/32 56 published July 30, 1998]. The DR4 was reported to contain a cytoplasmic death domain capable of engaging the cell suicide appa atus. Pan et al. disclose that DR4 is believed to be a receptor fcr the ligand known as Apo2L/TRAIL In Sheridan et al., Science, 277:818-821 (1997) and Pan et 4 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:27 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA Q010 al., science 277:815-818 (1997), another molecule believed to be a receptor for Ao2L/TRAIL is described [see also, W098/51793 Spublished Notember 19, 1998; W098/41629 published September 24, S1998] That molecule is referred to as DR5 (it has also been alternatively referred to as Apo-2; TRAIL-R, TR6, Tango-63, hAPO8.

TRICK2 or KILER [Screaton et al., Curr- Biol., 7:693-696 (1997); 0 0 Walczak et a EMBO 16:5386-5387 (1997); Wu et al., Nature Genetics, 17:141-143 (1997); W098/35986 published August 20, 1998; o EP870,827 published October 14, 1998; W098/46643 published October 22, 1998; O099/02653 published January 21, 1999; W099/09165 published Fe ruary 25, 1999; W099/11791 published March 11, 1999].

o Like DR4, DRM is reported to contain a cytoplasmic death domain and be capable of signaling apoptosis. The crystal structure of the complex formed between Apo-2L/TRAIL and DR5 is described in Hymowitz et Molecular Cell, 4:563-571 (1999).

A furthtr group of recently identified receptors are referred to as "decdy receptors," which are believed to function as inhibitors, 'ather than transducers of signaling. This group includes DCRI (also referred to as TRID, LIT or TRAIL-R3) [Pan et al., Scienc4, 276:111-113 (1997); Sheridan et al., Science, 277:818-821 t1997); McFarlane et al., J. Biol. Chem., 272:25417- 25420 (1997) Schneider et al-, FEES Letters, 416:329-334 (1997); Degli-Espost" et al., J. Exp. Med., 186:116S-1170 (1997); and Mongkolsapayl et al., J. Irnmunol., 160:3-6 (1998)1 and DCR2 (also called TRUNDI or TRAIL-R4) [Marsters et al., Curr. Biol., 7:1003- 1006 (1997); Pan et al., FEBS Letters, 424:41-45 (1998); Degli- Esposti et Immunity, 7:813-820 (1997)], both cell surface molecules, ak well as OPG [Simonet et al., supra; Emery et al., infra] and DR3 [Pitti et al., Nature, 396:699-703 (1998)], both of which are secreted, soluble proteins. Apo2L/TRAIL has been reported to bind those receptors referred to as DcR1, DcR2 and

OPG.

Apo2L/ThAIL is believed to act through the cell surface "death recepors" DR4 and DR5 to activate caspases, or enzymes that carry out the cell death program. Upon ligand binding, both DR4 and DR5 an trigger apoptosis independently by recruiting and activating the apoptosis initiator, caspase-8, through the deathdomain-containing adaptor molecule referred to as FADD/Mortl [Kischkel et al., Immunity, 12:611-620 (2000); Sprick et al., COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:27 FAX 61 3 92438333 GRIFFITH HACK -IPAUSTRALIA 0011 o O

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Immunity, 21:599-609 (2000); Bodmer et al., Nature Cell Biol., S2:241-243 (2400)]- In contrast to DR4 and DR5, the DRI and DcR2 Sreceptors do not signal apoptosis.

For a review of the TNF family of cytokines and their receptors, she Ashkenazi and Dixit, Science, 281:1305-1308 (1998); Ashkenazi ani Dixit, Curr. Opin. Cell Biol., 11:255-260 (2000); 00 Golstein, Curr. Biol., 7:750-753 (1997); Gruss and Dower, supra; Nagata, Cell 88:355-365 (1997); Locksley et al., Cell, 104:487- Sl501 (2001).

lN i SUMMARY OP THE INVENTION SCertaini proteins, such as Apo2L/TRAIL and other members of the TNF famity of cytokines, exhibit biological activity when the protein is i a trimer or trimeric form. Thus. for purposes of therapeutic r even diagnostic use, formulations of such proteins are desired herein the protein is stable and remains biologically active, particularly stable in a trimeric form. Applicants have found that dertain formulation components, or "excipients', can provide stablity for such proteins like Apo2L/TRAIL and enhance solubility to reduce aggregation or precipitation of the protein). Applicants also surprisingly found that, under certain conditions, !Apo2L/TRAIL can readily crystallize. Such crystal forms of Apd2L/TRAIL may be useful in preparation of suspension formulations of Apo2L/TRAIL and/or provide an effective and efficient pr cess for protein purification.

Accordi gly, the present invention provides compositions or formulations i comprising Apo2L/TRAIL and one or more excipients which provide sufficient ionic strength to enhance solubility and/or stability of the Apo2L/TRAIL, wherein the composition optionally s a pH of 6 (or about 6) to 9 (or about 9).

Optionally, Fhe excipient(s) providing sufficient ionic strength is sale, and may comprise an arginine salt or sulfate salt. In one embodime t, the compositions may further comprise a buffer.

Optionally, the concentration of the Apo2L/TRAIL protein in the composition ks about 1 to about 100 mg/mi, about 1 to about mg/ml, about 10 to about 20 mg/ml, or about 20 mg/ml. The compositions of the invention may comprise liquid formulations or lyophilized iformulations. The compositions may also comprise suspension firmulations in which the Apo2L/TRAIL protein is in the 6 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:27 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 0012- 0 0 form of crys als. Optionally, it may be desirable to include one or more surf ctants in the composition. Such surfactants may, for [X instance, comprise a polysorbate or poloxamer. Particularly l) desired formtlations are those in which the excipient(s) provide for optimized Apo2L/TRAIL trimer content and minimize the amount of Apo2L/TAL dimer or aggregate formation. Optionally, the 00 formulations contain no more than 10% Apo2L/TRAIL dimer or Apo2L/TRAIL 4ggregates (of the total amount of Apo2L/TRAL protein O in the formulation).

C 10 In optional embodiments, the present invention provides o compositions comprising about 1 to about 20 mg/ml of Apo2L/TRAIL o and arginine salt, wherein the composition has a pH of about to about 8. Optionally, the compositions further comprise a buffer such as Tris and a surfactant such as polysorbate.

Optionally, the Apo2L/TRAIL protein does not include is not linked or fu ed to) any epitope tag molecule(s) or leucine zipper molecule(s).

The pre ent invention provides compositions comprising about 1 to about 20 mg/ml of Apo2L/TRAIL, about 0.4 to about arginine sal and buffer, wherein the compositions have a pH of about 7 to about 7.5. The Apo2L/TRAIL protein may be human Apo2L/TRAIL protein comprising amino acid residues 114 to 281 of Figure 1. Dptionally, the Apo2L/TRAIL protein is recombinantly expressed in host cells such as E. coli.

In addi ion, the invention provides methods for preparing the compositions described above. In the methods, the compositions are prepared by admixing or combining Apo2L/TRAIL and one or more excipients hich provide sufficient ionic strength to enhance solubility Ind/or stability of the Apo2L/TRAIL, wherein the composition has a pH of 6 (or about 6) to 9 (or about 9)optionally, he excipient(s) providing sufficient ionic strength is salt, ana may comprise an arginine salt or sulfate salt. A buffer may also be included to maintain the pH of the composition, and optionally to maintain the pH at about 6.5 to about Optionally, :he concentration of the Apo2L/TRAIL protein in the formulation Ls about 1 to about 100 mg/ml, about 1 to about mg/ml, about 10 to about 20 mg/ml, or at least 20 mg/l. In particularly desirable embodiments, the resulting compositions are pharmaceutic lly acceptable formulations.

7 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:28 FAX 61 3 92438333 GRIFFITH HACK 4, IPAUSTRALIA 013 In further embodiments, the invention provides compositions comprising A o2L/TRAIL protein crystals.

In stilL further embodiments, the invention provides methods of making cojositions comprising Apo2L/TRAIL crystals.

In yet Eurther embodiments, the invention provides methods of making and pirifying Apo2L/TRAIL.

In ad 'tional embodiments, the invention provides kits comprising: a container comprising an Apo2L/TRAIL composition dscribed herein and ins ructions for using the Apo2L/TRAIL composition; such as for using the composition to treat a disorder against which the composition Is effective. Optionally, the disorder is cancer, and more particularly, is a breast, lung, or colon (or colorectal) cancer.

In sti further aspects, the invention provides methods for treating a disorder, such as cancer or an immune related disorder, in a mammal omprising administering to the mammal, optionally by either inje tion or infusion, an effective amount of an Apo2L/TRAIL Aomposition provided by the present invention.

In more particular embodiments of the invention the following are providedl A stabl formulation of Apo-2 ligand, comprising Apo-2 ligand and about 0.)M to about 0.5M salt, wherein said formulation has a pH of about 6 to about 9- Optionally, the salt is an arginine salt or sod.um sulphate. optionally, the concentration of the arginine sal in the formulation is about 0.4M to about 0.5 M.

The arginin salt may include arginine succinate, arginine sulphate, arginine malate, arginine citrate, arginine tartrate, or arginine phosphate. The Apo-2 ligand may optionally comprise crystallized protein. The formulation may comprise a lyophilized or suspension formulation. Optionally, the pH of the formulation is about 6. to about 8.5, and optionally, about 7 to about Optionally, he formulation further comprises surfactant, such as a polysorbat or poloxamer. Optionally, the concentration of the surfactant In the formulation is about 0.005% to about 0.2%.

Optionally, Ihe formulation further comprises buffer, such as Tris buffer or Hpes. Optionally, the formulation further comprises one or more divalent metal ions or a preservative. Optionally, 8 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:28 FAX 61 3 92438333 GRIFFITH HACK 4t IFAUSTRALIA 014

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0 the formulation is storage-stable for at least 12 months.

A stabl 1 lyophilized formulation of Apo-2 ligand, comprising Sabout 1 mg/m to about 20 mg/ml Apo-2 ligand, about 0.2 M to about arginine salt, buffer, and surfactant, wherein said formulation has a pH of about 6 to about 9. Optionally, the arginine sal is arginine succinate, and the concentration of the arginine suctinate may be about 0.4M to about 0.SM. Optionally, the buffer is Tris buffer, and the surfactant is a polysorbate.

O Optionally, dhe formulation further comprises one or more divalent l 10 metal ions.

o A stabl formulation of Apo-2 ligand, comprising about Img/ml Sto about 20 g/ml Apo-2 ligand, about 0.2M to about 0.5 M salt, buffer, and surfactant, wherein said Apo-2 ligand comprises crystallized protein and said formulation has a pH of about 6 to about 9. Op[ionally, the salt is sodium sulphate, and the.buffer is Tris buffer. Optionally, the surfactant is polysorbate, and the pH is about 7 to about A stable formulation of Apo-2 ligand, comprising about 0.1 mg/ml to aout 2 mg/ml Apo-2 ligand, sugar, and surfactant, wherein said formulation has a pH of about 6 to about 9.

Optionally, jhe sugar is trehalose, and the concentration of the sugar in the formulation may be about 1% to about Optionally, the formulat on is lyophilized.

A meth d of making a stable formulation of Apo-2 ligand, comprising s eps of providing about 1 mg/ml to about 20 mg/ml Apo-2 ligand about 0.2 M to about D.5M arginine salt, buffer, and surfactant, b) combining or mixing the ingredients of step to make a formulation, and adjusting the pH of the formulation of step to about 6 to about 9. Optionally, the arginine salt is arginine su cinate, and the concentration of the arginine succinate is about 0.4M to about 0.5M. optionally, the buffer is Tris buffer, and the surfactant is a polysorbate.

A methbd of making crystallized Apo-2 ligand, comprising steps of providing Apo-2 ligand, buffer, and monovalent cationic sal combining or mixing the ingredients of step (a) to make a formulation at a temperature of about 200 C to about 300 C, and 1 wering the temperature of the formulation of step (b) to about 20 to about 8B C; wherein Apo-2 ligand crystallization occurs as e temperature of the formulation of step is 9 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:28 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 0015 lowered. Oj chloride. T about 0.15M.

as the temper further compa are dried.

crystals are A metho providing ho Apo-2 ligand; conditions as expressed Ape formulating i chloride or s IS of about 200 said formulat Apo-2 ligand lowered. Opl is concentr.

centrifugatic Optionally, a chromatogr )tionally, the salt is sodium sulphate or sodium he concentration of the salt may be about 0.1M to Optionally, the formulation of step is agitated ature is lowered in step Optionally, the method 'ises a step in which the Apo-2 ligand crystals Optionally, prior to the step the Apo-2 ligand washed.

I of making Apo- 2 ligand, comprising the steps of: (a) t cells comprising a vector containing DNA encoding culturing the host cells in culture medium under fficient to express Apo-2 ligand; obtaining said -2 ligand from the host cells and culture medium; (d) paid Apo-2 ligand into a solution containing sodium odium sulphate to make a formulation at a temperature C to about 30° C, and lowering the temperature of ion of step to about 2° C to about 80 C, wherein crystals form when the temperature of step is ionally, prior to step the Apo-2 ligand protein ited, and the protein may be concentrated by n, column chromatography or ultrafiltration, tep is conducting by applying the Apo-2 ligand to phic column (such as a cation exchange column) and eluting the 4PO-2 ligand into a sodium chloride or sodium sulphate containing juffer. Optionally, the cation exchange column comprises S -Sepharose fast flow, CM-Sepharose fast flow, or Macro-prep c ramic HS, and the buffer solution contains 50 mM Hepes, 50 mM Tris, 50 mM triethanolamine, 0.05% Triton X 100, 1 mM DTT, pH 7.5 8.0- Optionally, the formulation is agitated during step Otionally, the pH of the formulation in step is about 6.5 to about 8.5. Optionally, the host cells are prokaryote cells, such Es E. coli.

A devic2 for administering a formulation of Apo-2 ligand to a mammal, compising a container holding at least one dosage unit of the Apo-2 Ij and formulations described herein. Optionally, the device is a pen injector device, and the container is a cartridge.

An article of manufacture, comprising a container which includes an po2L/TRAIL formulation described herein, and printed instructions for use of the Apo-2L/TRAIL formulation. Optionally, the container is a bottle, vial, syringe, or test tube.

COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:29 FAX 61 3 92438333 GRIFFITH BACK 4 IPAUSTRALIA 0016s Optionally, container wt solution, or A metho.

exposing mamm formulation cells.

A meth administerinc amount of an the article of manufacture comprises a second ich includes water-for-injection, saline, Ringer's dextrose solution.

i of inducing apoptosis in mammalian cells, comprising alian cells to an effective amount of an Apo-2 ligand lescribed herein. The mammalian cells may be cancer od of treating cancer in a mammal, comprising to a mammal diagnosed as having cancer an effective Apo-2 ligand formulation described herein.

BRIEF DESCRIPTION OF THE DRAWINGS Figure shows the nucleotide sequence of human Apo-2L/TRAIL cDNA (SEQ Id NO:2) and its derived amino acid sequence (SEQ ID NO:1). The IN" at nucleotide position 447 (in SEQ ID NO:2) is used to indicate the nucleotide base may be a or Figure shows data trimer remaining and IEX main peak remaining) for various Apo2L/TRAIL formulations after 1 week storage at 3d 0

C-

Figure remaining) f storage at 41 Figure IEX main pe after 1 mont] Figure for the desc: Figures different fo: A shows data trimer remaining and IEX main peak >r various Apo2L/TRAIL formulations after 4 months

C.

3B shows data trimer remaining, monomer, and ak remaining) for various Apo2L/TRAIL formulations storage at 50° c.

30 shows an Arrhenius plot predictive of shelf-life ibed Apo2L/TRAIL formulation.

4A-4B show graphs of bioactivity and %trimer of two mulations at varying pH.

Figure 5 illustrates the structure of Apo2L/TRAIL and coordination of the structure by an intrinsic zinc molecule.

Figure 6 shows the effects of varying concentrations of COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:29 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 1017 polysorbate 6n stability of an Apo2L/TRAIL formulation.

Figure 7 shows the effects of varying concentrations of zinc on stability of an Apo2L/TRAIL formulation.

Figure R shows the equilibrium solubility and crystallization of Apo2L/TRAIL in a sodium sulphate formulation.

Figure y shows the equilibrium solubility and crystallization of Apo2L/TRAIL in various salt concentrations.

Figure crystallizat 1QA shows the effects of agitation rates on ,on of Apo2L/TRAIL.

Figure 110B shows the dissolution profile of Apo2L/TRAIL crystals und4r agitation.

Figure FOC shows the effects of agitation rate On Apo2L/TRAIL crystal size distribution.

Figure 11A shows the IEX profile of the Apo2L/TRAIL after reconstituti4n of vacuum dried crystals.

Figure 11B shows the bioactivity of the Apo2L/TRAIL after reconstitution of the vacuum dried crystals.

Figure for the desci 12 shows an Arrhenius plot predictive of shelf-life ibed Apo2L/TRAIL formulation.

Figure |13 shows a SDS-PAGE silver stain gel illustrating purity of thq described Apo2L/TRAIL preparations.

Figure crystallizat: 14 shows the effects of various salts on on of Apo2L/TRAIL.

DETAILED DESCRIPTION OF THE INVENTION

B

mily member" is used in a broad sense to refer to leptides that share some similarity to tumor necrosis 12 A. Definitiol 'TNF fe various poly] COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:29 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 1&18s

O

factor (TNF) with respect to structure or function- certain structural a d functional characteristics associated with the TNF l family of pclypeptides are known in the art and described, for exanmle, in the above Background of the Invention- Such polypeptides include but are not limited to those polypeptides referred to n the art as TNF-alpha, TNF-beta. CD40 ligand, 0 0 ligand, CD27 ligand, OX-40 ligand, 4-1BB ligand, Apo-1 ligand (also referred to as Fas ligand or CD95 ligand), Apo-2L/TRAIL o (also referred to as TRAIL), Apo-3 ligand (also referred to as TWEAK), APRI., OPG ligand (also referred to as RANK ligand, ODF, or TRANCE), d TALL-1 (also referred to as BlyS, BAFF or THANK) o (See, Gruss and Dower, Blood 1995, 85:3378-3404; Pitti et al,, J. Biol Chem. 1996, 271:12687-12690; Wiley et al., Immunity 1995, 3:673-682; Browning et al., cell 1993, 72:847-856; Armitage et al. Natu 1992, 357:80-82, PCT Publication Nos. WO 97/01633; and wo 97/2428; Marsters et al., Curr. Biol. 1998, 8:525-528; Chicheportic e et al., Biol. Chem. 1997, 272:32401-32410; Hahne et al., J. SE Med. 1998, 188:1185-1190; PCT Publication Nos.

WO98/28426; W098/46751; and WO/98/18921; Moore et al., Science 1999, 285:26 -263; Shu et al., J. Leukocyte Biol. 1999, 65:680; Schneider et al., J. Exp. Med. 1999, 189:1747-1756; Mukhopadhyay et al., J. Bxol- Chem. 1999, 274:15978-15981).

The ter 4 s "Apo2L/TRAIL", "Apo2L', "Apo-2 ligand" and "TRAIL" are used her in to refer to a polypeptide sequence which includes amino acid residues 114-281, inclusive, 95-281, inclusive, residues 92-81, inclusive, residues 91-281, inclusive, residues 41-281, inclsive, residues 15-281, inclusive, or residues 1-281, inclusive, o the amino acid sequence shown in Figure 1 (SEQ ID NO:1), as well as biologically active fragments, deletional, insertional, or substitutional variants of the above sequences.

In one embo iment, the polypeptide sequence comprises residues 114-281 of igure 1 (SEQ ID NO:l), and optionally, consists of residues 114-281 of Figure 1 (SEQ ID NO:1). Optionally, the polypeptide equence comprises residues 92-281 or residues 91-281 of Figure 1 (SEQ ID NO:l). The Apo-2L polypeptides may be encoded by the nativL nucleotide sequence shown in Figure 1 (SEQ ID NO:2).

Optionally, ;he codon which encodes residue Proll9 (Figure 1; SEQ ID NO:2) may be "CCT" or "CCG". In other embodiments, the fragments or variants are biologically active and have at least 13 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:29 FAX 61 3 92438333 GRIFFITH HACK 4IPAUSTRALIA a ools 0 0 about 80% amino acid sequence identity, more preferably at least Sabout 90% sequence identity, and even more preferably, at least X 95%, 96%. 97 98%, or 99% sequence identity with any one of the above recited Apo2L/TRAIL sequences, optionally, the Apo2L/TRAIL polypeptide s encoded by a nucleotide sequence which hybridizes under strin ent conditions with the encoding polynucleotide 0 0 sequence pro ided in Figure 1 (SEQ ID NO:2). The definition encompasses substitutional variants of Apo2L/TRAIL in which at least one of its native amino acids are substituted by an alanine 10 residue. Perticular substitutional variants of the Apo2L/TRAIL o include those in which at least one amino acid is substituted by o an alanine r sidue. These substitutional variants include those identified, for example, as "D203A"; "D218A" and "D269A." This nomenclature is used to identify Apo2L/TRAIL variants wherein the aspartic aciI residues at positions 203, 218, and/or 269 (using the numbering shown in Figure 1 (SEQ ID No01)) are substituted by alanine residues. Optionally, the Apo2L variants may comprise one or more of the alanine substitutions which are recited in Table I of published PCT application WO 01/00832. Substitutional variants include one or more of the residue substitutions identified in Table I of W9 01/00832 published January 4, 2001. The definition also encompasses a native sequence Apo2L/TRAIL isolated from an Apo2L/TRAIL source or prepared by recombinant or synthetic methods. The Apo2L/TRAIL of the invention includes the polypeptides referred to as Apo2L/TRAIL or TRAIL disclosed in PCT Publication Nos. W097/01633 and W097/25428. The terms "Apo2L/TRAIL' or "Apo2L" are used to refer generally to forms of the Apo2L/TR IL which include monomer, dimer or trimer forms of the polypept de. All numbering of amino acid residues referred to in the Apo2L sequence use the numbering according to Figure 1 (SEQ ID NO:1), uless specifically stated otherwise. For instance, 'D203" or "A p203" refers to the aspartic acid residue at position 203 in the sequence provided in Figure 1 (SEQ ID NO:1).

The term "Apo2L/TRAIL extracellular domain" or "Apo2L/TRAIL ECD" refers to a form of Apo2L/TRAIL which is essentially free. of transmembran0 and cytoplasmic domains. Ordinarily, the ECD will have less thin 1% of such transmembrane and cytoplasmic domains, and preferably, will have less than 0.5% of such domains- It will be understood that any transmembrane domain(s) identified for the 14 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 18:30 FAX B1 3 92438333 GRIFFITH HACK -+IPAUSTRALIA 00200 0 o polypeptides of the present invention are identified pursuant to criteria rou inely employed in the art for identifying that type C4 of hydrophob c domain. The exact boundaries of a transmembrane domain may ry but most likely by no more than about 5 amino acids at either end of the domain as initially identified. In preferred eibodiments, the ECD will consist of a soluble, 0 extracellula domain sequence of the polypeptide which is free of the transmem rane and cytoplasmic or intracellular domains (and is o not membrane bound)- Particular extracellular domain sequences of C< 10 Apo-2L/TRAIL are described in PCT Publication Nos. W097/01633 and oW097/25428.

SThe ter "Apo2L/TRAIL monomer" or "Apo2L monomer" refers to a covalent chain of an extracellular domain sequence of Apo2L.

The tern 'Apo2L/TRAIL dimer" or "Apo2L dimer" refers to two Apo-2L monom.rs joined in a covalent linkage via a disulfide bond.

The term as used herein includes free standing Apo2L dimers and Apo2L dimers that are within trimeric forms of Apo2L associated w th another, third Apo2L monomer).

The tejm "Apo2L/TRAIL trimer" or "Apo2L trimer" refers to three Apo2L onomers that are non-covalently associated.

The teAm "Apo2L/TRAIL aggregate" is used to refer to selfassociated igher oligomeric forms of Apo2L/TRAIL, such as Apo2L/TRAIL trimers, which form, for instance, hexameric and nanomeric fo4ms of Apo2L/TRAIL- Determination of the presence and quantity of Apo2L/TRAIL monomer, di4r, or trimer (or other aggregates) may be made using methods and assays known in the art (and using commercially available ma erials), such as native size exclusion HPLC denaturing size exclusion using sodium dodecyl sulphate ('SDS- SEC"), reverse phase HPLC, capillary electrophoresis, and including tose methods described in further detail in the Examples belw.

The ce m "tagged" when used herein refers to a chimeric polypeptide qomprising Apo2L/TRAIL, or a portion thereof, fused to a "tag pol ptide". The tag polypeptide has enough residues to provide an pitope against which an antibody can be made or to provide some other function, such as metal ion chelation, yet is short enougl such that it generally does not interfere with activity of the TNF family cytokine. The tag polypeptide COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 08/02 21307 18:30 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 021 0 0 preferably a so is fairly unique so that a tag-specific antibody does not sub tantially cross-react with other epitopes. Suitable tag polypept des generally have at least six amino acid residues o l and usually between about 8 to about 50 amino acid residues (preferably, between about 10 to about 20 residues).

The ter t "divalent metal ion" refers to a metal ion having 0 two positive charges. Examples of divalent metal ions include but are not limi.ed to zinc, cobalt, nickel, cadmium, magnesium, and o manganese. articular forms of such metals that may be employed include salt forms pharmaceutically acceptable salt forms), such as chlo ide, acetate, carbonate, citrate and sulfate forms of the above me tioned divalent metal ions. optionally, a divalent metal ion for use in the present invention is zinc, and preferably, the salt form, zinc sulfate or zinc chloride.

"Isolatd, when used to describe the various proteins disclosed herein, means protein that has been identified and separated axd/or recovered from a component of its natural environment. contaminant components of its natural environment are material 6 that would typically interfere with diagnostic or therapeutic uses for the protein, and may include enzymes, hormones, ank other proteinaceous or non-proteinaceous solutes.

In preferred embodiments, the protein will be purified to a degree suffi ient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or to hcmogeneity by SDS-PAGE under non-reducing or reducing conditions u ing Coomassie blue or, preferably, silver stain, or to homcgeneity by mass spectroscopic or peptide mapping techniques. Isolated protein includes protein in situ within recombinant cells, since at least one component of the Apo2L/TRAIL natural environmenrt will not be present. Ordinarily, however, isolated prtein will be prepared by at least one purification step.

An "isolated" Apo2L/TRAIL nucleic acid molecule is a nucleic acid moleculL that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated i the natural source of the Apo2L/TRAIL nucleic acid.

An isolated po2L/TRAIL nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated Apo2L/TRAIL nucleic acid molecules therefore are distinguished 16 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:31 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 0022~ 0 from the Apo /TRAIL nucleic acid molecule as it exists in natural Scells. How ver, an isolated Apo2L/TRATL nucleic acid molecule includes Apo!L/TRAIL nucleic acid molecules contained in cells Sthat ordinalily express Apo2L/TRAIL where, for example, the nucleic acid molecule is in a chromosomal location different from that of natu al cells.

00"Percent amino acid sequence identity" with respect to the sequencei identified herein is defined as the percentage of o amino acid esidues in a candidate sequence that are identical C 10 with the amio acid residues in the Apo2L/TRAIL sequence, after o) aligning the sequences and introducing gaps, if necessary, to o achieve the Aaximum percent sequence identity, and not considering any conserva ive substitutions as part of the sequence identity.

Alignment fo purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art can determine appropriate parameters for measuring alignment, Including assigning algorithms needed to achieve maximal alig ent over the full-length sequences being compared.

For purposes herein, percent amino acid identity values can be obtained usilg the sequence comparison computer program, ALIGN-2, which was autlhored by Genentech, Inc. and the source code of which has been filed with user documentation in the US Copyright Office, Washington, DC, 20559, registered under the US Copyright Registration No- TXU510087. The ALIGN-2 program is publicly available th ough Genentech, Inc., South San Francisco, CA_ All sequence. co 4 arison parameters are set by the ALIGN-2 program and do not vary.

"String ncy" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require highr temperatures for proper annealing, while shorter probes need Ilower temperatures. Hybridization generally depends on the ability of denatured DNA to re-anneal when complementary strands arei present in an environment below their melting temperature. The higher the degree of desired identity between the probe nd hybridizable sequence, the higher the relative temperature khich can be used. As a result, it follows that higher relative temperatures would tend to make the reaction 17 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:31 FAX 61 3 92438333 GRIFFITH HACK 4 IAUSTRALIA Qii023 conditions m re stringent, while lower temperatures less so. For additional d tails and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology. Wily Interscience Publishers, (1995).

"High stringency conditions", as defined herein, are identified those that: employ low ionic strength and high temperature Ior washing; 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50C; employ during hybridizatior a denaturing agent; 50% formamide with 0.1% bovine seru albumin/0.1% Ficoll/0.1% sodium phosp4ate buffer at pH 6.5 with 750 mM sodium chloride, mM sodium citrate at 42°C; or employ 50% formamide, 5 x ssc (0.75 M NaCl 1 0.075 M sodium citrate), 50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ug/ml), 0.1% SDS, and 10% dextran sulfate at 42C, with washes at 42 0 C in 0.2 x SSC (sodium chloride/sod um citrate) and 50% formamide at 55Oc, followed by a high-stringe cy wash consisting of 0.1 x SSC containing EDTA at 0

C.

"Modera ely stringent conditions' may be identified as described Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include overnight ii cubation at 37DC in a solution comprising: formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosp ate (pH 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing tie filters in 1 x SSC at about 37-50'C. The skilled artisan will recognize how to adjust the temperature, ionic strength, et,. as necessary to accommodate factors such as probe length and tle like.

The t rm "control sequences" refers to DNA sequences necessary fol the expression of an operably linked coding sequence in a particj lar host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally operator sequence, and a ribosome binding site.

Eukaryotic c ls are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functional r lationship with another nucleic acid sequence. For 1 18 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:31 FAX 61 3 92438333 i GRIFFITH HACK 4, IPAUSTRALIA 024 example, DNA linked to DN that particil or enhancer the transcri] operably lin] facilitate t: the DNA sequ a secretory enhancers do for a presequence or secretory leader is operably for a polypeptide if it is expressed as a preprotein ates in the secretion of the polypeptide; a promoter s operably linked to a coding sequence if it affects tion of the sequence; or a ribosome binding site is ed to a coding sequence if it is positioned so. as to anslation. Generally, "operably linked" means that nces being linked are contiguous, and, in the case of leader, contiguous and in reading phase. However, not have to be contiguous- Linking is accomplished by ligation t convenient restriction sites. If such sites do not exist, the s thetic oligonucleotide adaptors or linkers are used in accordanc with conventional practice.

The term "storage-stable" is used to describe a formulation having a sheLf-life acceptable for a product in the distribution chain of correrce, for instance, at least 12 months at a given temperature, and preferably, at least 24 months at a given temperature. Optionally, such a storage-stable formulation contains no more than 5% aggregates, no more than 10% dimers, and/or mini al changes in charge heterogeneity or biological activity.

As used herein, "soluble" refers to polypeptides that, when in aqueous iolutions, are completely dissolved, resulting in a clear to slightly opalescent solution with no visible particulatesl as assessed by visual inspection. A further assay of the turbidity of the solution (or solubility of the protein) may be made By measuring UV absorbances at 340 nm to 360 nm with a 1 cm pathlen4th cell where turbidity at 20 mg/ml is less than 0.05 absorbance units.

An "osiolyte" refers to a tonicity modifier or osmotic adjuster tha lends osmolality to a solution. Osmolality refers to the total osmotic activity contributed by ions and nonionized molecules to a solution. Examples include inorganic salts such as sodium chloride, polyethylene glycols (PEGs), polypropylene glycol, sugars such as sucrose or trehalose, glycerol, amino acids, and stgar alcohols such as mannitol known to the art that are generall, regarded as safe (GRAS).

"Preservatives" can act to prevent bacteria, viruses, and fungi from ploliferating in the formulation, and anti-oxidants, or 19 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:32 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 025 0 0 other compo nds can function in various ways to preserve the stability of the formulation. Examples include L octadecyldime thylbenzyl ammonium chloride, hexamethonium chloride, I) benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of compounds include aromatic 00 alcohols suce as phenol and benzyl alcohol, alkyl parabens such as methyl or -ropyl paraben, and m-cresol. Optionally, such a Scompound is phenol or benzyl alcohol. The preservative or other C 10 compound wil optionally be included in a liquid or aqueous form Sof the Apo2 /TRAIL formulation, but not usually in a lyophilized o form of the Eormulation. In the latter case, the preservative or other compoind will typically be present in the water for injection (W4I) or bacteriostatic water for injection (BWFI) used for reconstitution.

A "sur~ctant" can act to decrease turbidity or denaturation of a proteid in a formulation. Examples of surfactants include non-ionic suifactant such as a polysorbate, polysorbates or 80, (a poloxamer, poloxamer 184 or 188, Pluronic polyols, eth lene/propylene block polymers or any others known to the art that are GRAS. Optionally, the surfactant is a polysorbate Jr poloxamer.

A "buffer" as used herein is any suitable buffer that is GRAS and generallr confers a pH from about 6 to about 9, optionally from about 6.5 to about 8.5, and optionally at about 7 to about if the polypeptide is Apo2L/TRAIL. Examples include Tris, Hepes, trietianolamine, histidine, or any others known to the art to have the .esired effect.

The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. .xamples of such cytokines are lymphokines, monokines, and traditi >nal polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, Nmethionyl h man growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH thyroid stimulating hormone (TSH), and luteinizing hormone hepatic growth factor; fibroblast growth factor; prolactin; lacental lactogen; tumor necrosis factor-a and -P; COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:32 FAX 61 3 92438333 GRIFFITH HACK -+IPAUSTRALIA 1026 mullerian-inlibiting substance; mouse gonadotropin-associated peptide, in1ibin; activin; vascular endothelial growth factor; integrin; thjcormbopoietin (TPO); nerve growth factors; plateletgrowth factoc; transforming growth factors (TGFs) such as TGF-c and TGF-j; Insulin-like growth factor-1 and -II erythropoietin (EPO); osteo$nductive factors: interferons such as interferon-a, and -gaima; colony stimulating factors (CSFs) such as macrophage-CF (M-CsF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte- SF (G-CSF); interleukins (ILa) such as IL-1, IL-2, IL-3, IL-4, L-5, IL-6, IL-7, IL-B, IL-9, IL-11, IL-12; and other polypeptide actors including LIF and kit ligand As used herein, the term cytokine includes proteins from natural sources or from r combinant cell culture and biologically active equivalents 4f the native sequence cytokines.

The tebm "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destraction of cells. The term is intended to include 1.31 123 90 an R186 radioactive isotopes I, Y and Re 6 chemotherape tic agents, and toxins such as enzymatically active toxins of ba terial fungal, plant or animal origin, or fragments thereof- A "chemotherapeutic agent" is a chemical compound useful in the treatmert of cancer. Examples of cheotherapeutic agents include alkyLating agents such as thiotepa and cyclosphosphamide (CYTOXAN alkyl sulfonates such as busulfan, improsulfan and piposulfan; ziridines such as benzodopa, carboquone, meturedopa, and uredop ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenetl iophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; cc-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin dolastatin; duocarmycin (including the synthetic analogues, Kw-2189 and CBI-TMI); eleutherobin pancratistatin; a sarcodictyin; spongistatin; nitrogen mistards such as chlorambucil, chlornaphazine, cholophosphaide, estramustine, ifosfamide, mechlorethamine, 21 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 4 IPAUSTRALIA Q027 05!/02 2007 1R:32 FAX 61 3 92438333 GRIFFITH HACK IASRLA1h2 mechiorethajne oxide hydrochloride, meiphalan, navembichin, phenesterine, prednimustine, trofosfamide, Uracil mustard; nitrosureas jsuch as carmustine, chiorozotocin, fotemustine, tf0lmustine, *±mustinb. ranimustine; antibiotics such as the 0 5 eneddyne antibiotics calicheni-cin, especially caJ-icheamicin gumwalI and dklicheandcin phill, see, Agnew, Chem Intl. Ed.

00 Engl., 33:143-186 (1994); dynearioin, including dynemicin A; bisphosphon'-es, such as clodronate; an esperamicin; as well as o neocarzinost tin chrornophore and related chromoprotein enedlynaantiobiotici authramycin, ELasenne, bleonmycins, cactinomycin, carabicin, ocazninomyciir carzinophilin, cbromomycinz, dactinomycin, daunorubicin, detoruhbicin, S-diazo-5-oxo-L-norleucine, doxorubicin (Adlriamycin t M (including morpholino-doxor-ubicin, cyanomorpholino- 1S doxorubicin,l 2 -pyrrolino-doxorubicin and deoxydoxorubicin), epiriubicin, E sorubicin, idarubicin, iftarcelloirycin, mitomycins such as mritomfycij, mycophenolic acid, nogalainycin, olivtozaycina, peplomycin, 1 potfiromycin, puromycin. cguelaniycin, rodorubicin, streptonigri~ streptozocin, tubercidin. ubenimex. zinostatin.

zorubicin; rti-metabolites such as zuethotrexate and Sfluorouracil folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate: purine analogs such as fludarabine,l 6-mercaptopurine, thiamiprine, thioguanine; pyrliridine anialogs such as ancitabine, az-acitidine, 6-azauridine.

carmofur, cyIqarabine. dideoxyuridine, doxifluridine, enocitabine.

floxuridine, androgens such as calusterone, dromostanolone propionate, lepitiostanol. rnepitiostane, testolactole; antiadrenals suc4l as aiinoglutethimide. mitotane, trilostane; foliLc acid replenhisher such as frolinic acid; aceglatone; @Lldophosphai4e glycoside; aninolevuliflic acid, eniluracil; ainsacrine; ~estrsbncil; bisantrene; edatraxate; defofamine; demecolcine; jdiaziqruone; elfernithine; elliptinium acetate; an epothilone; toglucjd; gallium nitrate; hydroxyurea; lentinan; 3.onidaxnine; itaytansinoids such as maytan-sine and ansamitocins; mitog-uazone; mitoxantrone; mopidlamol; nitracrinei pentoctatin; phenamet; pkarubicin; losoxantrone; podophyllinic acid; 2ethylhYdrazid p; procarbazine; Pst; ratoxane; rhizoxin; aizofiran; spirogermaniujn yltenuazonic acid; triazicuone; 2, 2t,2''trichlorotric hylmine; trichothecengs (especially T-2 toxin, 1 22 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:33 FAX 61 3 92438333 GRIFFITH BACK 4, IPAUSTRALIA 028 0 0 verracurin roridin A and anguidine); urethan; vindesine; dacarbazine; I mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside cyclophosphamide; thiotepa; tL taxoids, e.gj paclitaxel (TAXOLO, Bristol-Myers Squibb Oncology, Princeton, iJ) and doxetaxel (TAXOTERE, Rh6ne-Poulenc Rorer, Antony, Frapce); chlorabucil; gemcitabine (Gemzarw); 6- 00 thioguanine; tmercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide o(V-16); if ofamide; mitoxantrone; vincristine; vinorelbine (Navelbine"') novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibior RS 2000; ifluoromethylornithine (DMFO); retinoids such as retinoic ac d; capecitabine; and pharmaceutically acceptable salts, acids lor derivatives of any of the above- Also included in this definit on are anti-hormonal agents that act to regulate or inhibit horne action on tumors such as anti-estrogens and selective es rogen receptor modulators (SERMs), including, for example, tam cifen (including Nolvadex raloxifene, droloxifene, 4-hydroxytamomifen, trioxifene, keoxifene, LY117018, onapristone, and toremife e (Fareston"); aromatase inhibitors that inhibit the enzyme aromarase, which regulates estrogen production in the adrenal glainds, such as, for example, aminoglutethiide, megestrol acetate (Megace"

M

exemestane, formestane. Iadrozole, vorozole (Rivisor), letrozole (Femara m and anastrozoe (ArimidexT"); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutica ly acceptable salts, acids or derivatives of any of the above.

A "grow h inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially cafcer cell overexpressing any of the genes identified herein, either in vitro or in vivo. Thus, the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressin such genes in S phase. Examples of growth inhibitory agsts include agents that block cell cycle progression (at a place :ther than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vin ristine and vinblastine), taxol, and topo II inhibitors t uch as doxorubicin, epirubicin, daunorubicin, 1 23 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:33 FAX 61 3 92438333 GRIFFITH HACK -IPAUTSTRALIA Q029m etoposide, over into Sas tamoxifen methotrexate be found in eds., Chapte antineoplast Philadelphia, "Biolog purposes hel stimulate apI or virally-ij single agent capable of ri binding and receptors maJ receptor, ani native or nat determining conducted fragmentation (1996)), casl eg., WO 98/) WO 98/I WO 99/] described in| 01/00832, andl The ter broad sense e death in man characteristic loss of plasr degradation o This activity cell viabilit, FACS analysis example, Nicc (1991), and I known in the e id bleomycin. Those agents that arrest G1 also spill Dhase arrest, for example, DNA alkylating agents such prednisone, dacarbazine, mechlorethamine, cisplatin, 5-fluorouracil, and ara-C. Further information can he Molecular Basis of Cancer, Mendelsohn and Israel, r 1, entitled "cell cycle regulation, oncogens, and C drugs" by Murakami e al. (WB saunders: 1995), especially p. 13.

Lcally active' or "biological activity" for the ein means having the ability to induce or ptosis in at least one type of mamnalian cancer cell ifected cell in vivo or ex vivo, either alone as a or in combination with a chemotherapeutic agent (b) ising an antibody, immunogenic; capable of or stimulating a receptor for Apo2L/TRAIL (such Sinclude the DR4 receptor, DR5 receptor, OPG, DCRI DcR2 receptor); or retaining the activity of a rally-occurring Apo2L/TRAIL polypeptide. Assays for biological activity of the Apo2L/TRAIL can be ing methods known in the art, such as DNA (see, Marsters et al., Curr. Biology, 6: 1669 1ase inactivation, DR4 binding, DR5 binding (see, 51793, published Nov. 19, 1998), DcRl binding (see, p8062, published Dec. 23, 1998), DcR2 binding (see, 0484, published March 4, 1999) as well as the assays PCT Publication Nos. W097/01633, W097/25428, WO WO 01/22987.

"apoptosis" and "apoptotic activity" are used in a nd refer to the orderly or controlled form of cell as that is typically accompanied by one or more Scell changes, including condensation of cytoplasm, membrane microvilli, segmentation of the nucleus, chromosomal DNA or loss of mitochondrial function.

can be determined and measured, for instance, by assays (such as Alamar blue assays or MTT assays), Scaspase activation, DNA fragmentation (see, for letti et al., J. Immunol. Methods, 139:271-279 oly-ADP ribose polymerase, "PARP", cleavage assays rt.

24 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:33 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 1030 As used herein, the term "disorder" in general refers to any condition th t would benefit from treatment with the compositions described he:ein, including any disease or disorder that can be treated by e fective amounts of polypeptides such as Apo2L/TRAIL.

This includes chronic and acute disorders, as well as those pathological conditions which predispose the mammal to the disorder in uestion. Non-limiting examples of disorders to be treated herein include benign and malignant cancers; inflammatory, angiogenic, land inmunologic disorders, autoimmune disorders, arthritis (i cluding rheumatoid arthritis), multiple sclerosis, and HIV/AIDS.

The terms "cancer", "cancerous", or "malignant" refer to or describe the physiological condition in manmals that is typically characterize by unregulated cell growth. Examples of cancer include but 1 are not limited to, carcinoma, lymphoma, leukemia, blastoma, an4 sarcoma. More particular examples of such cancers include squamous cell carcinoma, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal

I

cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic Ileukemia, colorectal cancer, endometrial cancer.

kidney cance prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cane bladder cancer, hepatoma, breast cancer, colon carcinoma, ard head and neck cancer. Optionally, the cancer cells express DR4 4d/or DRS receptor(s).

The teims "treating", "treatment" and "therapy" as used herein refer to curative therapy, prophylactic therapy, and preventative therapy. Consecutive treatment or administration refers to tr atment on at least a daily basis without interruption in treatment by one or more days. Intermittent treatment or administratio or treatment or administration in an intermittent fashion, refers to treatment that is not consecutive, but rather cyclic in natUre.

The tet "mammal" as used herein refers to any mammal classified a4 a mammal, including humans, cows, horses, dogs and cats. In a jreferred embodiment of the invention, the mammal is a human.

COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:34 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 031 B. Exemplary The pr< methods for formulation formulations, pharmaceutica protein in activity. Fc various exci formulations Apo2L/TRAIL.

The u crystallizati purification particular, I by various n term stabili lyophilized c through a re employed in subcutaneous Examples, so provided reaC biological a protein cryst buffer, e.g.

suspended in properties t] activity.

General] polypeptides various excip Methodo and Materials for Carrying Out the Invention sent invention provides various formulations, and making such formulations, of Apo2L/TRAIL. Various xcipients can enhance solubility of Apo2L/TRAIL in for instance, which are acceptable for uses and/or enhance stability of the Apo2L/TRAIL Sform trimer form) which has biological r example, Applicants have found that the presence of pients (for instance, arginine salts) in such can markedly increase the solubility and stability of iexpected finding of the readily reversible on of Apo2L/TRAIL further provides basis for methods and stable formulations of Apo2L/TRAIL. In orming crystals and subsequently drying the material ethods (including lyophilization) may provide long y of bulk preparations of the protein. Further, rystal compositions are expected to retain stability nge of temperatures. Dried crystals can also be suspension formulations suitable for, e.g., or intramuscular administration. As described in the 3ium salts, particularly sodium sulfate (Na2S 4 y and reversible crystallization, with retention of ;tivity upon re-dissolution of the protein. The als then readily re-dissolved in water or an aqueous a carboxylic acid salt of an amino acid, or can be non-aqueous media without loss in physicochemical iat can be important for the protein's biological y, the formulations are prepared using Apo2L/TRAIL (proteins), at the desired degree of purity, and Lents or components, described below- Production of ADo2L/TRAIL The de.

Apo2L/TRAIL 1 with a vecto recovering th cription below relates to methods of producing )y culturing host cells transformed or transfected Scontaining Apo2L/TRAIL encoding nucleic acid and polypeptide from the cell culture.

26 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:34 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 032 0 SThe DN encoding Apo2L/TRAIL may be obtained from any cDNA t library prep red from tissue believed to possess the Apo2L/TRAIL 4 mRNA and to express it at a detectable level. Accordingly, human l) Apo2L/TRAIL DNA can be conveniently obtained from a cDNA library prepared fro human tissues, such as the bacteriophage library of human placen al cDNA as described in PCT Publication W097/25428.

00 The Apo2L/T L-encoding gene may also be obtained from a genomic library or bt oligonucleotide synthesis.

SLibraries can be screened with probes (such as antibodies to o 10 the Apo2L/TR.L or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded o by it. Screening the cDNA or genomic library with the selected Sprobe may be conducted using standard procedures (Sambrook et al., Molecular Cl ning: A Laboratory Manual; New York: Cold Spring Harbor Labor tory Press, 1989). An alternative means to isolate the gene enciding Apo2L/TRAIL is to use PCR methodology (Sambrook et al., supra; Dieffenbach et al., PCR Primer:A Laboratory Manual, Cold Spring arbor Laboratory Press, 1995).

Amino a id sequence fragments or variants of Apo2L/TRAIL can be prepared y introducing appropriate nucleotide changes into the Apo2L/TRAIL pNA, or by synthesis of the desired Apo2L/TRAIL polypeptide. Such fragments or variants represent insertions, substitutions, and/or deletions of residues within or at one or both of the ends of the intracellular region, the transmembrane region, or tde extracellular region, or of the amino acid sequence shown for the full-length Apo2L/TRAIL in Figure 1 (SEQ ID NO:1).

Any combinaci n of insertion, substitution, and/or deletion can be made to arrive at the final construct, provided that the final construct possesses, for instance, a desired biological activity or apoptoti activity as defined herein. In a preferred embodiment, he fragments or variants have at least about amino acid s quence identity, more preferably, at least about sequence ide tity, and even more preferably, at least 95%, 96%, 97%, 98% or 9 sequence identity with, for example, the sequences identified erein for the intracellular, transmembrane, or extracellular domains of Apo2L/TRAIL, or the full-length sequence for Apo-2L/TF IL. The amino acid changes also may alter posttranslational processes of the Apo-2L/TRAIL, such as changing the number or postion of glycosylation sites or altering the membrane 27 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:34 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 1033 anchoring characteristics- Variations in the Apo2L/TRAIL sequence as described above can Sbe made us .ng any of the techniques and guidelines for conservative and non-conservative mutations set forth in U.S. Pat.

No. 5,364,931. These include oligonucleotide-mediated (sitedirected) mu tgenesis, alanine scanning, and PCR mutagenesis.

00 Scanning amino acid analysis can be employed to identify one or more amlo acids along a contiguous sequence. Among the O preferred scakning amino acids are relatively small, neutral amino acids. SucI amino acids include alanine, glycine, serine and o cysteine. A anine is typically a preferred scanning amino acid o among this gtoup because it eliminates the side-chain beyond the Sbeta-carbon and is less likely to alter the main-chain conformation jof the variant. (Cunningham et al., Science 1989.

244:1081). Elanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H.

Freeman Co. NY); Chothia, J. Mol. Biol. 1976, 150:1).

Particu3ar Apo2L/TRAIL variants of the present invention include thos, Apo2L/TRAIL polypeptides which include one or more of the reciled alanine substitutions provided in TABLE I of published PC application WO 01/00832. Such Apo2L/TRAIL variants will typical y comprise a non-naturally occurring amino acid sequence which differs from a native Apo2L/TRAIL amino acid sequence (suca as provided in Figure 1; SEQ ID NO:1, for a full length or malure form of Apo2L/TRAIL or an extracellular domain sequence thereof) in at least one or more amino acids.

Optionally, the one or more amino acids which differ in the Apo2L/TRAIL variant as compared to a native Apo2L/TRAIL will comprise amiro acid substitution(s) such as those indicated in Table I of 01/00832. Apo2L/TRAIL variants of the invention include solub.e Apo2L/TRAIL variants comprising residues 91-281, 92-281, 95-281 or 114-281 of Figure 1 (SEQ ID NO:1) and having one or more amino acid substitutions. Preferred Apo2L/TRAIL variants will include those variants comprising residues 91-281, 92-281, 95-281 or 114 281 of Figure 1 (SEQ ID NO:1) and having one or more amino acid sdbstitutions which enhance biological activity, such as receptor kinding. A particularly preferred variant comprises residues 114-281 of Figure 1 (SEQ ID NO:1). In a specific 28 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:35 FAX 61 3 92438333 GRIFFITH HACK 4, IPAUSTRALIA 034 embodiment, (SEQ ID NO:1 As desc ray crystal identified, provide the structure obt which contai coordinates I three subuni, appears to monomers whi per monomer) core beta-st IS structurally TNF-beta, an or TNF-beta.

Variati the scope of modified form Apo2L/TRAIL I modified met methionyl spc The nuc or variant A for further Various vect generally in following: a more marker transcriptior below. Optii genes, enhan that may be E detail in PC] Express that is reco( the Apo2L/TRJ po-2L/TRAIL consists of residues 114-281 of Figure 1 :ibed in WO 01/00832 published January 4, 2001, the xstructure of the extracellular domain of Apo2L/TRAIL and alanine-scanning mutagenesis was performed to mapping of its receptor contact regions. The ained for Apo2L/TRAIL revealed a homotrimeric protein s a novel divalent metal ion (zinc) binding site that :he interaction of the Apo2L/TRAIL trimer molecule's s. Like other members of the TNF family, Apo2L/TRAIL amprise a compact trimer formed of three jelly roll 2 2 :h bury approximately 5100 Angstrom (1700 Angstrom to form the globular trimer. The position of the rands was well conserved compared to the other characterized members of the TNF family, TNF-alpha, CD40L when compared to the core strands of TNF-alpha ns in the Apo2L/TRAIL sequence also included within the invention relate to amino-terminal derivatives or s. Such Apo2L/TRAIL sequences may include any of the )olypeptides described herein having a methionine or lionine (such as formyl methionyl or other blocked cies) at the N-terminus of the polypeptide sequence.

Leic acid cDNA or genomic DNA) encoding native po2L/TRAIL may be inserted into a replicable vector ;loning (amplification of the DNA) or for expression.

>rs are publicly available. The vector components :lude, but are not limited to, one or more of the signal sequence, an origin of replication, one or genes, an enhancer element, a promoter, and a termination sequence, each of which is described onal signal sequences, origins of replication, marker :er elements and transcription terminator sequences mployed are known in the art and described in further Publication W097/25428.

Lon and cloning vectors usually contain a promoter nized by the host organism and is operably linked to .L nucleic acid sequence. Promoters are untranslated 29 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:35 FAX 61 3 92438333 GRIFFITH HACK IAUSTRALIA 035 0 0 sequences located upstream to the start codon of a structural C) gene (genera 1ly within about 100 to 1000 bp) that control the transcriptiod and translation of a particular nucleic acid o sequence, suqh as the Apo2L/TRAIL nucleic acid sequence, to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are 00 promoters tht initiate increased levels of transcription from DNA under their control in response to some change in culture Sconditions, the presence or absence of a nutrient or a change in temperature. At this time a large number of promoters o recognized b a variety of potential host cells are well known.

SThese promot rs are operably linked to Apo2L/TRAIL encoding DNA by removing the promoter from the source DNA by restriction enzyme digestion an inserting the isolated promoter sequence into the vector. Bot the native Apo2L/TRAIL promoter sequence and many heterologous promoters may be used to direct amplification and/or expression ol the Apo2L/TRAIL DNA.

Promote s suitable for use with prokaryotic and eukaryotic hosts are kn<wn in the art, and are described in further detail in PCT Publication No. W097/25428.

Preferred methods for the production of soluble Apo2L/TRAIL in E. coli ploy an inducible promoter for the regulation of product expr ssion. The use of a controllable, inducible promoter allows for culture growth to the desirable cell density before induction oft product expression and accumulation of significant amounts of p oduct which may not be well tolerated by the host- Three nducible promoter systems (T7 polymerase, trp and alkaline pho:phatase have been evaluated by Applicants for the expressiln of Apo2L/TRAIL (amino acids 114-281). The use of each of thes three promoters resulted in significant amounts of soluble, bio ogically active Apo2L/TRAIL trimer being recovered from the har4ested cell paste. The AP promoter is preferred among these three inducible promoter systems tested because of tighter promoter condrol and the higher cell density and titers reached in harvested cell paste- Construjtion of suitable vectors containing one or more of the above-lit ted components employs standard ligation techniques.

Isolated pla mids or DNA fragments are cleaved, tailored, and religated in t e form desired to generate the plasmids required.

COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:35 FAX 61 3 92438333 GRIFFITH HACK -IPAUSTRALIA 0036 0 0 constructed, the ligation mixtures can be used to transform E.

coli K12 st:ain 294 (ATCC 31.446) and successful transformants Sselected b m apicillin or tetracycline resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion, and/or sequenced 00 using standa d techniques known in the art. (see, Messing et al., Nucleic Acids Res. 1981, 9:309; Maxam et al., Methods in O Enzymology 1 80, 65:499).

C' 10 Express on vectors that provide for the transient expression in mammalian cells of MA encoding Apo2L/TRAIL may be employed.

o In general, ransient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the ho t cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide bncoded by the expression vector (Sambrook et al., supra). Transient expression systems, comprising a suitable expression vector and a host cell, allow for the convenient positive ide tification of polypeptides encoded by cloned DNAs, as well as for he rapid screening of such polypeptides for desired biological r physiological properties. Thus, transient expression s stems are particularly useful in the invention for purposes of Ldentifying analogs and variants of Apo2L/TRAIL that are biologically active Apo2L/TRAIL.

Other nethods, vectors, and host cells suitable for adaptation ao the synthesis of Apo2L/TRAIL in recombinant vertebrate c ll culture are described in Gething et al., Nature 1981, 293:620-625; Mantei et al., Nature 1979, 281:40-46; EP 117,060; and EP 117,058.

Suitable host cells for cloning or expressing the DNA in the vectors here n include prokaryote, yeast, or higher eukaryote cells. Suit ble prokaryotes for this purpose include but are not limited to ubacteria, such as Gram-negative or Gram-positive organisms, f r example, Enterobacteriaceae such as Escherichia, E. c li, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Salmonella typhimurium, Serratia, Serratia marcescans, and Shigella. as well as Bacilli such as B. subtilis and B. lich iformis B. licheniformis 41P disclosed in DD 266,710 pubj ished 12 April 1989), Pseudomonas such as P.

31 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:36 FAX 61 3 92438333 GRIFFITH HACK 4, IPAUSTRALIA 037 0 0 aeruginosa, d Streptomyces. Preferably, the host cell should t secrete minir al amounts of proteolytic enzymes.

ME, colr is the preferred host cel-l for use in the present t invention. A. coli is particularly well suited for the expression of Apo2L/TRAIL (comprising amino acids 114-281 of Figure a polypeptide of under 20kd in size with no glycosylation requirement. As a production host, E. coli can be cultured to relatively high cell density and is capable of producing o relatively h gh levels of heterologous proteins.

In addttion to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression Shosts for Ap 2L/TRAIL-encoding vectors. Suitable host cells for the expression of glycosylated Apo2L/TRAIL are derived from multicellula4 organisms. Examples of all such host cells, includind CH cells, are described further in PCT Publication No.

W097/25428.

Host C lls are transfected and preferably transformed with the above-deqcribed expression or cloning vectors for Apo2L/TRAIL production ad cultured in nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Transfe tion refers to the taking up of an expression vector by a host c 11 whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily s illed artisan,for example, CaPO 4 and electroporation.

Successful hransfection is generally recognized when any indication of the operation of this vector occurs within the host cell.

Transfomation means introducing DNA into an organism so that the DNA is r licable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformaticn is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described i Sambrook et al-, supra, or electroporation is generally used for prokaryotes or other cells that contain substantial ell-wall barriers. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described (Saw et al., Gene 1983, 23:315 and PCT Publication No.

32 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:36 FAX 61 3 92438333 GRIFFITH HACK -*IPAUSTRALIA 038 WO 89/05859) ultrasound tx January 1991.

For man phosphate pr 1978. 52:4561 cell host as Patent No.

carried out Bact. 1977, 1979, 76:3821 cells, suchi bacterial pr polybr techniques f Methods in Ei 1988, 336:348 Prokary in suitable al,, supra.

employed fot application J Apo2L/TRAIL n Example, Ham's F10 (S 1640 (Sigma) Sigma). Anj hormones an transferrin, chloride, ca HEPES), nucl (such as Gen compounds u micromolar r Any other appropriate the art. Th like, are t1 expression, In addition, plants may be transfected using eatment, PCT Publication No. WO 91/00358 published malian cells without such cell walls, the calcium !cipitation method (Graham and van der Eb, Virology 457) may be employed. General aspects of mammalian 'stem transformations have been described in U.S.

,399,216. Transformations into yeast are typically according to the method of Van Solingen et al.,J.

L30:946 and Hsiao et al. Proc, Natl. Acad. Sci. USA .However, other methods for introducing DNA into as by nuclear microinjection, electroporation, )toplast fusion with intact cells, or polycations, me, polyornithine, may also be used. For various )r transforming mammalian cells, see Keown et al.

LZymology 1990, 185:527-537 and Mansour et al. Nature -352.

>tic cells used to produce Apo2L/TRAIL may be cultured culture media as described generally in Sambrook et Particular forms of culture media that may be culturing E. coli are described further in PCT ?O 01/00832. Mammalian host cells used to produce ay be cultured in a variety of culture media.

Sof commercially available culture media include .gma), Minimal Essential Medium Sigma), RPMI- Sand Dulbecco's Modified Eagle's Medium ("DMEM", such media may be supplemented as necessary with d/or other growth factors (such as insulin, or epidermal growth factor), salts (such as sodium Icium, magnesium, and phosphate), buffers (such as !osides (such as adenosine and thymidine), antibiotics bamycin m drug), trace elements (defined as inorganic sually present at final concentrations in the ange) and glucose or an equivalent energy source.

necessary supplements may also be included at ;oncentrations that would be known to those skilled in Sculture conditions, such as temperature, pH, and the se previously used with the host cell selected for and will be apparent to the ordinarily skilled 33 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:36 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 1039 artisan.

In gene for maximizii found in Man Butler, ed. Express directly, fo3 blotting to Natl. Acad, analysis), labeled prob labels may particularly such as usin polynucleotie avidin or an, labels. sue ral, principles, protocols, and practical techniques ig the productivity of mamalian cell cultures can be malian Cell Biotechnology: A Practical Approach, M.

IRL Press, 1991).

Lon of the Apo2L/TRAIL may be measured in a sample example, by conventional Southern blotting, Northern quantitate the transcription of mRNA (Thomas, Proc.

Sci. USA 1980, 77:5201-5205), dot blotting (D&A r in situ hybridization, using an appropriately based on the sequences provided herein. Various be employed, most commonly radioisotopes, and 2P. However, other techniques may also be employed, biotin-modified nucleotides for introduction into a e. The biotin then serves as the site for binding to Libodies, which may be labeled with a wide variety of L as radionucleotides, fluorescers or enzymes.

Alternativel antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may e labeled and the assay may be carried out where the duplex is bo id to a surface, so that upon the formation of duplex on the surfa e, the presence of antibody bound to the duplex can be detected.

Gene (xpression, alternatively, may be measured by immunologica] methods, such as immunohistochemical staining of cells or tisjue sections and assay of cell culture or body fluids, to quantitat directly the expression of gene product. With immunohistoct mical staining techniques, a cell sample is prepared, typically by dehydration and fixation, followed by reaction witl labeled antibodies specific for the gene product coupled, whele the labels are usually visually detectable, such as enzymatic la 4 els, fluorescent labels, luminescent labels, and the like.

I

Antibodies useful for immunohistochemical staining and/or assay of samrle fluids may be either monoclonal or polyclonal, and may be prepa ed in any mammal. conveniently, the antibodies may be prepared Against a native Apo2L/TRAIL polypeptide or against a synthetic peitide based on the DNA sequences provided herein or 34 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:37 FAX 61 3 92438333 GRIFFITH HACK -*IPAUSTRALIA R040 against exogd specific ant 1 Apo2L/TI as a secrete hose cell 1l signal. If from the me Triton-X 100 enzymatic cle When Ap one of huma] polypeptides recover or polypeptides homogeneous medium or 1 debris.

soluble prot being exen fractionatior CM-sepharose; chromatograpl sulfate preci and die The Apo Apo2L/TRAIL deleted, ins fashion as n changes in p preparation polypeptide, purification; antigen can I A prote (PMSF) also during purif the growth o will appreci Apo2L/TRAIL r nous sequence fused to Apo2L/TRAIL DNA and encoding a body epitope.

AIL preferably is recovered from the culture medium polypeptide, although it also may be recovered from rsates when directly produced without a secretory :he Apo2L/TRAIL is membrane-bound, it can be released brane using a suitable detergent solution Sor its extracellular region may be released by avage.

i2L/TRAIL is produced in a recombinant cell other than I origin, the Apo2L/TRAIL is free of proteins or of human origin. However, it is usually necessary to Lrify ApoZL/TRAIL from recombinant cell proteins or to obtain preparations that are substantially is to Apo2L/TRAIL. As a first step, the culture sate may be centrifuged to remove particulate cell o2L/TRAIL thereafter is purified from contaminant ins and polypeptides, with the following procedures lary of suitable purification procedures: by on an ion-exchange column such as SP-sepharose or hydroxyapatite; hydrophobic interaction y; ethanol precipitation; chromatofocusing- ammonium pitation; gel filtration using, for example, Sephadex filtration.

2L/TRAIL can be isolated by affinity chromatography.

Eragments or variants in which residues have been erted, or substituted are recovered in the same itive Apo2L/TRAIL, taking account of any substantial roperties occasioned by the variation- For example, of an Apo2L/TRAIL fusion with another protein or a bacterial or viral antigen, facilitates an immunoaffinity column-containing antibody to the e used to adsorb the fusion polypeptide.

ase inhibitor such as phenyl methyl sulfonyl fluoride may be useful to inhibit proteolytic degradation Lcation, and antibiotics may be included to prevent E adventitious contaminants. One skilled in the art ate that purification methods suitable for native iay require modification to account for changes in the COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:37 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 0J041 character oj recombinant c During expose the containing chromatograp metal ions.

used during Optionally, I DTT or BME, Apo2L/TRAIL.

during recov of Apo2L/TRA] the cell cult Apo2L/TRAIL or its variants upon expression in ell culture.

any such purification steps, it may be desirable to recovered Apo2L/TRAIL to a divalent metal ionolution or to purification material (such as a y medium or support) containing one or more divalent The divalent metal ions and/or reducing agent may be recovery or purification of the Apo2L/TRAIL.

both divalent metal ions and reducing agent, such as may be used during recovery or purification of the It is believed that use of divalent metal ions !ry or purification will assist in providing stability L trimer or preserve Apo2L/TRAIL trimer formed during uring step.

PREPARATION OF FORMULATIONS In the preparation of the formulations herein, it is noted that the rec<mmended quality or "grade" of the components employed will depend on the ultimate use of the formulation. Fdr therapeutic ses, it is preferred that the component(s) are of an allowable grade (such as "GRAS") as an additive to pharmaceutical products.

In cer:ain embodiments, there are provided compositions comprising >o2L/TRATL and one or more excipients which provide sufficient i nic strength to enhance solubility and/or stability of the Apo2I /TRAIL, wherein the composition has a pH of 6 (or about 6) to 9 (or about The Apo2L/TRAIL protein may be prepared by ny suitable method to achieve the desired purity of the protein, for example, according to the above methods. In preferred embodiments, the Apo2LTRAIL protein comprises amino acids 114-281 of Figure 1, and more preferably, the Apo2L/TRAIL protein is recombinantly expressed in E. coli host cells. The concentratior of the Apo2L/TRAIL protein in the formulation may vary depending, for instance, on the intended use of the formulation. Those skilled in the art can determine without undue experimentation the desired concentration of the Apo2L/TRAIL protein. For therapeutic uses, the concentration of the Apo2L/TRAIL 4rotein in the formulation is optionally about 0.1 to about 100 mgjml, about 1 to about 20 mg/ml, about 10 to about 36 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:37 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 01042 ng/mil, or abcut 20 mr/ml.

2 The one or more excipients in the formulations which provide Ssufficient i nic strength to enhance solubility and/or stability 7 of the Apo2L TRAIL is optionally.a polyionic organic or inorganic acid, aspartate, sodium sulfate, sodium succinate, sodium acetate, sodium chloride, Captisoll Tris, arginine salt or other amino O acids, sugats and polyols such as trehalose and sucrose.

Preferably te one or more excipients in the formulations which Sprovide sufficient ionic strength is a salt. Salts which may be employed include but are not limited to sodium salts and arginine salts. The type of salt employed and the concentration of the salt is preferably such that the formulation has a relatively high ionic strength which allows the Apo2L/TRAIL in the formulation to be stable (i reduce precipitation and enhance trimer content) and/or which allows the soluble protein concentration to exceed 2 mg/ml, more preferably, exceed 5 mg/ml, even more preferably exceed 10 mg and most preferably to achieve a concentration of at least abo t 20 mg/ml. Optionally, the salt is present in the formulation t a concentration of about 20 mM to about 0.5 M. In more preferre embodiments, the salt is an arginine salt or sodium sulfate. O tionally, the arginine salt may comprise arginine citrate, arginine tartrate, arginine malate, arginine succinate, arginine pho phate, and arginine sulfate. More preferably, the arginine salt is present in a concentration of about 0.2 M to about 0-5 M. It is noted that while arginine tartrate is useful as an excipient in the formulations described herein, the use of tartaric acid as a vehicle at higher concentrations (such as hundreds of ml) may not be desirable for parenteral administration or human clinical applications. Applicants have observed in an in vivo animal tudy that vehicle concentrations of 0.5M arginine neutralized ith 0.25M tartrate administered intravenously at greater than 5 ml/kg/hr can have a deleterious effect on renal tissue. ccordingly, there may be an upper threshold concentration of tartaric acid beyond which one skilled in the art would not sel, If relat desired in th or less than excipient pro sct for clinical, therapeutic uses.

ively lower concentrations of Apo2L/TRAIL protein are 9 formulation, for instance, less than about 5 mg/ml, about 2mg/ml, or about 0.1 to about 2 mg/ml, the viding stability to the formulation may be a sugar, 37 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:38 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 0043 0 0 C- such as tiehalose, sucrose, glucose, lactitol, or lactose.

Optionally, the sugar may be employed in the formulations at a concentratio1 of about 1% to about The excipient may also be San arginine alt, as described above.

0 5 The co position preferably has a pH of 6 (or about 6) to 9 (or about 9 more preferably about 6.5 to about 8.5, and even 00 more prefera ly about 7 to about 7.5. In a preferred aspect of this embodim nt, the composition will further comprise a buffer to Smaintain the pH of the composition at least about 6 to about 8.

Examples of buffers which may be employed include but are not limited to is, HEPES, and histidine. When employing Tris, the SpH may optiolally be adjusted to about 7 to 8.5, When employing C Hepes or his idine, the pH may optionally be adjusted to about to 7. Opti nally, the buffer is employed at a concentration of about 5 mM t about 50 mM in the formulation, and preferably at a concentratior of about 10 mM to about 20 mM.

Particularly for liquid formulations (or reconstituted lyophilized formulations), it may be desirable to include one or more surfactants in the composition. Such surfactants may, for instance, c mprise a non-ionic surfactant like TWEENW or

PLURONICS

T polysorbate or poloxamer). Preferably, the surfactant ccprises polysorbate 20 ("Tween The surfactant will optionaly be employed at a concentration of about 0.005% to about 0.2%.

Preferred formulations are those in which the excipient(s) provide for ctimized Apo2L/TRAIL trimer content and minimize the amount of Ap I2L/TRAIL dimer formation (or aggregate formation) or changes in harge distribution. Optionally, the formulation contains no rre than 5% Apo2L/TRAIL aggregates, no more than Apo2L/TRAIL isulfide linked dimer (of the total amount of Apo2L/TRAIL p otein in the formulation), and/or no more than change in the initial charge distribution.

In pref rred embodiments, the present invention provides compositions comprising about 0.1 mg/ml to about 20 mg/ml of Apo2L/TRAIL a Ld an arginine salt, wherein the composition has a pH of about 6.5 to about 8.5 (optionally, 6.8 to Optionally, the compositions further comprise a buffer such as Tris and/or a surfactant su h as polysorbate 20. Preferably, the Apo2L/TRAIL protein does ot include is not linked or fused to) any 38 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:38 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 0044 0

O

C] epitope tag tolecule(s) or leucine zipper molecule(s).

d- In eve more preferred embodiments, the present invention T provides copositions comprising about 2 to about 20 mg/ml of Apo2L/TRAIL,I about 0.4 to about 0.5M arginine salt, and buffer, o 5 wherein the composition has a pH of about 6.5 to about Optionally, a divalent metal ion may be included in the 00 formulations The divalent metal ion may be a zinc molecule, such as zinc sullate, zinc chloride, or zinc acetate. The divalent o metal ion c optionally be included in the formulation at a concentratio of about 50 micromolar to about 400 micromolar.

The fomulations of the present invention may include, in Saddition to Apo2L/TRAIL and those components described above, C] further vari us other excipients or components- Optionally, the formulation may contain, for parenteral administration, a pharmaceutic lly or parenterally acceptable carrier, one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. Optionally, the carrier is a parenteral carrier, such as a ;olution that is isotonic 'with the blood of the recipient. Examples of such carrier vehicles include water, saline or a buffered solution such as phosphate-buffered saline (PBS), Ringer's solution, and dextrose solution, Various optional pharmaceutica ly acceptable carriers, excipients. or stabilizers are describe further in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980).

The fo mulations herein also may contain one or more preservatives. Examples include octadecyldimethylbenzyl ammonium chloride, hexmethonium chloride, benzalkonium chloride (a mixture of alkylbenzy dimethylammonium chlorides in which the alkyl groups are long-chail compounds), and benzethonium chloride. Other types of preservati es include aromatic alcohols, alkyl parabens such as methyl or propyl paraben, and m-cresol. Antioxidants include ascorbic acid and methionine; preservatives (such as octadecyldime hylbenzyl amfmonium chloride; hexamethonium chloride; benzalkonium :hloride, benzethonium chloride; butyl 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 39 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:38 FAX 61 3 92438333 GRIFFITH HACK 4, IPAUSTRALIA 045 such as pcLyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, manhose, or dextrins; sugars such as sucrose, mannitol, trehalose or sorbitol; or polyethylene glycol (PEG).

Additiohal examples of such carriers include lecithin, serum proteins, such as human serum albumin, buffer substances such as glycine. sotbic acid, potassium sorbate, partial glyceride mixtures of electrolytes pyrrolidone.

based form carboxymethyl polyacrylates polyethylene forms inclu liposomes, pl release prepa The coz formulations lyophilized which the A amorphous prel The finj frozen at

I

lyophilized an for injection The for must be steri! through sterij Therapeutic c having a steri bag or vial h needle.

saturated vegetable fatty acids, water, salts, or such as protamine sulfate, sodium chloride, polyvinyl and cellulose-based substances. Carriers for gel- S include polysaccharides such as sodium :ellulose or methylcellulose, polyvinylpyrrolidone, Spolyoxyethylene-polyoxypropylene-block polymers, glycol, and wood wax alcohols. Conventional depot le, for example, microcapsules, nano-capsules, asters, inhalation forms, nose sprays, and sustainedrations.

positions of the invention may comprise liquid (liquid solutions or liquid suspensions), and ormulations, as well as suspension formulations in o2L/TRAIL protein is in the form of crystals or ipitate.

Ll formulation, if a liquid, is preferably stored 200 c. Alternatively, the formulation can be id provided as a powder for reconstitution with water that optionally may be stored at 2-30" C.

lulation to be used for therapeutic administration Sterility is readily accomplished by filtration e filtration membranes 0.2 micron membranes).

rmpositions generally are placed into a container le access port, for example, an intravenous solution tving a stopper pierceable by a hypodermic injection The compbsition ordinarily will be stored in single unit or multi-dose con aqueous solu reconstitution the art and f tainers, for example, sealed ampules or vials, as an tion or as a lyophilized formulation for The containers may any available containers in illed using conventional methods. Optionally, the COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:39 FAX 61 3 92438333 GRIFFITH HACK 4IPAUSTRALIA 0046 formulation cartridge wh in the art for therapeu lyophilized sterile-filt; resulting mi prepared by using, for eJ In furt there are crystals, pF formulation surprisingly 5"C is crysta other protei precipitates the solid sta when brought without a los the biochemic quite diffe precipitation Optiona] a super-satur to about 30° and more prel carried out i from a few m crystallizati, agitation.

agitated or s control. Int enhance mixin! controlled h Perspectives, 239-245]. cooling rate, formation rate may be included in an injection pen device (or a Lch fits into a pen device), such as those available see, US Patent 5,370,629), which are suitable ;ic delivery of the formulation. As an example of a Eormulation, 10 mL vials are filled with 5.5 mL of red 2% aqueous Apo2L/TRAIL solution, and the cture is lyophilized. An injection solution can be -econstituting the lyophilized Apo2L/TRAIL formulation ample, Water-for-Injection.

her more particular embodiments of the formulations, provided compositions which include Apo2L/TRAIL r instance, the composition may comprise a suspension comprising Apo2L/TRAIL crystals. Applicants found that the solid state of Apo2L/TRAIL protein at Lline at moderate to low ionic conditions, unlike many s known in the art that are soluble or form amorphous under similar conditions Further, it was found that te of the Apo2L/TRAIL crystals reversibly solubilizes to ambient temperature room temperature) s in protein biological activity or adverse effect on al properties of the protein. This observation was rent from the denaturation or irreversible observed for other proteins known in the art.

ly, the Apo2L/TRAIL crystals are prepared by cooling ated solution of Apo-2L/TRAIL protein from about C to below about 150 C, preferably about 2 to 8" C, erably, below about 2-8* C. Crystallization can be i batch or semi-batch mode at a large range of scale, illiliters to hundreds of liters of solution. The )n rate can be controlled by programmed cooling and he equipment may include, but is not limited to, tatic tanks with surface and/or internal temperature ernal baffles and draft tubes may also be used to f in agitated tanks. Crystal nucleation can also be y seeding [Moore, AIChE Practical Engineering Distillation and Other Industrial Separations, pp.

he degree of super-saturation, salt composition, agitation rate, and seeding can affect crystal Scrystal size distribution, and crystal yield.

41 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:39 FAX 61 3 92438333 GRIFFITH HACK -*IPAUSTRALIA 0~i047 Options 2L/TRAIL prf Optionally, and optional about 6.5 to The Ap< salts. Opti Alternativel ionic strenc storage or drying metho drying, vact motion faciJ drying and f] The dr formulation Al ternatively vi-scosity bi administratic aqueous. Ex systems such polymer-based An example o (SAIB) predi carbonate, o distribution the viscous r or a microflu In one improved metl More partic crystallizatig storage. The saving alter requiring mul material can of bulk storz lly, to prepare the crystals, the solution of Apotein contains sodium sulphate or sodium chloride.

the salt concentration is about 100mM to about 150 mM ly the pH is about 6 to about 9 (preferably, pH of about ZL/TRAIL crystal slurry may be washed to remove the onally, the crystal slurry may be washed with water.

r, the crystal slurry may be equilibrated to a low th. Subsequently, the material may be dried for reparation for parenteral formulations. The crystal is may include but are not limited to static vacuum umn drying with vibration, rotation, or agitation itated by dry air/N2 flow, lyophilization, spray uidized bed drying.

led crystals can be reconstituted to a liquid and sterilized for parenteral injection.

the dried crystals can be suspended in a high compatible medium for subcutaneous or intramuscular The suspending medium may be aqueous or non- Lples of aqueous suspensions include cellulose-based as carboxymethyl cellulose, hydroxyethylcellulose, or systems like polylactic acid-glycolic acid (PGLA).

Snon-aqueous medium is sucrose acetate isobutyrate ssolved in solvents such as ethanol, propylene N-mathyl pyrrolidone. A suspension of uniform size :an be prepared by homogenizing the dried crystals in.

edium using, by way of example, a probe homogenizer METHODS OF USE AND OTHER APPLICATIONS embodiment of the invention, there is provided an od for purifying and storing Apo2L/TRAIL protein.

ilarly, the methods of purification employ n of Apo2L/TRAIL and the crystals can be dried for methods provide an effective, efficient, and cost lative to, for instance, purification protocols .iple column purifications. Drying the crystalline also provide a relatively low volume, effective way ge which avoids freezing the purified material in 42 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:39 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 048 0 0 r\ bulk contain rs and thawing the frozen bulk material.

C) In the methods, an Apo2L/TRAIL preparation, such as a cell CT paste containg recombinantly expressed Apo2L/TRAIL protein, is provided. Otionally, though not required, the cell paste may be O 5 processed (fr instance, may be exposed to one or more reducing agents such as DTT or BME) or partially purified using any 00 suitable me hods known in the art, such as cation exchange chromatograp y methods. Cation exchange chromatography materials may optiona ly be SP-sapharose, CM-Sepharose, or Macro-prep ceramic HS resin. The processed or partially purified Apo2L/TRAIL in the prep ration can be crystallized from, for instance, a supersaturat d solution by decreasing temperature and agitation ci using the thods described herein. The crystals may then be collected, a d washed with buffer (or water) (preferably a cold buffer at a eimperature of about 2 to 8O The washed crystals can be re-su pended or re-dissolved at ambient temperature.

Re-solu ]ilized Apo2L/TRAIL can be further purified by hydrophobic interaction chromatography, recrystallized, washed and stored as w t crystalline bulk material. Alternatively, the hydrophobic interaction or other chromatography step may be omitted in favor of simply recrystallizing. The wet crystalline bulk materia can be stored at -20°C or dried for storage at ambient temperature (room temperature) or at 2-8 0 C. Preferably, the dried crystalline material is re-solubilized in an arginine succinate-con aining formulation described above. Optionally, such a formlation can be sterile filtered and/or filled in individual dcsage vials, and lyophilized for later reconstitution or suspension. Optionally, the dried crystalline formulation can be filled a a powder in vials and made into a solution or suspension.

The Apo L/TRAIL formulations described herein can be employed in a varietj of therapeutic and non-therapeutic applications.

Among these pplications are methods of treating disorders, such as cancer, i mune related conditions, or viral conditions. Such therapeutic and non-therapeutic applications are further described, f r instance, in W097/25428, W097/01633. and WO 01/22987.

In the mthods of the invention for treating a disorder using a formulation disclosed herein, the formulation of Apo2L/TRAIL can 43 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:40 FAX 61 3 92438333 GRIFFITH HACK -*IPAUSTRALIA 049 be directly including i administratic patient, inc: Apo2L/TRAIL of parentera.

intravenous, the compositI repeated int injec aerosol form delivery (fox It is important in solutions, w patient upon formulations of the inject Apo2L/T sustained-re release prep hydrophobic pI the form of Examples of derivatives isobutyrate poly Biomed. Mater 12: 98-105 oi 3,773,919, EP ethyl-L-glutai non-degradabl degradable lac Depot (inject acid copolym hydroxybutyric delivery for continuous i minipumps sucl idministered to the mammal by any suitable technique, afusion or injection. The specific route of in will depend, on the medical history of the uding any perceived or anticipated side effects using nd the particular disorder to be corrected. Examples L administration include subcutaneous, intramuscular, intraarterial, and intraperitoneal administration of on. The formulations are preferably administered as ravenous subcutaneous intramuscular tions or infusions, intracranial infusions or as ilations suitable for intranasal or intrapulmonary intrapulmonary delivery see, EP 257,956).

noted that osmotic pressure of injections may be subcutaneous and intramuscular injection. Injectable en hypotonic or hypertonic, may cause pain to a infusion. Usually, for the therapeutic, injectable herein, it is preferred that the relative osmolarity able solution be about 300 mosm to about 600 mosm.

AIL can also be administered in the form of sase preparations. Suitable examples of sustainedarations include semipermeable matrices of solid 6lymers containing the protein, which matrices are in shaped articles, films, or microcapsules.

sustained-release matrices include cellulose carboxymethylcellulose), sucrose-acetate

SABER"

T

in non-aqueous media, polyesters, hydrogels 2-hydroxyethyl-methacrylate) (Langer et al., J.

Res. 1981, 15: 167-277; Langer, Chem. Tech. 1982, poly(vinylalcohol)), polylactides Patent No.

58,481), copolymers of L-glutamic acid and gamma ate (Sidman et al-, Biopolymers 1983, 22: 547-556), ethylene-vinyl acetate (Langer et al., supra), 'tic acid-glycolic acid copolymers such as the Lupron able microspheres composed of lactic acid-glycolic ar and leuprolide acetate), and poly-D-(-)-3acid (EP 133,988). One optional method of systemic-acting drugs involves administration by ifusion (using, slow-release devices or Sas osmotic pumps or skin patches), or by injection 44 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:40 FAX 61 3 924: '2 (using, e.g.

bolus adminis The con and dosed ii taking into patient, the 3 administrati factors known component f< consideratior \1 the Apo2L/TRa As a 1 effective amc be in the ra based on kg will be subje Althougl be employed may also be e It is employed irt include but therapy, cyto agent(s), cy farnesyl tra cyclin-depend defined furtl addition, the tumor antigen angiogenic an Apo2L receptoi Preparat may be used determined em and dosing sc Chemotherapy Baltimore, MD It may be de antigens, suc )8333 GRIFFITH HACK 4 IPAUSTRALIA 1 050 intravenous or subcutaneous means, including singleitration).

position to be used in the therapy will be formulated Sa fashion consistent with good medical practice, account the clinical condition of the individual site of delivery of the composition, the method of n, the scheduling of administration, and other to practitioners. The "effective amounts" of each ir purposes herein are thus determined by such s and are amounts that result in bioavailability of IL or other drugs to the mammal.

general proposition, the total pharmaceutically unt of the Apo2L/TRAIL polypeptides administered will age of from about 1 mg/kg/day to about 20 mg/kg/day ,f patient body weight although, as noted above, this -t to therapeutic discretion.

Sinjection is preferred, an infusion device may also or continuous infusions. An intravenous bag solution nployed.

contemplated that yet additional therapies may be :he methods. The one or more other therapies may are not limited to, administration of radiation tine(s), growth inhibitory agent(s), chemotherapeutic :otoxic agent(s), tyrosine kinase inhibitors, ras Isferase inhibitors, angiogenesis inhibitors, and nt kinase inhibitors which are known in the art and Ler with particularity in Section I above. In rapies based on therapeutic antibodies that target a such as Rituxan T or Herceptin" as well as anti- :ibodies such as anti-VEGF, or antibodies that target s, such as DR5 or DR4.

ion and dosing schedules for chemotherapeutic agents according to manufacturers' instructions or as irically by the skilled practitioner. Preparation ledules for such chemotherapy are also described in Service Ed., M.C. Perry, Williams Wilkins, (1992).

irable to also administer antibodies against other K as antibodies which bind to CD20, CDlla, CD18, COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:40 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 051 ErbB2, (VEGP), or TNFR1, TNFR antibodies b disclosed h Sometimes, ii cytokines tc formulations The Apo or sequentia Apo2L/TRAIL (prior to adm treatment of apoptotic eff The in described he preferably a described a label, direct formulation.

formulation.

treating can containers in test tubes.

materials sue an Apo2L/TRAlI treating the example, the vial having needle). Th indicates that the disorder c comprise a s pharmaceutical, or dextrose s desirable fron buffers, dilue with instructiq All pate EGPR, ErbB3, ErbB4, vascular endothelial factor ther TNFR family members (such as DR4, DR5, OPG, S Alternatively, or in addition, two or more .nding the same or two or more different antigens xein may be co-administered to the patient.

may be beneficial to also administer one or more the patient. In one embodiment, the Apo2L ire co-administered with a growth inhibitory agent.

L/TRAIL formulation may be administered concurrently Lly with such other agents. For example, the ormulation may be administered as a pre-treatment Lnistration of any such other agents), such as a pre- :ancer cells which may otherwise be resistant to the icts of Apo2L/TRAIL.

ation also provides kits which include a formulation ein. A typical kit will comprise a container, vial, for Apo2L/TRAIL in one or more excipients as re; and instructions, such as a product insert or ing the user as to how to employ the Apo2L/TRAIL This would preferably provide a pharmaceutical Preferably, the pharmaceutical formulation is for er or an immune related condition. Suitable :lude, for example, bottles, vials, syringes, and The containers may be formed from a variety of h as glass or plastic. The container holds formulation that is effective for diagnosing or disorder and may have a sterile access port (for container may be an intravenous solution bag or a a stopper pierceable by a hypodermic injection a label on, or associated with, the container the formulation is used for diagnosing or treating f choice. The article of manufacture may further acond container comprising water-for-injection, a Ly-acceptable solution, saline, Ringer's solution, Dlution. It may further include other materials a commercial and user standpoint, including other its, filters, needles, syringes, and package inserts )ns for use.

nts, patent applications, publications, product 46 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:41 FAX 61 3 92438333 GRIFFITH HACK 4, IPAUSTRALIA 052 descriptions the disclosu their entire The fol only, and a invention in in the examp unless other in the folli ATCC accessi Manassas, Vi: Formula identify Apo from a thera particular, and conditiol and protocols are cited throughout this application, -es of which are incorporated herein by reference in ;ies.

EXAMPLES

lowing examples are offered for illustrative purposes -e not intended to limit the scope of the present any way. commercially available reagents referred to .es were used according to manufacturer's instructions rise indicated. The source of those cells identified wing examples, and throughout the specification, by an numbers is the American Type Culture Collection, -ginia.

:ions were prepared and assays were conducted to L/TRAIL formulations having desirable characteristics )eutic, diagnostic, and/or commercial standpoint. In %pplicants sought to identify formulation components Is that, among other things, may enhance solubility of biologically active Apo2L/TRAIL, particularly at concentrations up to at least 0 mg/ml, and may provide stability upon storage at 2- 8" C or at aNmient temperature. Applicants also sought to identify Apo2L/TRAIL ormulations for use in the clinic that may preserve the protein's native non-covalent trimer content, charge distributioni and/or biological activity during storage.

EXAMPLE 1: Li quid Formulations of Apo2L/TRAIL with Enhanced Solubility aid Stability Apo2L/T AIL protein consisting of amino acids 114-281 (see Figure 1) was expressed in E. coli under the AP promoter control (preparation and expression described in Example 8 (Section A) of WO 01/00832 published January 4, 2001), and purified from the E.

coli cell l1sates by three chromatographic steps consisting of cation exchange, hydroxyapatite, and hydrophobic interaction chromatography (WO 01/00832, Example 8, Section In the third chromatographic separation, the Apo2L/TRAIL protein was eluted in 600 mM Na su.fate or 400 mM ammonium sulfate, 50 mM Tris, pH The protein as then buffer exchanged to the various formulation excipients .isted in Table 1 by dialysis, and was next concentrated at ambient temperature using Centricon-10 filtration 47 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:41 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA I053 up to concen through 0.22 assess solub: As shoi examined. H from coon otherwise ir (Pfanstiehl Arginine fre used for ini 8 0 C (the sto vials), 2) and diafilti freeze-thaw stability.

assessment o: spectroscopy rim.

Table ionic strengi Apo2L/TRAIL c erations of 20 mg/mL. The samples were then filtered micron filters and stored at either 2-8°C or 30 0 C to .lity and stability.

n in Table 1, about 20 different excipients were igh purity NF, USP, or EP grade excipients were used commercial sources (Sigma, Mallindkrodt) unless dicated as follows: alpha,alpha trehalose dihydrate or Senn), sucrose (Pfanstiehl), CaptisolT (Cydex), a base (Ajinomoto or Kyowa Hakko Kogyo). Criteria ;ial excipient screening included 1) solubility at 2rage condition of bulk preparations prior to fill in ;olubility under manufacturing scale ultrafiltration ,ation steps, 3) short term liquid stability and stability, and 4) lyophilized formulation physical Solubility at 2-8°c was evaluated by periodic visual Sprecipitation for up to 1 month and confirmed by UV scan using an extinction coefficient of 1.53 at 278 Sindicates that excipients having relatively high :h conditions provided solubility at concentrations of .bove 10 mg/ml at 2-80C.

TABLE 1 Excipiiet Solubility At 2-goC 8% TrehElose <3 mg/ml 8% Sucrose <3 mg/ml 16% Sucrose <2 mg/ml 6% Lact tol <2 mg/ml 4% Sucrse/4% Mannitol 2mg/ml PEG3 50 <5 mg/ml Glycerol <5 mg/ml 0.15 M iaC1 <5 mg/ml 0.25 M a phosphate <5 mg/ml M G ycine <12 mg/ml M Ajpartate 10-20 mg/ml M N sulfate 10-20 mg/ml COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:41 FAX 61 3 92438333 GRIFFITH HACK -*IPAUSTRALIA 1054 M N! acetate >20 mg/ml M Ni chloride >20 mg/ml 24% S1lfobutylether beta- >20 mg/ml cyclode:trin (CaptisolT) Tris >20 mg/ml Ar inine-tartrate >20 mg/ml Short-t 300C) was th in Table concentratiox assessed by (SEC) to det the preparat: charge distr: (Pharmacia) 4 mobile using a ProP.

run at a rat( The res arginine tar exhibited th (Figure 2).

Example 2; Arginine Salt Applica: arginine salt by ultrafiltj pharmacokinet form of th particularly mg/ml Apo2L/' To ic pharmaceutics physical sta: arginine saltitrating 0.

Table 2) to C arm liquid stability (1 week storage time period at en evaluated for several of those preparations shown L that provided solubility of Apo2L/TRAIL at Ls of about 20 mg/ml. The short-term stability was visual assessment of turbidity, size exclusion HPLC ermine the amount of native trimer and aggregates in .ons, and by ion exchange HPLC (IEX) to determine the .bution. SEC was conducted using a Superose 12 column ind a 13 mM Na phosphate, 400 mM ammonium sulfate (pH )hase run at a rate of 0.6 ml/min. IEX was conducted Lc WCX-10 column (Dionex) at 400C and a NaCL gradient Sof 0.5 ml/min.

ults are shown in Figure 2. The sodium sulfate, trate, Captisol"', and sodium acetate preparations e greatest stability in one or both of the assays Lyophilized Apo2L/TRAIL Formulations Containing ts found that Apo2L/TRAIL formulations containing s could be readily concentrated to 20 mg/mL protein ation and diafiltration. Because of certain in vivo ic properties of the Apo2L/TRAIL 114-281 amino acid r protein (described in Example Applicants sought to identify a stable formulation having RAIL protein.

entify those arginine salts that provide Lly and commercially viable lyophilized products, the >ility of vehicle formulations containing different Is was evaluated. Arginine salts were prepared by M Arginine free base with various acids (shown in ive a pH 7 solution in 20 mM Tris. 2 ml preparations 49 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:42 FAX 61 3 92438333 GRIFFITH HACK 4 IAUSTRALIA 055 were filled long freeze primary dryi of the solu using vapor preparations clinical seti Table containing p desirable ph acids. The as solid in rather than fragmented sl In add demonstrated administrati( arginine sal solution) an preparations 0.5 M in 5 cc glass vials and subjected to a conservative -drying (lyophilization) cycle (freezing at ig at 0°c and secondary drying at 42 0 Osmolality :ions (prior to lyophilization) was also determined pressure depression methods to identify those that may be suitable for IV administration in a :ing.

2 indicates that the lyophilized preparations flyanionic organic or inorganic acids exhibited more rsical stability than those prepared using monoanionic .yophilized products of the polyanionic salts appeared :act dried cakes (indicated in Table 2 as 'yes"), nelted, gelled, collapsed, fenestrated, egg-shaped or lells (indicated in Table 2 as ition, 0_5M arginine salts of polyanionic acids an osmolality which may be suitable for IV n (less than 2-fold hypertonic), unlike the monoionic s 0.5M arginine-lactate gives a 3.1x hypertonic i some of the other relatively high ionic strength that exhibited good solubility and liquid stability sodium acetate gives a 3.4 fold hypertonic solution).

TABLE 2 Arginine halts Acceptable lyo Osmolality of physical stability? M solution* Arg-citra:e Yes 505 Arg-tartr te Yes 530 Arg-malat Yes 573 Arg-succi ate Yes 630 Arg-oxala e Yes ND Arg-lacta e No 927 Arg-glyco ate No ND Arg-aceta e No 978 Arg-glut4 ate No 899 Arg-phosp ate Yes 465 Arg-sulfale Yes 462 Arg-nitra fe No 774 Arg-HCL No 830 *Val es are mosmol/kg. Isotonic solutions have an COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:42 FAX 61 3 92438333 GRIFFITH HACK -IPAUSTRALIA R056s osmoality of approximately 292-300.

indi ates value was not determined.

"ND"

Based c 2, several then evaluat storage at N prepared a! ultrafiltrat: tartrate or trehalose.

and 0.01% pc described ab native trime IS and charge di After 4 peak was det that was sto indicates formulations formulation I TO furt stability of lyophilized containing 2 biochemical monitored at n the results obtained in the study reported in Table lyophilized Apo2L/TRAIL-containing formulations were ed for biochemical stability of the protein after 'arious temperatures. Apo2L/TRAIL (residues 114-281; described in Example 1) was formulated by .on/diafiltration to 10 mg/ml in 0.5M argininearginine-citrate, or to 3 mg/ml in 8% sucrose or ai of these preparations contained 20 mM Tris, pH lysorbate 20. The samples were then lyophilized as we. Stability was assessed by measuring content of r and aggregates using SEC (described in Example 1) stribution using IEX (described in Example 1).

months storage at 40 0 C, the trimer and %IEX main rmnined relative to an unlyophilized control solution ted at -70 0 C (see Figure 3A). The data in Figure 3A :hat the arginine salt-containing lyophilized exhibited greater stability as compared to the reparations containing sucrose or trehalose.

er examine the effects of arginine-salt type on the Apo2L/TRAIL formulations, both the liquid and the formulations of four different arginine salt- 0 mg/ml Apo2L/TRAIL formulations were monitored for stability of the protein. Liquid stability was 2-80C and at ambient temperatures for up to 1 month I (Table 3) anl lyophilized stability was monitored at 50 0 C for one month (Figure 3B).

In addition to conducting SEC and IEX assays described in Example 1, covalent dimer formation was monitored by SEC under denaturing c nditions (SDS-SEC). The SDS-SEC assay was conducted using a TSK-000XL column (TosoHaas) run at 0.6 ml/min in a mobile phase consisting of 25 mM sodium phosphate, 0.1% SDS, 200 mM NaCl.

Samples were diluted to 1 mg/ml protein with a solution that gave mM Tris (pH 200 mM NaCI, 0.5% SDS, pH 9 and 5 mM iodoacetamide (Sigma) Samples were then incubated at 50 0 C for minutes prior to HPLC analysis.

Bioacti ity of the Apo2L/TRAIL in the various formulations 51 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:42 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 057 was also det for viable c uL at 2 Albumin, RPM well plates.

cell line, cells/well d 370C, and A] hour incubat: fluorescence and emission assay range concentratiol cells. Cell As show formulations formulations The arginine aggregate fo the highest and arginine IEX main p( After 2 sulfate and level of bioe Overall somewhat sup.

liquid state.

ermined using SK-MES-1 cells and Alamar Blue staining 11 counts. In the assay, the Apo2L/TRAIL formulation ug/mL) was added to assay medium Bovine serum E 1640) and 2-fold serial dilutions were made in 96- Then, 50 uL SK-MES-1 cells (human lung carcinoma ATCC HTB58) were added into the wells at 20000 ensity. The plates were incubated for 24 hours at amar Blue was added for the last 4 hours of the 24 Lon time. The staining intensity was determined on a plate reader with excitation wavelength set at 530 nm at 590 nm. A four-parameter fit to the data in the of 0.1 to 1000 ng/ml gives the ED50, or the Sof Apo2L/TRAIL that induces 50% killing of the killing potency increases with decreasing ,n in Table 3, after 1 month storage of the liquid at 2-80C, the four arginine-salt containing showed only small differences in Apo2L/TRAIL quality.

sulfate formulation exhibited the highest extent of rmation. The arginine-malate formulation exhibited axtent of dimer formation, and the arginine-phosphate malate formulations showed the largest change in the ak area.

weeks storage at ambient temperature, the argininerginine-succinate formulations retained the highest ctivity (Table 3).

the arginine-succinate formulation demonstrated ior stability characteristics for Apo2L/TRAIL in the Table 3 Formulations Trimer IEX main Monomer I% bioactivity peak remaining Argiie of control) Argininemalate 96.2 105.1 93.4 77.0 Argininesuccinate 96.2 94.7 94.9 68,5 Argininesulfate 95.4 93.4 93.3 90.5 Argininephophate 96.0 91.0 91.8 74.3 Data are for 1 month storage at 2-80C.

2 Data are for 2 weeks storage at ambient temperature.

COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:43 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA The st these argin: assessed by above). Nc observed(see of Apo2L/T Apo2L/TRAIL profile usin mg/ml Apo Tris, pH 7.0 Figure 3C, a a 2 year sl found that a Apo2L/TRAIL 0.02% polyso; for at least %main peak p 2-8 0 C (as we; described abc ability of the lyophilized formulations containing ne-salts after 1 month storage at 50°C was also SEC, IEX and SDS-sEC (using the protocols described changes in the physical-chemical properties ware Figure 3B), suggesting that significant stabilization AIL may be achieved through lyophilization of -ormulations containing arginine-salts. An Arrhenius y accelerated temperature stability of a lyophilized ZL/TRAIL formulation in 0.5M arginine-tartrate, 20 mM 0.01% polysorbate 20, 0.33 mM Zn sulfate is shown in d predicts a relatively long shelf life at 2-8 0 C, and elf-life at ambient temperature. Applicants have lyophilized formulation containing 20 mg/mL Ln 20 mM Tris, pH 7.2. 0.5 M arginine-succinate and kbate 20 was stable at temperatures as high as 12 months (Table The kinetics of change in IEX redicts a significantly long year) shelf life at Sas at ambient temperature), as with the formulation e in Figure 3C_ Table 4 Tempe ture IEX main %Monomer S__%Trimer peak 0 C liquid 97.4 46.1 99-0 contr Lyo, 2-80C, 12 mo 97.5 46.5 99.0 Lyo, 33 0 C, 12 mo 97.3 45.4 98-8 Lyo, 12 mo 97.4 44.6 98.8 Lyo, 500C, 12 mo 97.3 42.5 98.7 EXAMPLE 4: Ef As descj also, Hymow] Apo2L/TRAIL p at position c intramolecula: bioactivity c typically for groups, to pr and T. Crei Stability," M4 Humana Press rects of pH in Apo2L/TRAIL Formulations -ibed in WO 01/.00832 published January 4, 2001 (see .tz et al., Biochemistry, 39:633-640 (2000), rotein forms a homotrimer with a Zn-coordinated thiol ,steine 230 of each monomer, and the formation of an disulfide bond at cysteine 230 results in loss of If the protein. Cysteine-containing proteins are nulated at low pH, well below the pKa of the thiol event disulfide bond formation (see, N. Derby ;hton, "Disulfide Bonds in Protein Folding and thods in Enzymology, Vol 40 (Edited by B.A. Shirley; Inc, Totowa, NJ), Chapter 10 (1995)). surprisingly, 53 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:43 FAX 61 3 92438333 GRIFFITH HACK -*IPAUSTRALIA 059 as the resu non-covalent decreasing pI Apo2L/1 (referred tc prepared ess Ni-chelating the third c] dialysis intc phosphate (pi formulations to procedures Within formulations bioactivity observed witl poly-histidir acids 114-281 described by Recombinant prepared in 1 buffers (pH stability of for the formu 6.5 (see Fi Crystal coordination cysteine 230 stability of supra). Th.

although not loss in zn bi protonated at neutral pH ir preferred for stability of j EXAMPLE 5: El To exair .ts discussed below reveal, the stability of native, Apo2L/TRAIL trimer was found to decrease with

I.

RAIL consisting of amino acids 94-281 (Figure 1) herein and in Figute 4 as 'Apo2L/TRAIL.2") was sntially as described in WO 01/00832, except that a affinity chromatography, instead of HIC, was used as Lromatographic step. Formulations were prepared by 10 mM Na succinate (pH range 5.5 to 6.5) or 10 mM Na I range of 7 to Liquid stability of these was assessed by SEC and bioactivity assays accbrding described in the Examples above.

one day storage at ambient temperature, the having pH below about pH 6.5 lost trimer content and (see Figure 4A). This pH-stability profile was other Apo2L/TRAIL protein variants. For example, a e-tagged Apo2L/TRAIL formulation consisting of amino (Figure 1) (His-Apo2L/TRAIL; prepared essentially as Ashkenazi et al., "Safety and Antitumor Activity of 3oluble Apo2 ligand", JCI, 104:155-162 (1999) was 0 mM Na succinate (pH 5.5 to 6) or 10 mM Na phosphate 5.5 to After storage at 300C for 1 week, the trimer and bioactivity (determined as described above lations of Figure 4A) was found to be enhanced at pH gure 4B).

structure analysis of Apo2L/TRAIL reveals that of an intrinsic Zn atom to three free thiols of is needed for proper folding and native structural the protein (see WO 01/00832 and Hymowitz et al., unexpected pH-stability profile of Apo2L/TRAIL, fully understood, is believed to be associated with iding to the trimer as the thiol moiety becomes more lower pHs (see Figure It is believed that the range of about 6.5 to about 8.5 will be most maintaining the bioactivity and physical-chemical po2L/TRAIL formulations.

fect of surfactant on stability of Apo2L/TRAIL Ir Lne the effect of surfactant on stability of 54 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:43 FAX 61 3 92438333 GRIFFITH HACK 4 PAusTRALIA 0060 Apo2L/TRAIL Apo2L/TRAIL Example 1) i varying con (as control) temperature horizontally.

scattering (i observed as (Figure 6).

It is t! non-ionic s formulations handling thA interfaces.

EXAMPLE 6: Apo2L/TRAIL f A liqui residues 114was prepared mM Tris, p sulfate. Af stability was above in Exam] As show formulations linked intram affect the stability tow 7).

EXAMPLE 7: Apo2L/TRAIL Applicant Apo2L/TRAIL s strength), the the crystalli controlled to formulations, formulations containing 20 mg/ml (114-281 protein (Figure prepared as described in n 0.5 M arginine-succinate, 20 iM Tris, pH 7.2, and ;ntrations of Tween 20 (0.005%, 0.01%, 0.02%, or none were prepared and agitated at 70 rpm and ambient for up to 24 hours in glass vials positioned Within 1 hour of agitation, an increase in light ieasured by absorbance in the range of 340-360 nm) was the concentration of Tween 20 fell below 0.005% lerefore believed that it may be preferable to include irfactant(s) such as Tween 20 in Apo2L/TRAIL to enhance stabilization against agitation and .t can denature the bulk protein at air-water Effect of Zn sulfate on stability of liquid >rmulations I formulation of Apo2L/TRAIL protein consisting of 81 (Figure 1) (prepared as described in Example 1) using 20 mg/ml Apo2L/TRAIL, 0.5M arginine-tartrate, 7.0, in the presence of zero, 117 uM, or 330 uM Zn :er storage at 30 0 C or 2-80 for up to 2 months, evaluated by SEC, IEX, and SDS-SEC (as described les 1 and 2) relative to -70 0 C control samples.

i in Figure 7, addition of Zn sulfate to the brovided increased stabilization against disulfide- [lecular dimer formation. Though Zn sulfate did not stability of Apo2L/TRAIL at 2-8 0 C, it improved ards dimer formation at higher temperatures (Figure Reversible Crystallization Of Biologically Active s have found that under conditions of low 6 lubility (for example, at moderate to low ionic protein can be crystallized. As described herein, zation rate, particle size, and yield can be give useful industrial methods for purification, COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 nFi/n9 3007 1Rldd PAX 61 1 82dlRRd~ GRIFFITH HACK -IPAUSTRALIA 0061 0 bulk storage and controlled release suspension formulation of _QApo2L/TRAIL.

Apo2L/TRAIL protein consisting of residues 114-281 (Figure 1) (prepared as described in Example 1) was formulated at ambient o 5 temperature Using NAP5 column (Pharmacia) elution in 20 mM Tria, pH 7.2 and v rious concentrations of Na sulfate. After elution at o0 ambient temp rature, within hours, hexagonal shaped crystals of varying len ths were observed (see Figure Equilibrium o solubility w s reached when samples were continuously rotated for 3-4 days at a given temperature. There was a minimum in solubility at approximately 10-50 mM ionic strength. Solubility o increased to a maximum at approximately 0.3 M Na sulfate and then 0 decreased until limiting solubility of the salt was reached. The pattern was similar at higher temperatures, but solubility increased with increasing temperature. The observation of increasing p'otein solubility (hence decreased crystallization) with increasing salt concentration in the hundreds of millimolar salt range i unlike connon understanding with respect to other proteins, where crystallization propensity tends to increase with increasing salt concentration (see, A. Ducruix and MM Reis- Kautt, 'Solub lity Diagram Analysis and the Relative Effectiveness of Different Ions on Protein Crystallization," METHODS: A Companion to Methods in Enzymology, Vol. 1, pp. 25-30 (1990)).

Monovaleat cationic salts (such as sodium chloride) provided the greatest crystallization propensity as shown in Figure 9, while divalent cationic salts calcium chloride and magnesium chloride) siglificantly reduced crystallization. Crystallization also occurred in positively charged (lysine-salts and argininesalts) and ne atively charged (aspartic acid) amino acid solutions below 0.3 mo ar concentration (see Figure though arginine salts reduced crystallization propensity at the same ionic strength.

To identify various crystallization process parameters, a L solution containing 5 mg/ml Apo2L/TRAIL (residues 114-281; prepared as dfscribed in Example 0.1M NaSO, and 20mM Tris at pH 7.2 was subjected to a single step cooling from room temperature to 50C. Four experiments were performed with agitation ratas ranging from 0 to 200 rpm. The supernatant concentration of Apo2L/TRAIL was measured in 10-minute intervals 56 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:44 FAX 61 3 92438333 GRIFFITH HACK 4IPAUSTRALIA 0062f using UV spe Figure complete wit faster. Cry.

comparison.

until 2 days increase wit] Figure crystals und, to 30-35°C experiment.

measured in crystal diss increased fr( tank jacket agitation sp, dissolution temperature V Figure of agitatic distribution Size Analyze agitation spe 0.1] to D(v, Therefore, me appears to b Apo2L/TRIAL which may be :troscopy to monitor the progress of crystallization.

10A shows that crystallization was more than ain 2 hours when the bulk was agitated at 50 rpm or Itallization without agitation was much slower in Static crystallization did not reach 90% completion after cooling began. Crystallization rate appeared to k increasing agitation speed.

10B depicts the dissolution profile of Apo2L/TRAIL ar agitation. The crystal slurry was warmed from SC with the same heating rate in each dissolution Apo2L/TRAIL concentration in the supernatant was 5-minute intervals to monitor dissolution rate. The olution rate increased when agitation speed was m 50 rpm to 200 rpm. Heat transfer rate between the and the crystal slurry was also enhanced when ed increased. At 100 rpm agitation rate, complete Ias achieved within a half hour when the sample as at approximately 35 0

C-

OC shows the crystal size distribution as a function i speed during crystallization. Crystal size was measured using a Malvern MasterSizerX Particle r The mean diameter decreased as ed increased, and the crystal size distribution (D[v, became more uniform with faster agitation.

nipulating the agitation rate during crystallization effective in controlling the mean diameter of the irystals as well as the crystal size distribution, lesirable for controlled release formulations.

Example 8: Dring of Apo2L/TRAIL Crystals To asseE controlled re drying method The firs Apo2L/TRAIL described in sodium sulfat salt. The cr under 29-30 s the feasibility of drying-Apo2L/TRAIL crystals for lease formulation or bulk storage, three different were evaluated.

t drying method evaluated was static vacuum drying.

mino acids 114-281; purified according to the method Example 1) was crystallized in 20 mM Tris, 0.1 W pH 7.2 and washed with cold water to remove excess stal slurry was filled in open glass vials and dried inches of mercury vacuum at ambient temperature 57 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:44 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA U0633 overnight. i either water The sed drying (appa and decrease um filter washed with mixture (50% dried by pas; filter. A s i the filter cl filter chamI to break up process. T humidity sens port. The el flowing powd( turn increase in a buffere dried crystal The thirt method, the c in glass vial settled. TI secondary dry These crystal in buffered 0 The pro assessed usi discussed in that Apo2L/T remained bioc liquid contro: he dried crystals were dissolved to 2-3 mg/ml in or in 0.5 M arginine-salt, 20 mM Tris, pH 7.2.

ond drying method evaluated was vibrational vacuum atus obtained from SWECO Co.) to allow better flow solid clumping. The crystal slurry was loaded on a to remove the bulk liquid, and the wet crystals were cold Tris buffer (20mM, pH 7.5) or ethanol-water S63%, 75%, 100%, The washed crystals were ing dehumidified nitrogen gas from the bottom of the ight vacuum (8-10 inches of mercury) from the top of Lamber facilitated the drying rate. Furthermore, the !r containing the wet crystals vibrated at 1,800 rpm the wet cake into fine powder during the drying ie drying process was monitored using a relative or. Dried product was recovered through a discharge :hanol-water mixture washes produced finer and better ir than crystals washed with Tris buffer, which in d the process yield. These crystals were dissolved d 0.5M arginine salt, as described for the vacuum -s.

l drying method evaluated was lyophilization. In this rystal slurry was washed with cold water and filled s. Excess bulk water was removed after the crystals Le slurry was then frozen to -50 0 C. Primary and ing were carried out at -25'C and 0 0 C, respectively.

s were better flowing powders and dissolved readily .5M arginine-salts.

tein quality in the dissolved crystals was then ig SEC, SDS-SEC. IEX, and bioactivity assays as Examples 1 and 2. Table 5 and Figures 11A-11B show AIL crystals dried with the different methods hemically equivalent to the non-crystallized frozen .preparation.

Table COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:45 GRIFFITH HACK IPAUSTRALIA 064 The apparent impurities in

SDS-SEC.

The dat subsequent d protein struc Example 9: Apo2L/TRAIL i To as Apo2L/TRAIL, crystalline 7.5, 0.2-0.5M were stored reconstitutin for physicaldescribed in 20 mg/ml Apo2 pH 7-2, 0.01% lower monomer in control sample by SDS-SEC is due to the starting material which co-elute with Apo2L dimer on a suggest that crystallization of Apo2L/TRAIL and 'ying of the material does not adversely affect :ure or function.

Lyophilized Formulation of Crvstalline-containina I Sodium sulfate ress storage stability of crystal-containing lyophilized formulations were prepared with po2L/TRAIL (residues 114-281) in 20 mM Tris, pH 7sodium sulfate, and 0.01-0.05% Tween 20. The samples At various temperatures for up to 4 months. After g with sterile water, the formulations were tested shemical stability using SEC, IEX, and SDS-SEC assays Examples 1 and 2. Table 6 summarizes the data for a L/TRAIL formulation in 0.2 M Na sulfate, 20 mM Tris, Tween 20 after 3 months storage- Table 6 Temperature IEX main %Monomer %Trimer peak lIquid control 99.3 56-1 99.1 Lyo, 2-1oC, 3 mo 99.1 58.2 99.3 Lyo, 30'C, 3 mo 97.0 56.4 98.3 Lyo, 40C, 3 mo 94.1 52-9 95.9 Lyo, 50'C, 3 mo 90.1 44.8 91.7 Assuming profiles pred this formulat: though fille crystallize tc lyophilization a pseudo-first order degradation kinetics, Arrhenius ct significantly longer than 2 years shelf-life for .on at 2-8 0 C (see Figure 12). These preparations, 1 as clear liquid solutions of Apo2L/TRAIL, varying degrees during the freezing portion of the cycle, demonstrating that dried formulations 59 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:45 FAX 61 3 92438333 GRIFFITH HACK 4IPAusTRALIA 0065s containing c term storage Example 10: and Purificat The pr sulfate solut protein from employed for without adver The ha (described ii (or 1.5M Tr: corporation, 5mM DTT and hours. The (Alfa Laval clarified b) (extract) w concentration was then loa cation exchan mM Hepes Apo2L/TRAIL flowed throu equilibration The column wa equilibration NaCi (or 0.

Triethanolami The amb; SP column was jacket for h conical botto crystallized impeller unde skid was used to appro crystallizatic 4°C. After crystallizatic established.

'ystallized Apo2L/TRAIL and stability.

sodium sulfate have long Apo2L/TRAIL Crystallization as a Method of Recovery ion >pensity of crystallization of Apo2L/TRAIL in Na ions was used as a means of purifying the Apo2L/TRAIL E. coli extracts. The following protocol may be recovery and purification of recombinant Apo2L/TRAIL se effect on protein quality.

rvested whole cell broth derived from E. coli Example 1) was adjusted to pH 7.5 with 1.5 M Hepes and then homogenized in a homogenizer (Gaulin Everett, MA). The homogenate was conditioned with flocculated with 0.1 polyethyleneimine for 1-2 flocculated material was centrifuged by a BTPX205 eparation AB, Sweden) continuous feed centrifuge and depth filtration_ The clarified cell lysate as conditioned with Triton-X00 to a final of 0.05 The conditioned, clarified cell lysate 5ed onto a cation exchange column (SP-Sepharose FF je resin, Amersham Pharmacia, Sweden] equilibrated in (or 50 mM Tris)/0.05% Triton-X 100/1mM DTT, pH ound to the column while the non-binding proteins ;h the column and were removed by washing with buffer until absorbance at 280 nm reached baseline.

s then washed with 3 column volumes of 0.1 M Nacl in buffer. The Apo2L/TRAIL was step-eluted using 0-1 M 1M Na 2 SO,) in 50 mM each of Hepes, Tris and 0.05 Triton-X 100 and 1 mM DTT buffer, pH 7.8.

ent temperature Apo2L/TRAIL pool collected from the placed in a stainless steel tank with an insulated ating and cooling. The tank was outfitted with a Sand a flush bottom valve for maximal recovery of >rotein. The pool was agitated using a marine type L modest mixing conditions. A temperature control to linearly ramp the temperature from approximately limately 4°C over the course of 1 hour. Spontaneous n was observed within minutes after the pool reached more than 12 hours under these conditions, r was complete as equilibrium solubility was nearly The crystals were then captured on a filtration COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:45 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA a 066 assembly cont deposition o chilled 20-5 compared to t residual moti Following th< buffer throug of the cryst dissolved, pi container and The pur: the total E_ Lysate (LAL) performed by antibodies on horseradish i activity was absorbance at level was det SDS-PAGE sili polyacrylamid containing 0 constant curr gel. Gels we Merrill silve Protein bioactivity ai The pur; above crystal: in Table 7.

three-chromat( also shown.

aining a 2 0um polypropylene frit. Following crystal i the filter surface, the crystals were washed with imM Tris at pH 7.5. An equal volume of wash buffer he Apo2L/TRAIL SP pool volume was then used to remove er liquor (supernatant) from the deposited crystals.

wash, the crystals were dissolved in 100mM sodium Tris at pH 7.5 by recirculating the dissolution h the crystal bed at approximately 30 0 C. Dissolution lls was observed within approximately 4 hours. The rified Apo2L/TRAIL was then sterile filtered into a stored frozen at -70 0

C.

.ty of the Apo2L/TRAIL preparations was determined by coli protein (ECP) ELISA assays, Limulus Amebocyte assay, and SDS-PAGE silver stain. ECP ELISA was immobilizing affinity-purified goat anti-whole ECP microtiter plate wells, incubating samples and then 'eroxidase-conjugated ECPs. The peroxidase enzymatic then quantified with o-phenylenediamine by reading 490 nm in a microtiter plate reader. Endotoxin armined using the Limulus Amebocyte clot lysis assay.

'er stain was performed on a 10 to 20% gradient gel (Daiichi Pure Chemicals) in Tris-glycine buffer 1% SDS. Electrophoresis was conducted at 50 mA ent until dye front reached near the bottom of the re fixed and stained by Coomassie Brilliant Blue or stain methods.

quality was assessed by SEC, SDS-SEC, IEX,and .cording to methods described in Examples 1 and 2.

ty and quality of Apo2L/TRAIL recovered using the ization method at a 60 L fermentation scale is shown For comparison, a reference standard purified by a graphic step method as described in Example 1 is Table 7 Apo2L/ Pro ein Purity Protein Quality TRAIL ECP LAL SDS- Monomer Bioactivity IEX Prep. (ppm) (EU/mg) PAGE Trimer by SDS- of main peak by SEC SEC control I_ Apo2L/ 10 0.034 No band 99.0 99-0 126 63 TRAIL at purified by crystallization COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:46 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 0067 Reference material purified by standard chromatoraph

I

As shi preparation suitable for binding pro removed by t the "one-col 1 cryscallizati Apo2L/TRAIL method descri type on crysti "Poisoning" c for partiall observed for The bi adversely inr Apo2L/TRAIL of recombinan state, can be its purificat: preparation c formulations a The pres specific emh modifications herein will bi foregoing de.

modifications appended claim The ent our Australia] cross-referen 92 0.023 Band at 98.9 98-9 86 61 OkDa mn in Table 7 and Figure 13, the Apo2L/TRAIL .t a manufacturing scale had a high degree of purity therapeutic use. In particular, a 10 kDa E_ coli DNA sin that tends to co-purify with Apo2L/TRAIL was ie crystallization process. The data indicate that mn" step purified Apo2L/TRAIL protein is amenable to an and has a purity comparable to or better than the protein purified by the three-column purification :ed in Example 1. Figure 14 shows the effect of salt allization of a one-column step purified Apo2L/TRAIL.

f crystallization by divalent cations was observed purified Apo2L/TRAIL (Fig. 14), similar to that .99% purified Apo2L/TRAIL shown in Figure 9.

Themical properties of Apo2L/TRAIL were also not acted by crystallization of the partially purified see Table The data suggest that crystallization :-expressed Apo2L/TRAIL, when in a partially purified an effective, efficient and cost-effective means for .on. Optionally, such crystals can then be used for f dried bulk for storage or controlled release s described in Examples 8 and 9.

snt invention is not to be limited in scope by the odiments described herein- Indeed, various of the invention in addition to those described :come apparent to those skilled in the art from the cription and the accompanying figures. Such are intended to fall within the scope of the s.

ire disclosure in the complete specification of i Patent Application No. 2002346376 is by this :e incorporated into the present specification.

62 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05

Claims

1. A stabli and about 0.; pH of about 6 ED IS: formulation of Apo-2 M to about 0.5M salt, to about 9. ligand, comprising Apo-2 ligand wherein said formulation has a

2. salt. The formulation of claim 1 wherein said salt is arginine

3. The fon arginine salt

4. The for selected from sulphate, arc and arginine The f arginine succ

6. The for sulphate.

7. The for comprises cry. ulation of claim 2 wherein the concentration of said in the formulation is about 0.4M to about 0.5 M. nulation of claim 2 wherein the arginine salt is the group consisting of arginine succinate, arginine inine malate, arginine citrate, arginine tartrate, phosphate. rmulation of claim 2- wherein the arginine salt is nate. nulation of claim 1 wherein the salt is sodium ulation of claim 1 or 6 wherein the Apo-2 ligand ;tallized protein. ulation of claim 1 wherein said formulation is

8. The for lyophilized.

9. The fornlation of claim I wherein the is about 6.5 to about The is about

11. The ligand is

12. The comprises formlation of claim 9 wherein the 7 to about formilation of claim 1 wherein the abo t 1 mg/ml to about 20 mg/ml. pH of said formulation pH of said formulation concentration of Apo-2 for ulation of claim 1 wherein amio acids 114 to 281 of Figure 1. said Apo-2 ligand COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:46 FAX 61 3 92438333 GRIFFITH HACK -*IPAUSTRALIA 0069a

13. The fox linked or fu:

14. The fox comprises sm The for polysorbate c nulation of claim 12 wherein ed to an epitope tag. nulation of claim 1 wherein factant. said Apo-2 ligand is not said formulation further

16. The for surfactant ir

17. The for comprises buf

18. The for buffer.

19. The forn is about 7 to The forn comprises one ulation of claim 14 wherein said surfactant is a r poloxamer. lulation of claim 14 wherein the concentration of said the formulation is about 0.005% to about 0.2%. lulation of claim 1 wherein said formulation further Eer. lation of claim 17 wherein said buffer is Tris Llation of claim 18 wherein the pH of the formulation about ulation of claim 1 wherein said formulation further or more divalent metal ions.

21- The f omulation of claim 20 wherein said one or more divalent metal ions is zinc.

22. The formlation of claim I further comprising a preservative.

23. The form storage-stable

24. The forn storage-stable A stable, about 1 mg/ml arginine formulation ha lulation of claim 1 wherein said for at least 12 months. ulacion of claim 23 wherein said for at least 24 months. formulation is -formulation is lyophilized formulation of Apo-2 ligand, comprising :o about 20 mg/ml Apo-2 ligand, about 0.2 M to about salt, buffer, and surfactant, wherein said a pH of about 6 to about 9. 64 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:46 FAX 61 3 92438333

26. The forulat arginine succinat GRIFFITH HACK IPAUSTRALIA [1070 :ion of claim 25, wherein said arginine salt is e-

27. said The fo ulation of claim 26, wherein the concentration arginine succinate is about 0.4M to about 0.SM.

28. The buffer. fornulation of claim 25, wherein said buffer is Tris

29. The fon polysorbate. aulation of claim 25, wherein said surfactant is a The comprises to1ulation of claim 25, wherein amino acids 114 to 281 of Figure 1. said Apo-2 ligand

31. The form comprises one ulation of claim 25, wherein said formulation further or more divalent metal ions.

32. A stable formulation of Apo-2 ligand, comprising about Img/ml to about 20 dg/ml Apo-2 buffer, and crystallized about 9.

33. The forn sulphate.

34. The for buffer. The fort polysorbate.

36. The form pH of about 7

37. A stable mg/ml to abot wherein said f, surfactant, >rotein and ligand, about 0.2M to about 0.5 M salt, wherein said Apo-2 ligand comprises said formulation has a pH of about 6 to lation of claim 32, wherein said salt is sodium llation of claim 32, wherein said buffer is Tris ulation of claim 32, wherein said surfactant is lation of claim 32, wherein said formulation has a o about Eormulation of Apo-2 ligand, comprising about 0.1 t 2 mg/ml Apo-2 ligand, sugar, and surfactant, rmulation has a pH of about 6 to about 9. COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:47 FAX 61 3 92438333 GRIFFITH HACK 4, IPAUSTRALIA 071 The formulation of claim 37 wherein said sugar is trehalose.

39. The fox sugar in the The fo: lyophilized.

41. A methc comprising st Apo-2 ligand, surfactant, make a formul step to a nulation of claim 37 wherein the concentration of the formulation is about 1% to about 8%. mulation of claim 37 wherein said formulation is i of making a stable formulation of Apo-2 ligand, eps of providing about 1 mg/ml to about 20 mg/ml about 0.2 M to about 0.5M arginine salt, buffer, and 3) combining or mixing the ingredients of step to ation, and adjusting the pH of the formulation of >out 6 to about 9.

42. The method of claim 41, wherein said arginine salt is arginine succI nate.

43- The met] arginine suec: Lod of claim 42, wherein the concentration of said .nate is about 0.4M to about The methd of claim 41, wherein said buffer is Tris buffer-

45. The met polysorbate.

46. The meth amino acids 13 47 A methoc steps of (a) cationic salt, to make a forn C, and low -to about 20 C occurs as the lowered. aod of claim 41, wherein said surfactant is a 3d of claim 41, wherein said Apo-2 ligand comprises 4 to 281 of Figure 1. of making crystallized Apo-2 ligand, comprising providing Apo-2 ligand,. buffer, and monovalent combining or mixing the ingredients of step (a) ulation at a temperature of about 20" C to about 300 ering the temperature of the formulation of step (b) to about 8" C; wherein Apo-2 ligand crystallization temperature of the formulation of step is

48. The methcd of claim 47, wherein said salt is sodium sulphate COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:47 FAX 61 3 92438333 GRIFFITH HACK -4IPAUSTRALIA 072

49. The met is 0.1M to aj The met is agitated z

51. The met a step ir

52. The met Apo-2 ligand

53. A methcx providing ho Apo-2 ligand; conditions su expressed Apo formulating chloride or s of about 200 i said formulat Apo-2 ligand lowered.

54. The metj Apo-2 ligand

55. The meth concentrated ultrafiltratic

56. The metl applying the j the Apo-2 li containing buf hod of claim 48 wherein the concentration of the salt )out 0.15M. od of claim 47, wherein the formulation of step (b) .s the temperature is lowered in step od of claim 47, wherein the method further comprises which the Apo-2 ligand crystals are dried- lod of claim 51, wherein prior to said step the crystals are washed. of making Apo-2 ligand, comprising the steps of: (a) t cells comprising a vector containing INA encoding culturing the host cells in culture medium under fficient to express Apo-2 ligand; obtaining said -2 ligand from the host cells and culture medium; (d) aid Apo-2 ligand into a solution containing sodium )dium sulphate to make a formulation at a temperature to about 30° C, and lowering the temperature of on of step to about 20 c to about 8" C, wherein crystals form when the temperature of step is 3d of claim 53 wherein rotein is concentrated. Dd of claim 54 wherein by centrifugation, prior to said step the the Apo-2 ligand protein is column chromatography or n. :d of claim 53 wherein step is conducting by po-2 ligand to a chromatographic column and eluting and into a sodium chloride or sodium sulphate Cer solution.

57. The metho 1 of claim 56 a cation exchapge column. wherein said chromatographic column is 67 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:47 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA Ri073 58, The meth comprises SE Macro-prep ce

59. The methi mM Hepes, 100, 1 mM DTT )d of claim 56 wherein said cation exchange column -Sepharose fast flow, CM-Sepharose fast flow, or :amic HS. d of claim 56 wherein said buffer solution contains 50 mM Tris, 50 mM triethanolamine, 0.05% Triton X pH 7.5 od of claim 53 wherein the formulation is agitated k). od of claim 53 wherein the pH of the formulation in out 6-5 to about

60. The metl during step

61. The metI step is a

62. The cells methPd of claim 53 wherein said host cells are prokaryote

63. The met d of claim 62 wherein said prokaryote cells are E. coli.

64. A device maommal, conpri the Apo-2 liga for administering a formulation of Apo-2 ligand to a sing a container holding at least one dosage unit of nd formulation of claim 1, 25, 32, or 37. The device. devite of claim 64 wherein said device is a pen injector The devi4e of claim 64 wherein the container is a cartridge.

67. An artic includes the j and printed in

68. is a The artic bottle, v: le of manufacture, comprising a container which po2L/TRAIL formulation of claim 1, 25, 32, or 37, structions for use of said Apo-2L/TRAIL formulation. le of manufacture of claim 67 where said container .al, syringe, or test tube. :le of manufacture of claim 67 which comprises a aer which includes water-for-injection, saline, on, or dextrose solution.

69. The arti, second contai Ringer's solut: COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05 05/02 2007 16:47 FAX 61 3 92438333 GRIFFITH HACK 4 IPAUSTRALIA 0074 A metho exposing man ligand formul

71. The met cancer cells. Sof inducing apoptosis in mammalian cells, alian cells to an effective amount of ation of claim 1, 25, 32, or 37. comprising the Apo-2

72. A meth. administering amount of the aod of claim 70 wherein said mammalian cells are 'd of treating cancer in a mammal, comprising to a mammal diagnosed as having cancer an effective Apo-2 ligand formulation of claim 1, 25. 32, or 37- 69 COMS ID No: SBMI-06142977 Received by IP Australia: Time 16:51 Date 2007-02-05