U.S. patent application number 11/915110 was filed with the patent office on 2011-03-03 for method of purifying apo-2 ligand/trail usin crystallisation the cold.
Invention is credited to Heather Flores, Tanya Lin, Timothy Matthews, Roger Pai, Zahra Shahrokh.
Application Number | 20110054153 11/915110 |
Document ID | / |
Family ID | 37150024 |
Filed Date | 2011-03-03 |
United States Patent
Application |
20110054153 |
Kind Code |
A1 |
Flores; Heather ; et
al. |
March 3, 2011 |
METHOD OF PURIFYING APO-2 LIGAND/TRAIL USIN CRYSTALLISATION THE
COLD
Abstract
The present invention relates generally to Apo2L/TRAIL
purification involving crystallization.
Inventors: |
Flores; Heather; (Hayward,
CA) ; Lin; Tanya; (Tucson, AZ) ; Matthews;
Timothy; (San Mateo, CA) ; Pai; Roger; (Los
Altos Hills, CA) ; Shahrokh; Zahra; (Weston,
MA) |
Family ID: |
37150024 |
Appl. No.: |
11/915110 |
Filed: |
May 10, 2006 |
PCT Filed: |
May 10, 2006 |
PCT NO: |
PCT/US06/18137 |
371 Date: |
February 26, 2009 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11136842 |
May 24, 2005 |
7741285 |
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11915110 |
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10771254 |
Feb 3, 2004 |
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11136842 |
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PCT/US02/36251 |
Nov 12, 2002 |
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10771254 |
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60338249 |
Nov 13, 2001 |
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Current U.S.
Class: |
530/414 ;
530/416 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 47/183 20130101; A61K 38/16 20130101; A61K 47/26 20130101;
A61K 33/30 20130101; C07K 14/70575 20130101; A61K 47/02 20130101;
A61K 33/00 20130101; A61K 9/0019 20130101; A61K 33/30 20130101;
A61K 31/198 20130101; A61K 38/16 20130101; A61K 9/19 20130101; A61K
31/198 20130101; A61K 33/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
530/414 ;
530/416 |
International
Class: |
C07K 1/00 20060101
C07K001/00 |
Claims
1. A method of recovering Apo2L/TRAIL from a mixture comprising (a)
loading the mixture on a cation exchange column; (b) washing the
cation exchange column with an equilibration buffer whereby
non-binding components present in the mixture are removed; (c)
eluting Apo2L/TRAIL bound to the cation exchange column with an
elution buffer; (d) gradually cooling the eluate to a temperature
of about 2 to 4.degree. C., whereby Apo2L/TRAIL is spontaneously
precipitated in a crystalline form to yield a mixture of mother
liquor and Apo2L/TRAIL crystals, and (e) recovering Apo2L/TRAIL
from the mixture obtained in step (d) in a purity of at least about
99%.
2. The method of claim 1 wherein the mixture loaded on the cation
exchange column is a culture medium or cell lysate of Apo2L/TRAIL
producing cells.
3. The method of claim 2 wherein said mixture is the cell lysate of
Apo2L/TRAIL producing E. coli host cells.
4. The method of claim 3 wherein said lysate is clarified prior to
loading on the cation exchange column.
5. The method of claim 1 wherein the eluate obtained in step (c) is
subjected to the crystallization step of (d) without additional
purification.
6. The method of claim 1 wherein the cation exchange column is an
SP-Sepharose column.
7. The method of claim 6 wherein the pH of the mixture loaded on
said column is or is adjusted to about 7.5.
8. The method of claim 6 wherein elution of Apo2L/TRAIL is
performed in an elution buffer comprising 100-200 mM NaCl or
100-150 mM Na.sub.2SO.sub.4 in a buffer adjusting the pH to
7.5-7.8.
9. The method of claim 1 wherein in step (d) the eluate is cooled
from a temperature of about 15 to 30.degree. C. to a temperature of
about 2 to 8.degree. C. in about 1 to 60 hours.
10. The method of claim 9 wherein in step (d) the eluate is cooled
to a temperature of about 2 to 8.degree. C. in about 1 to 8
hours.
11. The method of claim 9 wherein in step (d) the eluate is cooled
to a temperature of about 2 to 8.degree. C. in about 1 hour.
12. The method of claim 11 wherein in step (d) the eluate is cooled
to a temperature of about 4.degree. C. in about 1 hour.
13. The method of claim 1 wherein the pH of the eluate is or is
adjusted to pH 7.0-8.0 prior to crystallization.
14. The method of claim 13 wherein the pH of the eluate is or is
adjusted to about 7.3 prior to crystallization.
15. The method of claim 13 wherein the pH of the eluate is or is
adjusted to about 7.5-8.0 after crystallization.
16. The method of claim 1 wherein in step (d) the temperature of
about 2 to 4.degree. C. is maintained until equilibrium solubility
of Apo2L/TRAIL is achieved or nearly achieved.
17. The method of claim 16 wherein in step (d) solubility of
Apo2L/TRAII is decreased by the addition of an anti-solvent.
18. The method of claim 17 wherein said anti-solvent is selected
from the group consisting of a polyethylene glycol (PEG), MPD,
ethanol, isopropanol, and dioxane.
19. The method of claim 18 wherein the molecular weight of the PEG
is between about 400 and about 10,000 daltons.
20. The method of claim 19 wherein the molecular weight of the PEG
is 400, 3,350 or 10,000 daltons.
21. The method of claim 1 wherein in step (e) Apo2L/TRAIL is
recovered in the form of crystals separated from the mother liquor
by filtration or centrifugation or a combination thereof.
22. The method of claim 21 wherein pH of the mother liquor is
adjusted to about 8.0 prior to filtration to decrease
solubility.
23. The method of claim 1 further comprising the steps of
dissolving the Apo2L/TRAIL crystals obtained in step (d) and
subjecting the solution obtained to a second chromatographic
purification step.
24. The method of claim 23 wherein said second chromatographic
purification step is hydrophobic interaction chromatography.
25. The method of claim 24 wherein hydrophobic interaction
chromatography is performed on a Phenyl-Sepharose column.
26. The method of claim 23 wherein said second chromatographic
purification step is cation exchange chromatography.
27. The method of claim 26 wherein said cation exchange
chromatography is performed on an CM-Sepharose or SP-Sepharose
column.
28. The method of claim 23 wherein Apo2L/TRAIL is recovered and
formulated following said second chromatographic purification step
by ultrafiltration-diafiltration.
29. The method of claim 1 wherein said purity is at least about
99.5%.
30. The method of claim 1 wherein said purity is at least about
99.9%.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to Apo2L/TRAIL
purification involving crystallization.
BACKGROUND OF THE INVENTION
[0002] Various molecules, such as tumor necrosis factor-alpha
("TNF-alpha"), tumor necrosis factor-beta ("TNF-beta" or
"lymphotoxin-alpha"), lymphotoxin-beta ("LT-beta"), CD30 ligand,
CD27 ligand, CD40 ligand, OX-40 ligand, 4-1 BB ligand, Apo-1 ligand
(also referred to as Fas ligand or CD95 ligand), Apo-2 ligand (also
referred to as Apo2L or TRAIL), Apo-3 ligand (also referred to as
TWEAK), APRIL, OPG ligand (also referred to as RANK ligand, ODF, or
TRANCE), and TALL-1 (also referred to as BlyS, BAFF or THANK) have
been identified as members of the tumor necrosis factor ("TNF")
family of cytokines [See, e.g., Gruss and Dower, Blood,
85:3378-3404 (1995); Schmid et al., Proc. Natl. Acad. Sci., 83:1881
(1986); Dealtry et al., Eur. J. Immunol., 17:689 (1987); Pitti et
al., J. Biol. Chem., 271:12687-12690 (1996); Wiley et al.,
Immunity, 3:673-682 (1995); Browning et al., Cell, 72:847-856
(1993); Armitage et al. Nature, 357:80-82 (1992), WO 97/01633
published Jan. 16, 1997; WO 97/25428 published Jul. 17, 1997;
Marsters et al., Curr. Biol., 8:525-528 (1998); Chicheportiche et
al., Biol. Chem., 272:32401-32410 (1997); Hahne et al., J. Exp.
Med., 188:1185-1190 (1998); WO98/28426 published Jul. 2, 1998;
WO98/46751 published Oct. 22, 1998; WO/98/18921 published May 7,
1998; Moore et al., Science, 285:260-263 (1999); Shu et al., J.
Leukocyte Biol., 65:680 (1999); Schneider et al., J. Exp. Med.,
189:1747-1756 (1999); Mukhopadhyay et al., J. Biol. Chem.,
274:15978-15981 (1999)]. Among these molecules, TNF-alpha,
TNF-beta, CD30 ligand, 4-1 BB ligand, Apo-1 ligand, Apo-2 ligand
(Apo2L/TRAIL) and Apo-3 ligand (TWEAK) have been reported to be
involved in apoptotic cell death.
[0003] Apo2L/TRAIL was identified several years ago as a member of
the TNF family of cytokines [see, e.g., Wiley et al., Immunity,
3:673-682 (1995); Pitti et al., J. Biol. Chem., 271:12697-12690
(1996)]. The full-length human Apo2L/TRAIL polypeptide is a 281
amino acid long, Type II transmembrane protein. Some cells can
produce a natural extracellular region [Mariani et al., J. Cell.
Biol., 137:221-229 (1997)]. Crystallographic studies of soluble
forms of Apo2L/TRAIL reveal a homotrimeric structure similar to the
structures of TNF and other related proteins [Hymowitz et al.,
Molec. Cell, 4:563-571 (1999); Hymowitz et al., Biochemistry,
39:633-644 (2000)]. Apo2L/TRAIL, unlike other TNF family members
however, was found to have a unique structural feature in that
three cysteine residues (at position 230 of each subunit in the
homotrimer) together coordinate a zinc atom, and that the zinc
binding is important for trimer stability and biological activity.
[Hymowitz et al., supra; Bodmer et al., J. Biol. Chem.,
275:20632-20637 (2000)].
[0004] It has been reported in the literature that Apo2L/TRAIL may
play a role in immune system modulation, including autoimmune
diseases such as rheumatoid arthritis, and in the treatment of HIV
[see, e.g., Thomas et al., J. Immunol., 161:2195-2200 (1998);
Johnsen et al., Cytokine, 11:664-672 (1999); Griffith et al., J.
Exp. Med., 189:1343-1353 (1999); Song et al., J. Exp. Med.,
191:1095-1103 (2000); Jeremias et al., Eur. J. Immunol., 28:143-152
(1998); Katsikis et al., J. Exp. Med., 186:1365-1372 (1997); Miura
et al., J. Exp. Med., 193:651-660 (2001)].
[0005] Soluble forms of Apo2L/TRAIL have also been reported to
induce apoptosis in a variety of cancer cells in vitro, including
colon, lung, breast, prostate, bladder, kidney, ovarian and brain
tumors, as well as melanoma, leukemia, and multiple myeloma [see,
e.g., Wiley et al., supra; Pitti et al., supra; Rieger et al., FEBS
Letters, 427:124-128 (1998); Ashkenazi et al., J. Clin. Invest.,
104:155-162 (1999); Walczak et al., Nature Med., 5:157-163 (1999);
Keane et al., Cancer Research, 59:734-741 (1999); Mizutani et al.,
Clin. Cancer Res., 5:2605-2612 (1999); Gazitt, Leukemia,
13:1817-1824 (1999); Yu et al., Cancer Res., 60:2384-2389 (2000);
Chinnaiyan et al., Proc. Natl. Acad. Sci., 97:1754-1759 (2000)]. In
vivo studies in murine tumor models further suggest that
Apo2L/TRAIL, alone or in combination with chemotherapy or radiation
therapy, can exert substantial anti-tumor effects [see, e.g.,
Ashkenazi et al., supra; Walzcak et al., supra; Gliniak et al.,
Cancer Res., 59:6153-6158 (1999); Chinnaiyan et al., supra; Roth et
al., Biochem. Biophys. Res. Comm 265:1999 (1999)]. In contrast to
many types of cancer cells, most normal human cell types appear to
be resistant to apoptosis induction by certain recombinant forms of
Apo2L/TRAIL [Ashkenazi et al., supra; Walzcak et al., supra]. Jo et
al. has reported that a polyhistidine-tagged soluble form of
Apo2L/TRAIL induced apoptosis in vitro in normal isolated human,
but not non-human, hepatocytes [Jo et al., Nature Med., 6:564-567
(2000); see 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 a 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)].
[0006] Induction of various cellular responses mediated by such TNF
family cytokines is believed to be initiated by their binding to
specific cell receptors. Previously, two distinct TNF receptors of
approximately 55-kDa (TNFR1) and 75-kDa (TNFR2) were identified
[Hohman et al., J. Biol. Chem., 264:14927-14934 (1989); Brockhaus
et al., Proc. Natl. Acad. Sci., 87:3127-3131 (1990); EP 417,563,
published Mar. 20, 1991; Loetscher et al., Cell, 61:351 (1990);
Schall et al., Cell 61:361 (1990); Smith et al., Science,
248:1019-1023 (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 of
cell surface receptors including extracellular, transmembrane and
intracellular regions. The extracellular portions of both receptors
were found naturally also as soluble TNF-binding proteins [Nophar,
Y. et al., EMBO J., 9:3269 (1990); and Kohno, T. et al., Proc.
Natl. Acad. Sci. U.S.A., 87:8331 (1990); Hale et al., J. Cell.
Biochem. Supplement 15F, 1991, p. 113 (P424)].
[0007] The extracellular portion of type 1 and type 2 TNFRs (TNFR1
and TNFR2) contains a repetitive amino acid sequence pattern of
four cysteine-rich domains (CRDs) designated 1 through 4, starting
from the NH.sub.2-terminus. [Schall et al., supra; Loetscher et
al., supra; Smith et al., supra; Nophar et al., supra; Kohno et
al., supra; Banner et al., Cell, 73:431-435 (1993)]. A similar
repetitive pattern of CRDs exists in several other cell-surface
proteins, including the p75 nerve growth factor receptor (NGFR)
[Johnson et al., Cell, 47:545 (1986); Radeke et al., Nature,
325:593 (1987)], the B cell antigen CD40 [Stamenkovic et al., EMBO
J, 8:1403 (1989)], the T cell antigen OX40 [Mallet et al., EMBO J.,
9:1063 (1990)] 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 [Upton et al., Virology, 160:20-29 (1987); Smith et al.,
Biochem. Biophys. Res. Commun., 176:335 (1991); Upton et al.,
Virology, 184:370 (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 members of the TNF/NGF receptor superfamily.
[0008] The TNF family ligands identified to date, with the
exception of lymphotoxin-beta, are typically type II transmembrane
proteins, whose C-terminus is extracellular. In contrast, most
receptors in the TNF receptor (TNFR) family identified to date are
typically type I transmembrane proteins. In both the TNF ligand and
receptor families, however, homology identified between family
members has been found mainly in the extracellular domain ("ECD").
Several of the TNF family cytokines, including TNF-alpha, Apo-1
ligand and CD40 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 family proteins are also usually cleaved proteolytically
to release soluble receptor ECDs that can function as inhibitors of
the cognate cytokines.
[0009] Pan et al. have disclosed another TNF receptor family member
referred to as "DR4" [Pan et al., Science, 276:111-113 (1997); see
also WO98/32856 published Jul. 30, 1998]. The DR4 was reported to
contain a cytoplasmic death domain capable of engaging the cell
suicide apparatus. Pan et al. disclose that DR4 is believed to be a
receptor for the ligand known as Apo2L/TRAIL.
[0010] In Sheridan et al., Science, 277:818-821 (1997) and Pan et
al., Science, 277:815-818 (1997), another molecule believed to be a
receptor for Apo2L/TRAIL is described [see also, WO98/51793
published Nov. 19, 1998; WO98/41629 published Sep. 24, 1998]. 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
KILLER [Screaton et al., Curr. Biol., 7:693-696 (1997); Walczak et
al., EMBO J., 16:5386-5387 (1997); Wu et al., Nature Genetics,
17:141-143 (1997); WO98/35986 published Aug. 20, 1998; EP870,827
published Oct. 14, 1998; WO98/46643 published Oct. 22, 1998;
WO99/02653 published Jan. 21, 1999; WO99/09165 published Feb. 25,
1999; WO99/11791 published Mar. 11, 1999]. Like DR4, DR5 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 al.,
Molecular Cell, 4:563-571 (1999).
[0011] A further group of recently identified receptors are
referred to as "decoy receptors," which are believed to function as
inhibitors, rather than transducers of signaling. This group
includes DCR1 (also referred to as TRID, LIT or TRAIL-R3) [Pan et
al., Science, 276:111-113 (1997); Sheridan et al., Science,
277:818-821 (1997); McFarlane et al., J. Biol. Chem.,
272:25417-25420 (1997); Schneider et al., FEBS Letters, 416:329-334
(1997); Degli-Esposti et al., J. Exp. Med., 186:1165-1170 (1997);
and Mongkolsapaya et al., J. Immunol., 160:3-6 (1998)] and DCR2
(also called TRUNDD or TRAIL-R4) [Marsters et al., Curr. Biol.,
7:1003-1006 (1997); Pan et al., FEBS Letters, 424:41-45 (1998);
Degli-Esposti et al., Immunity, 7:813-820 (1997)], both cell
surface molecules, as well as OPG [Simonet et al., supra; Emery et
al., infra and DCR3 [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.
[0012] Apo2L/TRAIL is believed to act through the cell surface
"death receptors" DR4 and DR5 to activate caspases, or enzymes that
carry out the cell death program. Upon ligand binding, both DR4 and
DR5 can trigger apoptosis independently by recruiting and
activating the apoptosis initiator, caspase-8, through the
death-domain-containing adaptor molecule referred to as FADD/Mort1
[Kischkel et al., Immunity, 12:611-620 (2000); Sprick et al.,
Immunity, 12:599-609 (2000); Bodmer et al., Nature Cell Biol.,
2:241-243 (2000)]. In contrast to DR4 and DR5, the DcR1 and DcR2
receptors do not signal apoptosis.
[0013] For a review of the TNF family of cytokines and their
receptors, see Ashkenazi and Dixit, Science, 281:1305-1308 (1998);
Ashkenazi and Dixit, Curr. Opin. Cell Biol., 11:255-260 (2000);
Golstein, Curr. Biol., 7:750-753 (1997); Gruss and Dower, supra;
Nagata, Cell, 88:355-365 (1997); Locksley et al., Cell, 104:487-501
(2001).
SUMMARY OF THE INVENTION
[0014] Certain proteins, such as Apo2L/TRAIL and other members of
the TNF family of cytokines, exhibit biological activity when the
protein is in a trimer or trimeric form. Thus, for purposes of
therapeutic or even diagnostic use, formulations of such proteins
are desired wherein the protein is stable and remains biologically
active, particularly stable in a trimeric form.
[0015] Applicants surprisingly found that the unique molecular
structure of APO2L/TRAIL, under certain conditions, allows it to
spontaneously crystallize. This property enabled the development of
an efficient and scaleable recovery/purification process for
APO2L/TRAIL that utilizes crystallization as a purification step.
In addition, the experience obtained with APO2L/TRAIL allowed the
development of a recovery and purification process involving
crystallization that can be used to proteins capable of
crystallization in general.
[0016] In one aspect, the present invention relates to a method of
recovering Apo2L/TRAIL from a mixture comprising
[0017] (a) loading the mixture on a cation exchange column;
[0018] (b) washing the cation exchange column with an equilibration
buffer whereby non-binding components present in the mixture are
removed;
[0019] (c) eluting Apo2L/TRAIL bound to the cation exchange column
with an elution buffer;
[0020] (d) gradually cooling the eluate to a temperature of about 2
to 4.degree. C., whereby Apo2L/TRAIL is spontaneously precipitated
in a crystalline form to yield a mixture of mother liquor and
Apo2L/TRAIL crystals, and
[0021] (e) recovering Apo2L/TRAIL from the mixture obtained in step
(d) in a purity of at least about 99%.
[0022] In a particular embodiment, the mixture loaded on the cation
exchange column is a culture medium or cell lysate of Apo2L/TRAIL
producing cells.
[0023] In another embodiment, the mixture is the cell lysate of
Apo2L/TRAIL producing E. coli host cells.
[0024] In yet another embodiment, the lysate is clarified prior to
loading on the cation exchange column.
[0025] In a further embodiment, the eluate obtained in step (c) is
subjected to the crystallization step of (d) without additional
purification.
[0026] The cation exchange column may, for example, be an
SP-Sepharose column.
[0027] In a still further embodiment, pH of the mixture loaded on
the cation exchange column (e.g. SP-Sepharose) is or is adjusted to
about 7.5. The elution of Apo2L/TRAIL may, for example, be
performed in an elution buffer comprising 100-200 mM NaCl or
100-150 mM Na.sub.2SO.sub.4 in a buffer adjusting the pH to
7.5-7.8.
[0028] In further embodiments, in step (d) the eluate is cooled
from a temperature of about 15 to 30.degree. C. to a temperature of
about 2 to 8.degree. C. in about 1 to 60 hours, or to a temperature
of about 2 to 8.degree. C. in about 1 to 8 hours, or to a
temperature of about 2 to 8.degree. C. in about 1 hour, or to a
temperature of about 4.degree. C. in about 1 hour.
[0029] In yet another embodiment, the pH of the eluate is or is
adjusted to pH 7.0-8.0, such as pH 7.3, prior to
crystallization.
[0030] In another embodiment, the pH of the eluate is or is
adjusted to about 7.5-8.0 after crystallization.
[0031] In an additional embodiment, in step (d) the temperature of
about 2 to 4.degree. C. is maintained until equilibrium solubility
of Apo2L/TRAIL is achieved or nearly achieved.
[0032] In the course of performing the method of the invention, in
step (d), solubility of Apo2L/TRAII may be decreased by the
addition of an anti-solvent, such as, for example, polyethylene
glycol (PEG), MPD, ethanol, isopropanol, and/or dioxane.
[0033] Thus, for example, PEG having a molecular weight of the PEG
between about 400 and about 10,000 daltons is used as an
anti-solvent. In other representative embodiments, the molecular
weight of PEG is 400, 3,350 or 10,000 daltons.
[0034] In a further embodiment, in step (e) Apo2L/TRAIL is
recovered in the form of crystals separated from the mother liquor
by filtration or centrifugation or a combination thereof. The pH of
the mother liquor may be adjusted to about 8.0 prior to filtration
to decrease solubility.
[0035] In a further aspect, the recovery/purification method of the
present invention further comprises the steps of dissolving the
Apo2L/TRAIL crystals obtained in step (d) of the above-described
method, and subjecting the solution obtained to a second
chromatographic purification step
[0036] In one embodiment, the second chromatographic purification
step is hydrophobic interaction chromatography, which may, for
example, be performed on a Phenyl-Sepharose column.
[0037] In another embodiment, the second chromatographic
purification step is cation exchange chromatography performed, for
example, on an CM-Sepharose or SP-Sepharose column.
[0038] In a further embodiment, Apo2L/TRAIL is recovered and
formulated following the second chromatographic purification step
by ultrafiltration-diafiltration.
[0039] In additional embodiments, the purity of the purified
protein is at least about 99.5%, or at least about 99.9%
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 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 "N" at nucleotide position 447 (in SEQ ID NO:2) is used
to indicate the nucleotide base may be a "T" or "G".
[0041] FIG. 2 shows a SDS-PAGE silver stain gel illustrating purity
of the described Apo2L/TRAIL preparations.
[0042] FIG. 3 shows the effects of various salts on crystallization
of Apo2L/TRAIL.
[0043] FIG. 4 shows equilibrium crystal size distributions for
linear temperature ramps between 22.degree. C. and 2.degree. C.
over 1, 4, 8, and 24 hour cooling periods.
[0044] FIG. 5 shows the effect of the addition of PEG on
APO2L/TRAIL solubility: 5 days of agitation at 2-8.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
[0045] "TNF family member" is used in a broad sense to refer to
various polypeptides that share some similarity to tumor necrosis
factor (TNF) with respect to structure or function. Certain
structural and functional characteristics associated with the TNF
family of polypeptides are known in the art and described, for
example, in the above Background of the Invention. Such
polypeptides include but are not limited to those polypeptides
referred to in the art as TNF-alpha, TNF-beta, CD40 ligand, CD30
ligand, CD27 ligand, OX-40 ligand, 4-1BB ligand, Apo-1 ligand (also
referred to as Fas ligand or CD95 ligand), Apo-2L/TRAIL (also
referred to as TRAIL), Apo-3 ligand (also referred to as TWEAK),
APRIL, OPG ligand (also referred to as RANK ligand, ODF, or
TRANCE), and TALL-1 (also referred to as BlyS, BAFF or THANK) (See,
e.g., 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.
Nature 1992, 357:80-82, PCT Publication Nos. WO 97/01633; and WO
97/25428; Marsters et al., Curr. Biol. 1998, 8:525-528;
Chicheportiche et al., Biol. Chem. 1997, 272:32401-32410; Hahne et
al., J. Exp. Med. 1998, 188:1185-1190; PCT Publication Nos.
WO98/28426; WO98/46751; and WO/98/18921; Moore et al., Science
1999, 285:260-263; Shu et al., J. Leukocyte Biol. 1999, 65:680;
Schneider et al., J. Exp. Med. 1999, 189:1747-1756; Mukhopadhyay et
al., J. Biol. Chem. 1999, 274:15978-15981).
[0046] The terms "Apo2L/TRAIL", "Apo2L", "Apo-2 ligand" and "TRAIL"
are used herein to refer to a polypeptide sequence which includes
amino acid residues 114-281, inclusive, 95-281, inclusive, residues
92-281, inclusive, residues 91-281, inclusive, residues 41-281,
inclusive, residues 15-281, inclusive, or residues 1-281,
inclusive, of the amino acid sequence shown in FIG. 1 (SEQ ID
NO:1), as well as biologically active fragments, deletional,
insertional, or substitutional variants of the above sequences. In
one embodiment, the polypeptide sequence comprises residues 114-281
of FIG. 1 (SEQ ID NO:1), and optionally, consists of residues
114-281 of FIG. 1 (SEQ ID NO:1). Optionally, the polypeptide
sequence comprises residues 92-281 or residues 91-281 of FIG. 1
(SEQ ID NO:1). The Apo-2L polypeptides may be encoded by the native
nucleotide sequence shown in FIG. 1 (SEQ ID NO:2). Optionally, the
codon which encodes residue Pro 119 (FIG. 1; SEQ ID NO:2) may be
"CCT" or "CCG". In other embodiments, the fragments or variants are
biologically active and have at least about 80% amino acid sequence
identity, more preferably at least about 90% sequence identity, and
even more preferably, at least 95%, 96%, 97%, 98%, or 99% sequence
identity with any one of the above recited Apo2L/TRAIL sequences.
Optionally, the Apo2L/TRAIL polypeptide is encoded by a nucleotide
sequence which hybridizes under stringent conditions with the
encoding polynucleotide sequence provided in FIG. 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 residue. Particular substitutional variants of the
Apo2L/TRAIL include those in which at least one amino acid is
substituted by an alanine residue. 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 acid residues at positions 203, 218, and/or
269 (using the numbering shown in FIG. 1 (SEQ ID NO:1) 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 WO 01/00832 published Jan. 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. WO97/01633 and WO97/25428. The terms "Apo2L/TRAIL" or "Apo2L"
are used to refer generally to forms of the Apo2L/TRAIL which
include monomer, dimer or trimer forms of the polypeptide. All
numbering of amino acid residues referred to in the Apo2L sequence
use the numbering according to FIG. 1 (SEQ ID NO:1), unless
specifically stated otherwise. For instance, "D203" or "Asp203"
refers to the aspartic acid residue at position 203 in the sequence
provided in FIG. 1 (SEQ ID NO:1).
[0047] The term "Apo2L/TRAIL extracellular domain" or "Apo2L/TRAIL
ECD" refers to a form of Apo2L/TRAIL which is essentially free of
transmembrane and cytoplasmic domains. Ordinarily, the ECD will
have less than 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
polypeptides of the present invention are identified pursuant to
criteria routinely employed in the art for identifying that type of
hydrophobic domain. The exact boundaries of a transmembrane domain
may vary but most likely by no more than about 5 amino acids at
either end of the domain as initially identified. In preferred
embodiments, the ECD will consist of a soluble, extracellular
domain sequence of the polypeptide which is free of the
transmembrane and cytoplasmic or intracellular domains (and is not
membrane bound). Particular extracellular domain sequences of
Apo-2L/TRAIL are described in PCT Publication Nos. WO97/01633 and
WO97/25428.
[0048] The term "Apo2L/TRAIL monomer" or "Apo2L monomer" refers to
a covalent chain of an extracellular domain sequence of Apo2L.
[0049] The term "Apo2L/TRAIL dimer" or "Apo2L dimer" refers to two
Apo-2L monomers 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 (i.e.,
associated with another, third Apo2L monomer).
[0050] The term "Apo2L/TRAIL trimer" or "Apo2L trimer" refers to
three Apo2L monomers that are non-covalently associated.
[0051] The term "Apo2L/TRAIL aggregate" is used to refer to
self-associated higher oligomeric forms of Apo2L/TRAIL, such as
Apo2L/TRAIL trimers, which form, for instance, hexameric and
nanomeric forms of Apo2L/TRAIL.
[0052] Determination of the presence and quantity of Apo2L/TRAIL
monomer, dimer, or trimer (or other aggregates) may be made using
methods and assays known in the art (and using commercially
available materials), such as native size exclusion HPLC ("SEC"),
denaturing size exclusion using sodium dodecyl sulphate
("SDS-SEC"), reverse phase HPLC, capillary electrophoresis, and
including those methods described in further detail in the Examples
below.
[0053] The term "tagged" when used herein refers to a chimeric
polypeptide comprising Apo2L/TRAIL, or a portion thereof, fused to
a "tag polypeptide". The tag polypeptide has enough residues to
provide an epitope against which an antibody can be made or to
provide some other function, such as metal ion chelation, yet is
short enough such that it generally does not interfere with
activity of the TNF family cytokine. The tag polypeptide preferably
also is fairly unique so that a tag-specific antibody does not
substantially cross-react with other epitopes. Suitable tag
polypeptides generally have at least six amino acid residues and
usually between about 8 to about 50 amino acid residues
(preferably, between about 10 to about 20 residues).
[0054] The term "divalent metal ion" refers to a metal ion having
two positive charges. Examples of divalent metal ions include but
are not limited to zinc, cobalt, nickel, cadmium, magnesium, and
manganese. Particular forms of such metals that may be employed
include salt forms (e.g., pharmaceutically acceptable salt forms),
such as chloride, acetate, carbonate, citrate and sulfate forms of
the above mentioned 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.
[0055] "Isolated," when used to describe the various proteins
disclosed herein, means protein that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would typically interfere with diagnostic or
therapeutic uses for the protein, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. In
preferred embodiments, the protein will be purified (1) to a degree
sufficient to obtain at least 15 residues of N-terminal or internal
amino acid sequence by use of a spinning cup sequenator, or (2) to
homogeneity by SDS-PAGE under non-reducing or reducing conditions
using Coomassie blue or, preferably, silver stain, or (3) to
homogeneity 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 environment
will not be present. Ordinarily, however, isolated protein will be
prepared by at least one purification step.
[0056] An "isolated" Apo2L/TRAIL nucleic acid molecule is a nucleic
acid molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the Apo2L/TRAIL nucleic acid.
An isolated Apo2L/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 from
the Apo2L/TRAIL nucleic acid molecule as it exists in natural
cells. However, an isolated Apo2L/TRAIL nucleic acid molecule
includes Apo2L/TRAIL nucleic acid molecules contained in cells that
ordinarily express Apo2L/TRAIL where, for example, the nucleic acid
molecule is in a chromosomal location different from that of
natural cells.
[0057] "Percent (%) amino acid sequence identity" with respect to
the sequences identified herein is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the Apo2L/TRAIL sequence, after aligning
the sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity, and not considering any
conservative substitutions as part of the sequence identity.
Alignment for 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
alignment over the full-length sequences being compared. For
purposes herein, percent amino acid identity values can be obtained
using the sequence comparison computer program, ALIGN-2, which was
authored by Genentech, Inc. and the source code of which has been
filed with user documentation in the US Copyright Office,
Washington, D.C., 20559, registered under the US Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available through Genentech, Inc., South San Francisco, Calif. All
sequence comparison parameters are set by the ALIGN-2 program and
do not vary.
[0058] "Stringency" 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 higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to re-anneal when complementary
strands are present in an environment below their melting
temperature. The higher the degree of desired identity between the
probe and hybridizable sequence, the higher the relative
temperature which can be used. As a result, it follows that higher
relative temperatures would tend to make the reaction conditions
more stringent, while lower temperatures less so. For additional
details and explanation of stringency of hybridization reactions,
see Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, (1995).
[0059] "High stringency conditions", as defined herein, are
identified by those that: (1) employ low ionic strength and high
temperature for washing; 0.015 M sodium chloride/0.0015 M sodium
citrate/0.1% sodium dodecyl sulfate at 50.degree. C.; (2) employ
during hybridization a denaturing agent; 50% (v/v) formamide with
0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50
mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,
75 mM sodium citrate at 42.degree. C.; or (3) employ 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's
solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree. C.
in 0.2.times.SSC (sodium chloride/sodium citrate) and 50% formamide
at 55.degree. C., followed by a high-stringency wash consisting of
0.1.times.SSC containing EDTA at 55.degree. C.
[0060] "Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0061] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0062] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0063] The term "storage-stable" is used to describe a formulation
having a shelf-life acceptable for a product in the distribution
chain of commerce, 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 minimal
changes in charge heterogeneity or biological activity. Degradation
pathways for proteins can involve chemical instability (i.e. any
process which involves modification of the protein by bond
formation or cleavage resulting in a new chemical entity) or
physical instability (i.e. changes in the higher order structure of
the protein). Chemical instability can result from, for example,
deamidation, racemization, hydrolysis, oxidation, beta elimination
or disulfide exchange. Physical instability can result from, for
example, denaturation, aggregation, precipitation or adsorption.
The three most common protein degradation pathways are protein
aggregation, deamidation and oxidation. Cleland et al. Critical
Reviews in Therapeutic Drug Carrier Systems 10(4): 307-377
(1993).
[0064] As used herein, "soluble" refers to polypeptides that, when
in aqueous solutions, are completely dissolved, resulting in a
clear to slightly opalescent solution with no visible particulates,
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 pathlength
cell where turbidity at 20 mg/ml is less than 0.05 absorbance
units.
[0065] An "osmolyte" refers to a tonicity modifier or osmotic
adjuster that 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
sugar alcohols such as mannitol known to the art that are generally
regarded as safe (GRAS).
[0066] "Preservatives" can act to prevent bacteria, viruses, and
fungi from proliferating in the formulation, and anti-oxidants, or
other compounds can function in various ways to preserve the
stability of the formulation. Examples include
octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,
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
alcohols such as phenol and benzyl alcohol, alkyl parabens such as
methyl or propyl paraben, and m-cresol. Optionally, such a compound
is phenol or benzyl alcohol. The preservative or other compound
will optionally be included in a liquid or aqueous form of the
Apo2L/TRAIL formulation, but not usually in a lyophilized form of
the formulation. In the latter case, the preservative or other
compound will typically be present in the water for injection (WFI)
or bacteriostatic water for injection (BWFI) used for
reconstitution.
[0067] A "surfactant" can act to decrease turbidity or denaturation
of a protein in a formulation. Examples of surfactants include
non-ionic surfactant such as a polysorbate, e.g., polysorbates 20,
60, or 80, a poloxamer, e.g., poloxamer 184 or 188, Pluronic
polyols, ethylene/propylene block polymers or any others known to
the art that are GRAS. Optionally, the surfactant is a polysorbate
or poloxamer.
[0068] A "buffer" as used herein is any suitable buffer that is
GRAS and generally 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
7.5, if the polypeptide is Apo2L/TRAIL. Examples include Tris,
Hepes, triethanolamine, histidine, or any others known to the art
to have the desired effect.
[0069] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
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 (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor- and -; mullerian-inhibiting
substance; mouse gonadotropin-associated peptide; inhibin; activin;
vascular endothelial growth factor; integrin; thrombopoietin (TPO);
nerve growth factors; platelet-growth factor; transforming growth
factors (TGFs) such as TGF- and TGF-; insulin-like growth factor-I
and -II; erythropoietin (EPO); osteoinductive factors; interferons
such as interferon-, -, and -gamma; colony stimulating factors
(CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF
(GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12;
and other polypeptide factors including LIF and kit ligand (KL). As
used herein, the term cytokine includes proteins from natural
sources or from recombinant cell culture and biologically active
equivalents of the native sequence cytokines.
[0070] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., I.sup.131, I.sup.125, Y.sup.90 and
Re.sup.186), chemotherapeutic agents, and toxins such as
enzymatically active toxins of bacterial, fungal, plant or animal
origin, or fragments thereof.
[0071] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclosphosphamide
(CYTOXAN.TM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphoramide 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 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CBI-TMI);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, ranimustine; antibiotics such as the enediyne
antibiotics (e.g. calicheamicin, especially calicheamicin gamma1I
and calicheamicin phil1, see, e.g., Agnew, Chem. Intl. Ed. Engl.,
33:183-186 (1994); dynemicin, including dynemicin A;
bisphosphonates, such as clodronate; an esperamicin; as well as
neocarzinostatin chromophore and related chromoprotein enediyne
antiobiotic chromomophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(Adriamycin.TM.) (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidamine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM.; razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,
2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g. paclitaxel (TAXOL.RTM., Bristol-Myers Squibb
Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE.RTM.,
Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine
(Gemzar.TM.); 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;
vinorelbine (Navelbine.TM.); novantrone; teniposide; edatrexate;
daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; capecitabine; and pharmaceutically acceptable
salts, acids or derivatives of any of the above. Also included in
this definition are anti-hormonal agents that act to regulate or
inhibit hormone action on tumors such as anti-estrogens and
selective estrogen receptor modulators (SERMs), including, for
example, tamoxifen (including Nolvadex.TM.), raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and toremifene (Fareston.TM.); aromatase inhibitors
that inhibit the enzyme aromatase, which regulates estrogen
production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide, megestrol acetate (Megace.TM.),
exemestane, formestane, fadrozole, vorozole (Rivisor.TM.),
letrozole (Femara.TM.), and anastrozole (Arimidex.TM.); and
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; and pharmaceutically acceptable salts,
acids or derivatives of any of the above.
[0072] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
cancer 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
overexpressing such genes in S phase. Examples of growth inhibitory
agents include agents that block cell cycle progression (at a place
other than S phase), such as agents that induce G1 arrest and
M-phase arrest. Classical M-phase blockers include the vincas
(vincristine and vinblastine), taxol, and topo II inhibitors such
as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
Those agents that arrest G1 also spill over into S-phase arrest,
for example, DNA alkylating agents such as tamoxifen, prednisone,
dacarbazine, mechlorethamine, cisplatin, methotrexate,
5-fluorouracil, and ara-C. Further information can be found in The
Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1,
entitled "Cell cycle regulation, oncogens, and antineoplastic
drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995),
especially p. 13.
[0073] "Biologically active" or "biological activity" for the
purposes herein means (a) having the ability to induce or stimulate
apoptosis in at least one type of mammalian cancer cell or
virally-infected cell in vivo or ex vivo, either alone as a single
agent or in combination with a chemotherapeutic agent (b) capable
of raising an antibody, i.e., immunogenic; (c) capable of binding
and/or stimulating a receptor for Apo2L/TRAIL (such receptors may
include the DR4 receptor, DR5 receptor, OPG, DcR1 receptor, and
DcR2 receptor); or (d) retaining the activity of a native or
naturally-occurring Apo2L/TRAIL polypeptide. Assays for determining
biological activity of the Apo2L/TRAIL can be conducted using
methods known in the art, such as DNA fragmentation (see, e.g.,
Marsters et al., Curr. Biology, 6: 1669 (1996)), caspase
inactivation, DR4 binding, DR5 binding (see, e.g., WO 98/51793,
published Nov. 19, 1998), DcR1 binding (see, e.g., WO 98/58062,
published Dec. 23, 1998), DcR2 binding (see, e.g., WO 99/10484,
published Mar. 4, 1999) as well as the assays described in PCT
Publication Nos. WO97/01633, WO97/25428, WO 01/00832, and WO
01/22987.
[0074] The terms "apoptosis" and "apoptotic activity" are used in a
broad sense and refer to the orderly or controlled form of cell
death in mammals that is typically accompanied by one or more
characteristic cell changes, including condensation of cytoplasm,
loss of plasma membrane microvilli, segmentation of the nucleus,
degradation of chromosomal DNA or loss of mitochondrial function.
This activity can be determined and measured, for instance, by cell
viability assays (such as Alamar blue assays or MTT assays), FACS
analysis, caspase activation, DNA fragmentation (see, for example,
Nicoletti et al., J. Immunol. Methods, 139:271-279 (1991), and
poly-ADP ribose polymerase, "PARP", cleavage assays known in the
art.
[0075] As used herein, the term "disorder" in general refers to any
condition that would benefit from treatment with the compositions
described herein, including any disease or disorder that can be
treated by effective 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 question. Non-limiting examples of disorders to be treated
herein include benign and malignant cancers; inflammatory,
angiogenic, and immunologic disorders, autoimmune disorders,
arthritis (including rheumatoid arthritis), multiple sclerosis, and
HIV/AIDS.
[0076] The terms "cancer", "cancerous", or "malignant" refer to or
describe the physiological condition in mammals that is typically
characterized by unregulated cell growth. Examples of cancer
include but are not limited to, carcinoma, lymphoma, leukemia,
blastoma, and 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
cancer, ovarian cancer, liver cancer, lymphoblastic leukemia,
lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney
cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic
cancer, glioblastoma multiforme, cervical cancer, stomach cancer,
bladder cancer, hepatoma, breast cancer, colon carcinoma, and head
and neck cancer. Optionally, the cancer cells express DR4 and/or
DR5 receptor(s).
[0077] The terms "treating", "treatment" and "therapy" as used
herein refer to curative therapy, prophylactic therapy, and
preventative therapy. Consecutive treatment or administration
refers to treatment on at least a daily basis without interruption
in treatment by one or more days. Intermittent treatment or
administration, or treatment or administration in an intermittent
fashion, refers to treatment that is not consecutive, but rather
cyclic in nature.
[0078] The term "mammal" as used herein refers to any mammal
classified as a mammal, including humans, cows, horses, dogs and
cats. In a preferred embodiment of the invention, the mammal is a
human.
[0079] The term "polyol" when used herein refers broadly to
polyhydric alcohol compounds. Polyols can be any water-soluble
poly(alkylene oxide) polymer for example, and can have a linear or
branched chain. Preferred polyols include those substituted at one
or more hydroxyl positions with a chemical group, such as an alkyl
group having between one and four carbons. Typically, the polyol is
a poly(alkylene glycol), preferably poly(ethylene glycol) (PEG).
However, those skilled in the art recognize that other polyols,
such as, for example, poly(propylene glycol) and
polyethylene-polypropylene glycol copolymers, can be employed for
conjugation to proteins and other biomolecules. Polyols include
those known in the art and those publicly available, such as from
commercially available sources.
B. Exemplary Methods and Materials for Carrying Out the
Invention
[0080] The present invention provides methods for recovery and
purification of Apo2L/TRAIL. In particular, the invention provides
methods, involving crystallization, to recover and purify
Apo2L/TRAIL from mixtures in which it is accompanied by other
contaminants, such as contaminating proteins and other impurities.
In a specific embodiment, the invention provides methods to recover
and purify Apo2L/TRAIL from recombinant host cultures or cell
lysates, such as cell lysates of Apo2L/TRAIL producing E. coli
recombinant host cells.
[0081] The basis for these purification methods is the unexpected
finding that Apo2L/TRAIL readily and spontaneously crystallizes in
certain buffer systems. This finding allows using crystallization
as an efficient purification step in the purification scheme of
Apo2L/TRAIL. In particular, experimental work underlying the
present invention has shown that crystallization can be implemented
as a step in the purification process of APO2L/TRAIL and other
proteins showing a similar tendency of spontaneous crystallization.
The incorporation of a crystallization step in the purification
scheme allows the reduction of purification process steps while
maintaining comparable yields to traditional purification schemes
using multiple chromatographic purification steps, without
crystallization. Accordingly, implementing crystallization into the
purification process may result in marked time and cost savings,
without compromising efficiency, product yields or product
quality.
[0082] B.1 Production of Apo2L/TRAIL
[0083] The description below relates to methods of producing
Apo2L/TRAIL by culturing host cells transformed or transfected with
a vector containing Apo2L/TRAIL encoding nucleic acid and
recovering the polypeptide from the cell culture.
[0084] The DNA encoding Apo2L/TRAIL may be obtained from any cDNA
library prepared from tissue believed to possess the Apo2L/TRAIL
mRNA and to express it at a detectable level. Accordingly, human
Apo2L/TRAIL DNA can be conveniently obtained from a cDNA library
prepared from human tissues, such as the bacteriophage library of
human placental cDNA as described in PCT Publication WO97/25428.
The Apo2L/TRAIL-encoding gene may also be obtained from a genomic
library or by oligonucleotide synthesis.
[0085] Libraries can be screened with probes (such as antibodies to
the Apo2L/TRAIL or oligonucleotides of at least about 20-80 bases)
designed to identify the gene of interest or the protein encoded by
it. Screening the cDNA or genomic library with the selected probe
may be conducted using standard procedures (Sambrook et al.,
Molecular Cloning: A Laboratory Manual; New York: Cold Spring
Harbor Laboratory Press, 1989). An alternative means to isolate the
gene encoding Apo2L/TRAIL is to use PCR methodology (Sambrook et
al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, 1995).
[0086] Amino acid sequence fragments or variants of Apo2L/TRAIL can
be prepared by introducing appropriate nucleotide changes into the
Apo2L/TRAIL DNA, 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 the extracellular region, or of the amino acid sequence
shown for the full-length Apo2L/TRAIL in FIG. 1 (SEQ ID NO:1). Any
combination 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 apoptotic
activity as defined herein. In a preferred embodiment, the
fragments or variants have at least about 80% amino acid sequence
identity, more preferably, at least about 90% sequence identity,
and even more preferably, at least 95%, 96%, 97%, 98% or 99%
sequence identity with, for example, the sequences identified
herein for the intracellular, transmembrane, or extracellular
domains of Apo2L/TRAIL, or the full-length sequence for
Apo-2L/TRAIL. The amino acid changes also may alter
post-translational processes of the Apo-2L/TRAIL, such as changing
the number or position of glycosylation sites or altering the
membrane anchoring characteristics.
[0087] Variations in the Apo2L/TRAIL sequence as described above
can be made using any of the techniques and guidelines for
conservative and non-conservative mutations set forth in U.S. Pat.
No. 5,364,934. These include oligonucleotide-mediated
(site-directed) mutagenesis, alanine scanning, and PCR
mutagenesis.
[0088] Scanning amino acid analysis can be employed to identify one
or more amino acids along a contiguous sequence. Among the
preferred scanning amino acids are relatively small, neutral amino
acids. Such amino acids include alanine, glycine, serine and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant. (Cunningham et al., Science 1989, 244:1081).
Alanine 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).
[0089] Particular Apo2L/TRAIL variants of the present invention
include those Apo2L/TRAIL polypeptides which include one or more of
the recited alanine substitutions provided in TABLE I of published
PCT application WO 01/00832. Such Apo2L/TRAIL variants will
typically comprise a non-naturally occurring amino acid sequence
which differs from a native Apo2L/TRAIL amino acid sequence (such
as provided in FIG. 1; SEQ ID NO:1, for a full length or mature
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 amino acid substitution(s) such as
those indicated in Table I of WO 01/00832. Apo2L/TRAIL variants of
the invention include soluble Apo2L/TRAIL variants comprising
residues 91-281, 92-281, 95-281 or 114-281 of FIG. 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 FIG. 1 (SEQ ID NO:1)
and having one or more amino acid substitutions which enhance
biological activity, such as receptor binding. A particularly
preferred variant comprises residues 114-281 of FIG. 1 (SEQ ID
NO:1). In a specific embodiment, Apo-2L/TRAIL consists of residues
114-281 of FIG. 1 (SEQ ID NO:1).
[0090] As described in WO 01/00832 published Jan. 4, 2001, the
x-ray crystal structure of the extracellular domain of Apo2L/TRAIL
identified, and alanine-scanning mutagenesis was performed to
provide the mapping of its receptor contact regions. The structure
obtained for Apo2L/TRAIL revealed a homotrimeric protein which
contains a novel divalent metal ion (zinc) binding site that
coordinates the interaction of the Apo2L/TRAIL trimer molecule's
three subunits. Like other members of the TNF family, Apo2L/TRAIL
appears to comprise a compact trimer formed of three jelly roll
monomers which bury approximately 5100 Angstrom.sup.2 (1700
Angstrom.sup.2 per monomer) to form the globular trimer. The
position of the core beta-strands was well conserved compared to
the other structurally characterized members of the TNF family,
TNF-alpha, TNF-beta, and CD40L when compared to the core strands of
TNF-alpha or TNF-beta.
[0091] Variations in the Apo2L/TRAIL sequence also included within
the scope of the invention relate to amino-terminal derivatives or
modified forms. Such Apo2L/TRAIL sequences may include any of the
Apo2L/TRAIL polypeptides described herein having a methionine or
modified methionine (such as formyl methionyl or other blocked
methionyl species) at the N-terminus of the polypeptide
sequence.
[0092] The nucleic acid (e.g., cDNA or genomic DNA) encoding native
or variant Apo2L/TRAIL may be inserted into a replicable vector for
further cloning (amplification of the DNA) or for expression.
Various vectors are publicly available. The vector components
generally include, but are not limited to, one or more of the
following: a signal sequence, an origin of replication, one or more
marker genes, an enhancer element, a promoter, and a transcription
termination sequence, each of which is described below. Optional
signal sequences, origins of replication, marker genes, enhancer
elements and transcription terminator sequences that may be
employed are known in the art and described in further detail in
PCT Publication WO97/25428.
[0093] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the Apo2L/TRAIL nucleic acid sequence. Promoters are untranslated
sequences located upstream (5') to the start codon of a structural
gene (generally within about 100 to 1000 bp) that control the
transcription and translation of a particular nucleic acid
sequence, such 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
promoters that initiate increased levels of transcription from DNA
under their control in response to some change in culture
conditions, e.g., the presence or absence of a nutrient or a change
in temperature. At this time a large number of promoters recognized
by a variety of potential host cells are well known. These
promoters are operably linked to Apo2L/TRAIL encoding DNA by
removing the promoter from the source DNA by restriction enzyme
digestion and inserting the isolated promoter sequence into the
vector. Both the native Apo2L/TRAIL promoter sequence and many
heterologous promoters may be used to direct amplification and/or
expression of the Apo2L/TRAIL DNA.
[0094] Promoters suitable for use with prokaryotic and eukaryotic
hosts are known in 15, the art, and are described in further detail
in PCT Publication No. WO97/25428.
[0095] Preferred methods for the production of soluble Apo2L/TRAIL
in E. coli employ an inducible promoter for the regulation of
product expression. The use of a controllable, inducible promoter
allows for culture growth to the desirable cell density before
induction of product expression and accumulation of significant
amounts of product which may not be well tolerated by the host.
[0096] Various inducible promoter systems (including T7 polymerase,
trp and alkaline phosphatase (AP)) have been evaluated by
Applicants for the expression of Apo2L/TRAIL (amino acids 114-281).
The use of each of the T7 polymerase, trp and alkaline phosphatase
promoters resulted in significant amounts of soluble, biologically
active Apo2L/TRAIL trimer being recovered from the harvested cell
paste. Another optional promoter is a glycerol-phosphate promoter
system.
[0097] Construction of suitable vectors containing one or more of
the above-listed components employs standard ligation techniques.
Isolated plasmids or DNA fragments are cleaved, tailored, and
relegated in the form desired to generate the plasmids
required.
[0098] For analysis to confirm correct sequences in plasmids
constructed, the ligation mixtures can be used to transform E. coli
K12 strain 294 (ATCC 31,446) and successful transformants selected
by ampicillin or tetracycline resistance where appropriate.
Plasmids from the transformants are prepared, analyzed by
restriction endonuclease digestion, and/or sequenced using standard
techniques known in the art. (See, e.g., Messing et al., Nucleic
Acids Res. 1981, 9:309; Maxam et al., Methods in Enzymology 1980,
65:499).
[0099] Expression vectors that provide for the transient expression
in mammalian cells of DNA encoding Apo2L/TRAIL may be employed. In
general, transient expression involves the use of an expression
vector that is able to replicate efficiently in a host cell, such
that the host cell accumulates many copies of the expression vector
and, in turn, synthesizes high levels of a desired polypeptide
encoded 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
identification of polypeptides encoded by cloned DNAs, as well as
for the rapid screening of such polypeptides for desired biological
or physiological properties. Thus, transient expression systems are
particularly useful in the invention for purposes of identifying
analogs and variants of Apo2L/TRAIL that are biologically active
Apo2L/TRAIL.
[0100] Other methods, vectors, and host cells suitable for
adaptation to the synthesis of Apo2L/TRAIL in recombinant
vertebrate cell 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.
[0101] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes for this purpose include but are not
limited to eubacteria, such as Gram-negative or Gram-positive
organisms, for example, Enterobacteriaceae such as Escherichia,
e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,
Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia
marcescans, and Shigella, as well as Bacilli such as B. subtilis
and B. lichenifonnis (e.g., B. lichenifonnis 41P disclosed in DD
266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa,
and Streptomyces. Preferably, the host cell should secrete minimal
amounts of proteolytic enzymes.
[0102] E. coli is the preferred host cell for use in the present
invention. E. coli is particularly well suited for the expression
of Apo2L/TRAIL (comprising amino acids 114-281 of FIG. 1), a
polypeptide of under 20 kd 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 relatively
high levels of heterologous proteins.
[0103] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for Apo2L/TRAIL-encoding vectors. Suitable host cells for the
expression of glycosylated Apo2L/TRAIL are derived from
multicellular organisms. Examples of all such host cells, including
CHO cells, are described further in PCT Publication No.
WO97/25428.
[0104] Host cells are transfected and preferably transformed with
the above-described expression or cloning vectors for Apo2L/TRAIL
production and cultured in nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0105] Transfection refers to the taking up of an expression vector
by a host cell whether or not any coding sequences are in fact
expressed. Numerous methods of transfection are known to the
ordinarily skilled artisan, for example, CaPO.sub.4 and
electroporation. Successful transfection is generally recognized
when any indication of the operation of this vector occurs within
the host cell.
[0106] Transformation means introducing DNA into an organism so
that the DNA is replicable, either as an extrachromosomal element
or by chromosomal integrant. Depending on the host cell used,
transformation is done using standard techniques appropriate to
such cells. The calcium treatment employing calcium chloride, as
described in Sambrook et al., supra, or electroporation is
generally used for prokaryotes or other cells that contain
substantial cell-wall barriers. Infection with Agrobacterium
tumefaciens is used for transformation of certain plant cells, as
described (Shaw et al., Gene 1983, 23:315 and PCT Publication No.
WO 89/05859). In addition, plants may be transfected using
ultrasound treatment, PCT Publication No. WO 91/00358 published 10
Jan. 1991.
[0107] For mammalian cells without such cell walls, the calcium
phosphate precipitation method (Graham and van der Eb, Virology
1978, 52:456-457) may be employed. General aspects of mammalian
cell host system transformations have been described in U.S. Pat.
No. 4,399,216. Transformations into yeast are typically carried out
according to the method of Van Solingen et al., J. Bact. 1977,
130:946 and Hsiao et al. Proc. Natl. Acad. Sci. USA 1979, 76:3829.
However, other methods for introducing DNA into cells, such as by
nuclear microinjection, electroporation, bacterial protoplast
fusion with intact cells, or polycations, e.g., polybrene,
polyornithine, may also be used. For various techniques for
transforming mammalian cells, see Keown et al. Methods in
Enzymology 1990, 185:527-537 and Mansour et al. Nature 1988,
336:348-352.
[0108] Prokaryotic cells used to produce Apo2L/TRAIL may be
cultured in suitable culture media as described generally in
Sambrook et al., supra. Particular forms of culture media that may
be employed for culturing E. coli are described further in PCT
application WO 01/00832. In a particularly preferred process,
APO2L/TRAIL (comprising amino acids 114-281 of FIG. 1 produced in
E. coli is fermented using a zinc supply and glycerophosphate. The
fermentation titers preferably range from about 4 to about 6
g/l.
[0109] Mammalian host cells used to produce Apo2L/TRAIL may be
cultured in a variety of culture media.
[0110] Examples of commercially available culture media include
Ham's F10 (Sigma), Minimal Essential Medium ("MEM", Sigma),
RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ("DMEM",
Sigma). Any such media may be supplemented as necessary with
hormones and/or other growth factors (such as insulin, transferrin,
or epidermal growth factor), salts (such as sodium chloride,
calcium, magnesium, and phosphate), buffers (such as HEPES),
nucleosides (such as adenosine and thymidine), antibiotics (such as
Gentamycin.TM. drug), trace elements (defined as inorganic
compounds usually present at final concentrations in the micromolar
range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
[0111] In general, principles, protocols, and practical techniques
for maximizing the productivity of mammalian cell cultures can be
found in Mammalian Cell Biotechnology: A Practical Approach, M.
Butler, ed. (IRL Press, 1991).
[0112] Expression of the Apo2L/TRAIL may be measured in a sample
directly, for example, by conventional Southern blotting, Northern
blotting to quantitate the transcription of mRNA (Thomas, Proc.
Natl. Acad. Sci. USA 1980, 77:5201-5205), dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Various labels may
be employed, most commonly radioisotopes, and particularly
.sup.32P. However, other techniques may also be employed, such as
using biotin-modified nucleotides for introduction into a
polynucleotide. The biotin then serves as the site for binding to
avidin or antibodies, which may be labeled with a wide variety of
labels, such as radionucleotides, fluorescers or enzymes.
Alternatively, 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 be labeled and the assay may be carried out where the
duplex is bound to a surface, so that upon the formation of duplex
on the surface, the presence of antibody bound to the duplex can be
detected.
[0113] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. With
immunohistochemical staining techniques, a cell sample is prepared,
typically by dehydration and fixation, followed by reaction with
labeled antibodies specific for the gene product coupled, where the
labels are usually visually detectable, such as enzymatic labels,
fluorescent labels, luminescent labels, and the like.
[0114] Antibodies useful for immunohistochemical staining and/or
assay of sample fluids may be either monoclonal or polyclonal, and
may be prepared in any mammal. Conveniently, the antibodies may be
prepared against a native Apo2L/TRAIL polypeptide or against a
synthetic peptide based on the DNA sequences provided herein or
against exogenous sequence fused to Apo2L/TRAIL DNA and encoding a
specific antibody epitope.
[0115] The Apo-2L polypeptide may be covalently attached
(hereinafter "conjugated") to one or more chemical groups. Chemical
groups suitable for use in an Apo-2L conjugate are preferably not
significantly toxic or immunogenic. A variety of exemplary chemical
groups that can be conjugated to polypeptides are known in the art
and include for example carbohydrates, such as those carbohydrates
that occur naturally on glycoproteins, polyglutamate, and
non-proteinaceous polymers, such as polyols (see, e.g., U.S. Pat.
No. 6,245,901).
[0116] A polyol, for example, can be conjugated to polypeptides
such as an Apo-2L at one or more amino acid residues, including
lysine residues, as is disclosed in WO 93/00109, supra. The polyol
employed can be any water-soluble poly(alkylene oxide) polymer and
can have a linear or branched chain. Suitable polyols include those
substituted at one or more hydroxyl positions with a chemical
group, such as an alkyl group having between one and four carbons.
Typically, the polyol is a poly(alkylene glycol), such as
poly(ethylene glycol) (PEG), and thus, for ease of description, the
remainder of the discussion relates to an exemplary embodiment
wherein the polyol employed is PEG and the process of conjugating
the polyol to a polypeptide is termed "pegylation." However, those
skilled in the art recognize that other polyols, such as, for
example, poly(propylene glycol) and polyethylene-polypropylene
glycol copolymers, can be employed using the techniques for
conjugation described herein for PEG.
[0117] The average molecular weight of the PEG employed in the
pegylation of the Apo-2L can vary, and typically may range from
about 500 to about 30,000 daltons (D). Preferably, the average
molecular weight of the PEG is from about 1,000 to about 25,000 D,
and more preferably from about 1,000 to about 5,000 D. In one
embodiment, pegylation is carried out with PEG having an average
molecular weight of about 1,000 D. Optionally, the PEG homopolymer
is unsubstituted, but it may also be substituted at one end with an
alkyl group. Preferably, the alkyl group is a C1-C4 alkyl group,
and most preferably a methyl group. PEG preparations are
commercially available, and typically, those PEG preparations
suitable for use in the present invention are nonhomogeneous
preparations sold according to average molecular weight.
Optionally, an Apo-2L trimer will be pegylated in a manner such
that a PEG molecule is linked or conjugated to one, two or each of
the three monomers that make up the trimeric Apo-2L. In such an
embodiment, it is preferred that the PEG employed have an average
molecular weight of about 1,000 to about 5,000 D. It is also
contemplated that the Apo-2L trimers may be "partially" pegylated,
i.e., wherein only one or two of the three monomers that make up
the trimer are linked or conjugated to PEG.
[0118] A variety of methods for pegylating proteins are known in
the art. Specific methods of producing proteins conjugated to PEG
include the methods described in U.S. Pat. No. 4,179,337, U.S. Pat.
No. 4,935,465 and U.S. Pat. No. 5,849,535. Typically the protein is
covalently bonded via one or more of the amino acid residues of the
protein to a terminal reactive group on the polymer, depending
mainly on the reaction conditions, the molecular weight of the
polymer, etc. The polymer with the reactive group(s) is designated
herein as activated polymer. The reactive group selectively reacts
with free amino or other reactive groups on the protein. The PEG
polymer can be coupled to the amino or other reactive group on the
protein in either a random or a site specific manner.
[0119] B.2 Crystallization of Apo2L/TRAIL
[0120] Crystallization is widely used for purification of small
molecules. However, generally, crystallization techniques have not
been widely applied for proteins as various parameters may affect
the protein crystallization, including, for example, solubility,
nucleation and growth rate, and crystal size distribution (each
being a function of further parameters, such as solubility,
temperature, pH, buffer, impurities, and the like). Since proteins
are generally more difficult to crystallize than small molecules,
the recovery and purification of therapeutic proteins to date has
rarely involved a crystallization step(s).
[0121] Applicants surprisingly found that the solid state of
Apo2L/TRAIL protein at 5.degree. C. is crystalline at moderate to
low ionic strength conditions, unlike many other proteins known in
the art that are soluble or form amorphous precipitates under
similar conditions. Further, it was found that the solid state of
the Apo2L/TRAIL crystals reversibly solubilizes when brought to
ambient temperature (i.e., room temperature) without a loss in
protein biological activity or adverse effect on the biochemical
properties of the protein. This observation was quite different
from the denaturation or irreversible precipitation observed for
other proteins known in the art.
[0122] Optionally, the Apo2L/TRAIL crystals are prepared by cooling
a super-saturated solution of Apo-2L/TRAIL protein from about 20 to
about 30.degree. C. to below about 15.degree. C., preferably about
2 to 8.degree. C., more preferably, below about 2-8.degree. C.,
even more preferably below about 4.degree. C., most preferably to
about 2 to 4.degree. C. Optionally, the Apo2L/TRAIL concentration
can be above 3 g/L in order to initiate spontaneous
crystallization. Antisolvents can be used to initiate spontaneous
crystallization at lower protein concentrations. Crystallization
can be carried out in batch or semi-batch mode at a large range of
scale, from a few milliliters to hundreds of liters of solution.
The crystallization rate can be controlled by programmed cooling
and agitation. The equipment may include, but is not limited to,
agitated or static tanks with surface and/or internal temperature
control. Internal baffles and draft tubes may also be used to
enhance mixing in agitated tanks. Crystal nucleation can also be
controlled by seeding [Moore, AIChE Practical Engineering
Perspectives, Distillation and Other Industrial Separations, pp.
239-245]. The degree of super-saturation, salt composition, cooling
rate, agitation rate, and seeding, among other parameters, can
affect crystal formation rate, crystal size distribution, and
crystal yield.
[0123] Optionally, to prepare the crystals, the solution of
Apo-2L/TRAIL protein contains sodium sulphate or sodium chloride.
Optionally, the salt concentration is about 100 mM to about 200 mM
and optionally the pH is about 6 to about 9 (preferably, pH of
about 6.5 to about 8.5).
[0124] B.3 Use of Crystallization in the Recovery and Purification
of APO2L/TRAIL
[0125] In the methods of the present invention, crystallization is
a step in the recovery and purification of Apo2L/TRAIL, and
optionally is a step in a one-column or a two-column scheme for the
recovery and purification of Apo2L/TRAIL.
[0126] In a particular embodiment, Apo2L/TRAIL is purified from a
recombinant host culture or cell lysate, or clarified cell lysate
using a purification process including a crystallization step. If
Apo2L/TRAIL is produced in E. coli, typically the whole cell broth
is harvested and homogenized to break open the E. coli cells and
release soluble Apo2L/TRAIL within the cytoplasm. After removing
the solid debris, e.g. by centrifugation, the mixture is loaded
onto a cation exchange chromatographic resin, such as, for example,
SP-Sepharose Fast Flow or CM-Sepharose Fast Flow (Amersham
Pharmacia, Sweden). Typical protocols for purifying Apo2L/TRAIL
from cell broth obtained by fermentation of E. coli are provided in
Examples 2 and 3.
[0127] In a typical protocol, the pH of the whole cell broth
obtained by fermentation of the E. Coli cells is adjusted to about
7.5, e.g. by addition of sodium HEPES or any other appropriate
buffer. Preferably, a reducing agent, such as 1,4-dithio-threitol
(DTT) or .alpha.-mercaptoethanol is added, to prevent the formation
of disulfide bonds between the non-covalently bound monomers of
Apo2L/TRAIL. The cells are burst open by one or more passes on a
commercially available high pressure homogenizer, the cell debris
is removed, and the cell lysate is clarified. Specific treatment
parameters, such as selection and concentration of reagents, depend
on the composition of the starting whole cell broth, such as, for
example, cell density.
[0128] The Apo2L/TRAIL-containing mixture, such as a clarified cell
lysate, is then loaded on a first chromatographic column, using a
cation exchange resin. Cation exchange chromatography retains
biomolecules by the interaction of charged groups that are acidic
in nature on the surface of the resin with histidine, lysine and
arginine. Cation exchange resins are commercially available from
the product lines of various manufacturers, such as, for example,
Sigma Aldrich. Cation exchangers include resins carrying, for
example, carboxymethyl functional groups (weak cation exchanger,
such as, CM cellulose/Sephadex) or sulfonic acid functional groups
(strong cation exchanger, such as, SP Sephadex). In the first
chromatographic purification step of the methods of the present
invention, strong cation exchange columns, e.g. SP-Sepharose.RTM.,
Spectra/Gel.RTM. strong cation exchangers, etc. TSKgel strong
cation exchangers, etc. are preferred. In the case of an
SP-Sepharose.RTM. column, the cross-linked agarose matrix with
negatively charged functional groups binds to Apo2L/TRAIL while
allowing the majority of the impurities and Apo2L/TRAIL variants to
pass through the column. Elution can be performed using salt
gradient elution or step elution, step elution being preferred
since it provides better conditions for the subsequent
crystallization step, without compromising yields. The elution
buffer usually contains sodium chloride or sodium sulfate, and salt
concentration is selected to meet the demands of the cation
exchange column and the subsequent crystallization step. The
SP-Sepharose.RTM. column needs a fairly high salt concentration to
remove the bound Apo2L/TRAIL protein, while for the subsequent
crystallization step relatively low salt concentrations are
preferred, in order to lower protein solubility. Typically, about
100-150 mM Na.sub.2SO.sub.4 or 100-200 mM NaCl concentrations are
used. A typical elution buffer consists of 200 mM NaCl, 50 mM
HEPES, 0.05% Triton X-100, 1 mM DTT, pH 7.5.
[0129] The concentration of Apo2L/TRAIL in the cation exchange,
e.g. SP-Sepharose.RTM. elution pool, influences the theoretical
yield for the following crystallization step. Concentration must be
high enough to maximize the solubility differences at lower
temperatures, but not too high to trigger spontaneous
crystallization at or around room temperature.
[0130] In a representative protocol, two wash steps are employed
between loading and eluting the Apo2L/TRAIL protein. The first wash
uses equilibration buffer, and the second is a salt wash, using a
buffer identical to the subsequent elution buffer, except using a
lower salt concentration (e.g. 100 mM NaCl instead of 200 mM
NaCl).
[0131] The SP elution step, including the two wash steps, typically
produces Apo2L/TRAIL concentrations around 3-6 g/L, such as about 5
g/L with yields around 80-90%. The salt wash step results in loss
of the active protein, therefore, removing this step, the yield can
be increased over 95%. However, elimination of this step also
decreases the column's ability to remove endotoxins and
extracellular proteins, thereby lowering purity.
[0132] The elution pool leaving the cation exchange column is
subjected to crystallization directly without any further
additional purification step, but optionally including sterile
filtration. Crystallization is typically performed by gradually
decreasing the temperature from about 15-30.degree. C. to about 2
to 8.degree. C. in a time frame that can extend as long as 60
hours, but typically is shorter, such as, for example, about 1 to 8
hours.
[0133] In a typical crystallization process, the elution pool
leaving the cation exchange column is transferred into a
temperature-controlled tank with adequate agitation. It is
important to ensure that the vessel and protein solution are free
from any particulates prior to crystallization, in order to avoid
nucleation based on such solid particulates, which would influence
the crystallization kinetics. For small scale applications, for
example, a 1 or 2 liter Applikon.RTM. reaction vessel can be used.
In the 1 L vessel, temperature is controlled via cooling coils
immersed into the vessel. The 2 L reaction vessel contains a heat
exchange jacket. A linear temperature ramp can be produced in both
vessels by using a programmable heat exchange bath (e.g.
PolyScience Programmable Temperature Circulator Model 1157). The
vessel is usually equipped by an agitator to thoroughly mix the
solution, and suspend the crystals once formed. The agitation rate
is typically around 250 rpm for 0.4 L scale and is scaled for
larger pools by keeping a constant power to volume ratio,
proportional to N.sup.3N/V (constant diameter agitator).
[0134] It has been found that the solubility of Apo2L/TRAIL
increases with increasing salt concentration, and Apo2L/TRAIL is
approximately equally soluble in sodium sulfate and sodium
chloride. Crystals formed in sodium chloride have a more
exaggerated thickness compared to crystals formed in sodium
sulfate, which are more flat in appearance. As a result, crystals
produced in sodium chloride are easier to separate by filtration,
which makes sodium chloride the preferred salt. As background
buffers, HEPES and TRIS typically provide comparable results.
[0135] Apo2L/TRAIL solubility decreases with increasing pH within a
range of about pH 7.0 and 8.0. Higher pH tends to increase yields
but can make the crystals more amorphous in appearance. In
addition, the crystals are larger at higher pH, but also more
fragile. In view of these considerations, a preferred pH, producing
desired crystal morphology is 7.3.+-.0.1.
[0136] The temperature ramp used during crystallization (typically
from about ambient temperature to about 2.degree. C.) had no
significant effect on average crystal size or size distribution
between about 1 and 24 hours. The temperature ramp may be linear,
but non-linear cooling rate may also be used to further improve the
crystal size profile by maintaining a constant supersaturation
level as the crystallization progresses. Since Apo2L/TRAIL does not
spontaneously crystallize in the buffers systems of the present
invention until the temperature is below about 8.degree. C.,
preferably below about 5.degree. C., it is possible to quickly drop
the temperature to around 10.degree. C. and then slowly cool the
pool to allow for crystallization.
[0137] Crystal size is influenced by the rate of agitation. By
testing three different agitation rates (100 rpm, 175 rpm and 250
rpm), crystallization was found to be fastest with the greatest
agitations rate, but crystal size distribution and the appearance
of crystals were very similar for the 175 rpm and 250 rpm agitation
rates. At lower rates, crystals are not completely suspended, and
crystal aggregation may take place. At higher agitation rates care
must be taken not to damage the soluble protein by exposure to
shear effects at the air/liquid interface.
[0138] Crystallization efficiency may be improved by lowering the
solubility of Apo2L/TRAIL. Thus, the overall yield of the
crystallization step is controlled in part by the solubility of
Apo2L/TRAIL in the chilled pool collected from the first cation
exchange chromatography column. The two factors that affect yield
are in initial concentration of Apo2L/TRAIL in the elution pool
collected from the first cation exchange chromatography column
(e.g. SP column), and the concentration of soluble Apo2L/TRAIL in
the crystal slurry (i.e. the amount of Apo2L/TRAIL that does not
crystallize). Apo2L/TRAIL which is still in solution following
crystallization will be lost during filtration. The addition of
anti-solvents can change the solution chemistry to lower the
equilibrium solubility:
Percent theoretical
yield=[Apo2L].sub.22C-[Apo2L].sub.4C/[aPO2I].sub.22C.times.100%,
where the subscripted numbers indicate temperature values.
[0139] By reducing the Apo2L/TRAIL in solution, less protein is
removed when the mother liquor is filtered off. Anti-solvents, also
known as precipitating agents, are well known in the art and can
work in a variety of ways. Some anti-solvents dehydrate the
solution by absorbing water. This essentially reduces the activity
of water available to dissolve the protein (see, e.g. McPherson,
A., 1998, Crystallization of Biological Macromolecules. Cold Spring
Harbor Laboratory Press. Plainview N.Y.).
[0140] A widely used anti-solvent is polyethylene glycol (PEG), a
polymer available in a wide range of molecular weight. As shown in
the Examples, in the methods of the present invention PEG of higher
molecular weight (3350 and 10000) provided better results. Other
polymers that can be used as anti-solvents include, for example,
Eudragit RS, ethylcellulose, isopropyl alcohol, ethanol, dioxane,
and 2-methyl-2,4-pentanediol (MPD).
[0141] When crystallization is complete, the Apo2L/TRAIL crystals
are removed, for example by filtration. The crystals may be kept
suspended throughout filtration, using a built-in agitator, or can
be deposited in a packed bed. It is important to avoid the
formation of a compressed crystal cake, which could make it
difficult to achieve the desired flow rate. Therefore, differential
pressures across the packed bed must be minimized. Flow rates may
vary, and typically are between about 200 cm/hr and about 100
cm/hr. The flow rate may depend on the equipment used, and the
applied differential pressure during filtration. Filtration may be
performed batch-wise or continuously. Further purification can be
achieved, for example, by washing the deposited crystal bed with a
solution that does not substantially dissolve Apo2L/TRAIL crystals,
such as a chilled solution (2-8 C) of low molarity TRIS at about pH
7.5.
[0142] Following crystallization and separation, the Apo2L/TRAIL
crystals can be dissolved and stored or converted into a
formulation suitable for the intended use.
[0143] Alternatively, a further chromatography purification step
can be added to further improve purity by removing the anti-solvent
(PEG) residues and buffer components, and reduce the levels of
residual extracellular proteins, endotoxin, dimers, and aggregates.
The second chromatographic column, used following crystallization,
can be a cation exchange column, or a hydrophobic interaction
column. Since the crystallization pool is very pure, it is
typically not necessary to use a bind-and-elute mode of separation
(such as typically used with SP-Sepharose or CM-Sepharose), a
flow-through column, such as Phenyl Sepharose HIC resin, will
typically show a good performance. The use of both types of resins,
cation exchange in a bind-and-elute mode and HIC in flow-through
mode, have been tested and the results are discussed in the
Examples. It was found that while a bind and step elution
chromatography step provides a very powerful tool for initial
purification, in the second chromatography purification step,
hydrophobic interaction chromatography on Phenyl-Sepharose is
sufficient to provide the desired purity and yields. Since this is
a flow-through step, it provides excellent yields and reduces the
number of solutions required to complete the operation compared to
bind and elute chromatography.
[0144] B.4 Use of Apo2L/TRAIL
[0145] The methods of the present invention provide an effective,
efficient, and cost saving alternative to, for instance,
purification protocols requiring multiple column purifications. As
discussed above, in one embodiment, the purification scheme of the
present invention involves the use of a single cation exchange
column, followed by crystallization. The Apo2L/TRAIL crystals
obtained by the method of the present invention can be dried for
storage. Drying the crystalline material can also substantially
reduce storage volume, and provide an effective way of bulk storage
which avoids freezing the purified material at low concentration in
formulation solution. The crystal slurry at very high protein
concentration can be frozen in smaller volume containers.
[0146] In another embodiment, the Apo2L/TRAIL crystals are
collected, and washed with buffer (or water) (preferably a cold
buffer at a temperature of about 2 to 8.degree. C.). The washed
crystals can be re-suspended or re-dissolved at ambient
temperature. Re-solubilized Apo2L/TRAIL can be further purified by
hydrophobic interaction chromatography or a second step of cation
exchange chromatography as described above, recrystallized, washed
and stored as wet crystalline bulk material. Alternatively, the
hydrophobic interaction or other chromatography step may be omitted
in favor of simply recrystallizing.
[0147] The wet crystalline bulk material can be stored at
-20.degree. C. or dried for storage at ambient temperature (room
temperature) or at 2-8.degree. C. Preferably, the dried crystalline
material is re-solubilized in an arginine succinate-containing
formulation. Optionally, such a formulation can be sterile filtered
and/or filled in individual dosage vials, and lyophilized for later
reconstitution or suspension. Optionally, the dried crystalline
formulation can be filled as a powder in vials and made into a
solution or suspension. It may be desirable to achieve a water
content of about 5% to about 10% in the dried Apo2L/TRAIL
crystals.
[0148] The Apo2L/TRAIL formulations can be employed in a variety of
therapeutic and non-therapeutic applications. Among these
applications are methods of treating disorders, such as cancer,
immune related conditions, or viral conditions. Such therapeutic
and non-therapeutic applications are further described, for
instance, in WO97/25428, WO97/01633, and WO 01/22987.
[0149] In the methods of the invention for treating a disorder
using a formulation disclosed herein, the formulation of
Apo2L/TRAIL can be directly administered to the mammal by any
suitable technique, including infusion or injection. The specific
route of administration will depend, e.g., on the medical history
of the patient, including any perceived or anticipated side effects
using Apo2L/TRAIL and the particular disorder to be corrected.
Examples of parenteral administration include subcutaneous,
intramuscular, intravenous, intraarterial, and intraperitoneal
administration of the composition. The formulations are preferably
administered as repeated intravenous (i.v.), subcutaneous (s.c.),
intramuscular (i.m.) injections or infusions, intracranial
infusions or as aerosol formulations suitable for intranasal or
intrapulmonary delivery (for intrapulmonary delivery see, e.g., EP
257,956).
[0150] It is noted that osmotic pressure of injections may be
important in subcutaneous and intramuscular injection. Injectable
solutions, when hypotonic or hypertonic, may cause pain to a
patient upon infusion. Usually, for the therapeutic, injectable
formulations herein, it is preferred that the relative osmolarity
of the injectable solution be about 300 mosm to about 600 mosm.
[0151] Apo2L/TRAIL can also be administered in the form of
sustained-release preparations. Suitable examples of
sustained-release preparations include semipermeable matrices of
solid hydrophobic polymers containing the protein, which matrices
are in the form of shaped articles, e.g., films, or microcapsules.
Examples of sustained-release matrices include cellulose
derivatives (e.g., carboxymethylcellulose), sucrose-acetate
isobutyrate (SABER.TM.) in non-aqueous media, polyesters, hydrogels
(e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed.
Mater. Res. 1981, 15:167-277; Langer, Chem. Tech. 1982, 12: 98-105
or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919, EP
58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate
(Sidman et al., Biopolymers 1983, 22: 547-556), non-degradable
ethylene-vinyl acetate (Langer et al., supra), degradable lactic
acid-glycolic acid copolymers such as the Lupron Depot (injectable
microspheres composed of lactic acid-glycolic acid copolymer and
leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP
133,988). One optional method of delivery for systemic-acting drugs
involves administration by continuous infusion (using, e.g.,
slow-release devices or minipumps such as osmotic pumps or skin
patches), or by injection (using, e.g., intravenous or subcutaneous
means, including single-bolus administration).
[0152] The composition to be used in the therapy will be formulated
and dosed in a fashion consistent with good medical practice,
taking into account the clinical condition of the individual
patient, the site of delivery of the composition, the method of
administration, the scheduling of administration, and other factors
known to practitioners. The "effective amounts" of each component
for purposes herein are thus determined by such considerations and
are amounts that result in bioavailability of the Apo2L/TRAIL or
other drugs to the mammal.
[0153] As a general proposition, the total pharmaceutically
effective amount of the Apo2L/TRAIL polypeptides administered will
be in the range of from about 1 mg/kg/day to about 20 mg/kg/day
based on kg of patient body weight although, as noted above, this
will be subject to therapeutic discretion.
[0154] Although injection is preferred, an infusion device may also
be employed for continuous infusions. An intravenous bag solution
may also be employed.
[0155] It is contemplated that yet additional therapies may be
employed in the methods. The one or more other therapies may
include but are not limited to, administration of radiation
therapy, cytokine(s), growth inhibitory agent(s), chemotherapeutic
agent(s), cytotoxic agent(s), tyrosine kinase inhibitors, ras
farnesyl transferase inhibitors, angiogenesis inhibitors, and
cyclin-dependent kinase inhibitors which are known in the art and
defined further with particularity in Section I above. In addition,
therapies based on therapeutic antibodies that target tumor
antigens such as Rituxan.TM. or Herceptin.TM. as well as
anti-angiogenic antibodies such as anti-VEGF, or antibodies that
target Apo2L receptors, such as DR5 or DR4.
[0156] Preparation and dosing schedules for chemotherapeutic agents
may be used according to manufacturers' instructions or as
determined empirically by the skilled practitioner. Preparation and
dosing schedules for such chemotherapy are also described in
Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,
Baltimore, Md. (1992).
[0157] It may be desirable to also administer antibodies against
other antigens, such as antibodies which bind to CD20, CD11a, CD18,
CD40, ErbB2, EGFR, ErbB3, ErbB4, vascular endothelial factor
(VEGF), or other TNFR family members (such as DR4, DR5, OPG, TNFR1,
TNFR2). Alternatively, or in addition, two or more antibodies
binding the same or two or more different antigens disclosed herein
may be co-administered to the patient. Sometimes, it may be
beneficial to also administer one or more cytokines to the patient.
In one embodiment, the Apo2L formulations are co-administered with
a growth inhibitory agent.
[0158] The Apo2L/TRAIL formulation may be administered concurrently
or sequentially with such other agents. For example, the
Apo2L/TRAIL formulation or a chemotherapeutic agent may be
administered as a pre-treatment (prior to administration of any
such other agents), such as a pre-treatment of cancer cells which
may otherwise be resistant to the apoptotic effects of
Apo2L/TRAIL.
[0159] The invention also provides kits which include a formulation
described herein. A typical kit will comprise a container,
preferably a vial, for Apo2L/TRAIL in one or more excipients as
described above; and instructions, such as a product insert or
label, directing the user as to how to employ the Apo2L/TRAIL
formulation. This would preferably provide a pharmaceutical
formulation. Preferably, the pharmaceutical formulation is for
treating cancer or an immune related condition. Suitable containers
include, for example, bottles, vials, syringes, and test tubes. The
containers may be formed from a variety of materials such as glass
or plastic. The container holds an Apo2L/TRAIL formulation that is
effective for diagnosing or treating the disorder and may have a
sterile access port (for example, the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). The label on, or associated with, the
container indicates that the formulation is used for diagnosing or
treating the disorder of choice. The article of manufacture may
further comprise a second container comprising water-for-injection,
a pharmaceutically-acceptable solution, saline, Ringer's solution,
or dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, syringes, and package inserts
with instructions for use.
[0160] All patents, patent applications, publications, product
descriptions, and protocols are cited throughout this application,
the disclosures of which are incorporated herein by reference in
their entireties.
EXAMPLES
[0161] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way. Commercially available reagents referred to
in the examples were used according to manufacturer's instructions
unless otherwise indicated. The source of those cells identified in
the following examples, and throughout the specification, by ATCC
accession numbers is the American Type Culture Collection,
Manassas, Va.
Example 1
Production of Apo2L/TRAIL in E. coli and Purification by Multiple
Chromatographic Steps (Without Crystallization)
[0162] A. Apo2L/TRAIL protein consisting of amino acids 114-281
(see FIG. 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 Jan. 4, 2001), and purified from the E. coli
cell lysates by three chromatographic steps consisting of cation
exchange, hydroxyapatite, and hydrophobic interaction
chromatography (WO 01/00832, Example 8, Section C). In the third
chromatographic separation, the Apo2L/TRAIL protein was eluted in
600 mM Na sulfate or 400 mM ammonium sulfate, 50 mM Tris, pH
7.5.
[0163] B. Another method for purification of Apo2L/TRAIL consisted
of four chromatography step and two ultrafiltration/diafiltration
(UFDF) steps. The whole cell broth obtained from the E. coli
production process was homogenized to break open the E. coli cells
and release the soluble APO2L/TRAIL held within the cytoplasm. The
solid cell debris was then removed by centrifugation.
[0164] Primary isolation was performed by binding and gradient
elution on a cation exchange (CEX) column (SP-Sepharose Fast Flow
column). The eluate was then transferred to a hydroxyapatite (HA)
chromatography column, followed by hydrophobic interaction
(Phenyl-Sepharose) chromatography. After an
ultrafiltration/diafiltration (UFDF) step, the mixture was loaded
onto a CM-Sepharose Fast Flow column, and the eluted protein
concentrated by a final UFDF step.
Example 2
Apo2L/TRAIL Crystallization as a Method of Recovery and
Purification Following One-Column Purification
[0165] The propensity of crystallization of Apo2L/TRAIL in Na
sulfate solutions was used as a means of purifying the Apo2L/TRAIL
protein from E. coli extracts. The following protocol was employed
for recovery and purification of recombinant Apo2L/TRAIL without
adverse effect on protein quality.
[0166] The harvested whole cell broth derived from E. coli
(described in Example 1) was adjusted to pH 7.5 with 1.5 M Hepes
(or 1.5M Tris) and then homogenized in a homogenizer (Gaulin
corporation, Everett, Mass.) at 6,500 psi. The homogenate was
diluted one to one with 5 mM DTT in pure water. Once the solution
reaches room temperature, 5% polyethyleneimine (PEI) was added to
give a final concentration of 0.1%, and the solution was
flocculated for 1-2 hours. The flocculated material was centrifuged
by a BTPX205 (Alfa Laval Separation AB, Sweden) continuous feed
centrifuge and clarified by depth filtration. The clarified cell
lysate (extract) was conditioned with Triton-X100 to a final
concentration of 0.05%. The conditioned, clarified cell lysate was
then loaded onto a cation exchange column (SP-Sepharose FF cation
exchange resin, Amersham Pharmacia, Sweden) equilibrated in 50 mM
Hepes (or 50 mM Tris)/0.05% Triton-X 100/1 mM DTT, pH 7.5.
Apo2L/TRAIL bound to the column while the non-binding proteins
flowed through the column and were removed by washing with
equilibration buffer until absorbance at 280 nm reached baseline.
The column was then washed with 3 column volumes of 0.1 M NaCl in
equilibration buffer. The Apo2L/TRAIL was step-eluted using 0.1 M
NaCl (or 0.1 M Na.sub.2SO.sub.4) in 50 mM each of Hepes, Tris and
Triethanolamine, 0.05% Triton-X 100 and 1 mM DTT buffer, pH
7.8.
[0167] The ambient temperature Apo2L/TRAIL pool collected from the
SP column was placed in a stainless steel tank with an insulated
jacket for heating and cooling. The tank was outfitted with a
conical bottom and a flush bottom valve for maximal recovery of
crystallized protein. The pool was agitated using a marine type
impeller under modest mixing conditions. A temperature control skid
was used to linearly ramp the temperature from approximately
25.degree. C., to approximately 4.degree. C. over the course of 1
hour. Spontaneous crystallization was observed within minutes after
the pool reached 4.degree. C. After more than 12 hours under these
conditions, crystallization was complete as equilibrium solubility
was nearly established. The crystals were then captured on a
filtration assembly containing a 20 .mu.m polypropylene frit.
Following crystal deposition on the filter surface, the crystals
were washed with chilled 20-50 mM Tris at pH 7.5. An equal volume
of wash buffer compared to the Apo2L/TRAIL SP pool volume was then
used to remove residual mother liquor (supernatant) from the
deposited crystals. Following the wash, the crystals were dissolved
in 100 mM sodium sulfate/20 mM Tris at pH 7.5 by recirculating the
dissolution buffer through the crystal bed at approximately
30.degree. C. Dissolution of the crystals was observed within
approximately 4 hours. The dissolved, purified Apo2L/TRAIL was then
sterile filtered into a container and stored frozen at -70.degree.
C.
[0168] The purity of the Apo2L/TRAIL preparations was determined by
the total E. coli protein (ECP) ELISA assays, Limulus Amebocyte
Lysate (LAL) assay, and SDS-PAGE silver stain. ECP ELISA was
performed by immobilizing affinity-purified goat anti-whole ECP
antibodies on microtiter plate wells, incubating samples and then
horseradish peroxidase-conjugated ECPs. The peroxidase enzymatic
activity was then quantified with o-phenylenediamine by reading
absorbance at 490 nm in a microtiter plate reader. Endotoxin level
was determined using the Limulus Amebocyte clot lysis assay.
SDS-PAGE silver stain was performed on a 10 to 20% gradient
polyacrylamide gel (Daiichi Pure Chemicals) in Tris-glycine buffer
containing 0.1% SDS. Electrophoresis was conducted at 50 mA
constant current until dye front reached near the bottom of the
gel. Gels were fixed and stained by Coomassie Brilliant Blue or
Merrill silver stain methods.
[0169] Protein quality was assessed by SEC, SDS-SEC, IEX, and
bioactivity according to methods described in Example 1.
[0170] The purity and quality of Apo2L/TRAIL recovered using the
above crystallization method at a 60 L fermentation scale is shown
in Table 1. For comparison, a reference standard purified by a
three-chromatographic step method as described in Example 1 is also
shown.
TABLE-US-00001 TABLE 1 Protein Quality Apo2L/ Protein Purity
Bioactivity TRAIL ECP LAL SDS- % Trimer % Monomer % of control %
IEX Prep. (ppm) (EU/mg) PAGE by SEC by SDS-SEC (.+-.20%) main peak
Apo2L/ 10 0.034 No band at 99.0 99.0 126 63 TRAIL 10 kDa purified
by crystal- lization Reference 0.82 0.023 Band at 98.9 98.9 86 61
material ~10 kDa purified by standard chroma- tography
[0171] As shown in Table 1, the Apo2L/TRAIL preparation at a
manufacturing scale had a high degree of purity suitable for
therapeutic use. The data indicate that the "one-column" step
purified Apo2L/TRAIL protein is amenable to crystallization and has
a purity comparable to or better than the Apo2L/TRAIL protein
purified by the three-column purification method described in
Example 1. FIG. 3 shows the effect of salt type on crystallization
of a one-column step purified Apo2L/TRAIL. "Poisoning" of
crystallization by divalent cations was observed for partially
purified Apo2L/TRAIL (FIG. 3).
[0172] The biochemical properties of Apo2L/TRAIL were also not
adversely impacted by crystallization of the partially purified
Apo2L/TRAIL (see Table 1). The data suggest that crystallization of
recombinant-expressed Apo2L/TRAIL, when in a partially purified
state, can be an effective, efficient and cost-effective means for
its purification. Optionally, such crystals can then be used for
preparation of dried bulk for storage or controlled release
formulations.
Example 3
Method for Recovery and Purification of Apo2L/TRAIL Using
Crystallization Including a Second Chromatography Step Following
Crystallization (Two-Column Purification)
[0173] The harvested whole cell broth derived from E. coli
(described in Example 1) was adjusted to pH 7.5 with 1.5 M Hepes
(or 1.5M Tris). DTT was added to 5 mM to prevent formation of
disulfide bonds between the non-covalently bound monomers. Two
passes on a homogenizer (Gaulin Corporation, Everett, Mass.) at
6,500 psi burst the E. coli cells. The lysate was then diluted one
to one with 5 mM DTT in pure water. Once the solution reached room
temperature, 5% PEI was added to give a final concentration of 0.2%
PEI. PEI caused flocculation of the cell solids, and the material
was mixed for at least 30 minutes before centrifuging to allow
complete flocculation. After centrifugation, the clarified lysate
was filtered using a Cuno Maximizer 30/60SP depth filter (Cuno
Incorporated, Meriden, Conn.). Before loading the clarified lysate
onto an SP Sepharose column, the pH was adjusted to 7.5 using 1M Na
HEPES and the conductivity was adjusted below 9.5 mS/cm using 5 mM
DTT in water.
[0174] As in the purification method described in Example 2,
SP-Sepharose resin, a strong cation exchange resin, was chosen for
the primary capture step. The cross-linked agarose matrix with
negatively charged functional groups bound to APO2L/TRAIL, while
allowing a majority of impurities and APO2L/TRAIL variants to pass
through the column. The following buffer conditions were used: 200
mM NaCl, 50 mM HEPES, 0.05% Triton X-100, 1 mM DTT, pH 7.5.
[0175] Crystallization of the SP elution pool was achieved by a
controlled temperature ramp from 22.degree. C. to 4.degree. C. over
a span of four hours. The SP elution pool was sterile filtered and
transferred to a temperature controlled tank with good agitation.
It was important to ensure that the vessel and the protein solution
were free from any particulars prior to crystallization. As the SP
elution pool cooled, crystals formed spontaneously with an average
chord length of 44 .mu.m as determined by Lasantec's Focused Beam
Reflectance Measurement technology. The crystal morphology was
hexagonal faces with depth approximately half of the largest chord
length. After holding the pool for approximately 1 to 2 hours a
4.degree. C. to allow the crystal growth rate to slow, 50% PEG 3350
was added to give a final concentration of 5% PEG 3350. The
addition of PEG 3350 (an anti-solvent) lowered the solubility of
APO2L/TRAIL, and promoted further crystal growth.
[0176] The crystals formed were then removed by filtration, either
batch-wise or continuously. In both cases, the mother liquor was
removed, and the crystals washed to remove impurities and residual
solvent. Filtration was performed at 2-8.degree. C. The crystal
slurry was transferred to a Buchner or Nutsche type filter
containing 5-20 .mu.m sintered steel, sintered polypropylene or
steel mesh filter either by siphoning or pressurizing the tank
containing the crystal slurry. The crystals then were either
manually scraped from the filter, or dissolved in a buffer system
suitable for the next purification step.
[0177] Before loading on a CM-Sepharose column, the crystals were
dissolved in 0.5 M arginine-succinate/20 mM TRIS/pH 7.2. Before
loading on a Phenyl-Sepharose column, the crystals were dissolved
in 0.6 mM Na.sub.2SO.sub.4/50 mM TRIS/pH 7.5
[0178] The chromatography step following crystallization served to
remove the PEG and buffer components from the crystal protein pool,
and to provide at least moderate removal of ECP's, endotoxin,
dimers, and aggregates.
[0179] In one set of experiments, a CM-Sepharose bind-and-elute
column was used in this step. Before loading this column, the
APO2L/TRAIL crystals were dissolved in formulation buffer, 0.5 M
arginine-succinate/20 mM TRIS pH 7.2, and the dissolved pool
diluted 5 fold with 20 mM TRIS. The dissolved crystal pool was
loaded onto the column and eluted with 125 mM NaCl/50 mM TRIS/1 mM
DTT/pH 7.5. The column operation was repeated various times to show
consistent recovery (85-95%) and purity.
[0180] In another set of experiments, a flow-through column, Phenyl
Sepharose HIC, was used. In this case, the crystals were dissolved
into 0.6 M NaSO.sub.4/50 mM TRIS/1 mM DTT/pH 7.5. The solubility of
APO2L/TRAIL was very high in this solution because of the high salt
concentration. Three runs consistently had 98% yields and the
chromatograms were nearly identical. The level of purity was high,
as was observed using the CM-Sepharose bind-and-elute column.
Example 4
Selection of Crystallization Conditions
[0181] The SP-Sepharose elution pool from the first chromatography
purification step described in Example 3 was cooled to produce
crystals and then heated to dissolve the crystals multiple
times.
[0182] A real time particle size analyzer (Lasentec Focused Beam
Reflectance Measurement--FBRM) was used to monitor the crystal
chord length and distribution throughout the crystallization
process. In the FBRM method, a laser is rotated quickly on a
circular path. As the laser passes over the crystal, the beam of
light is reflected for a certain duration which is multiplied by
the speed of the rotating laser to give a "chord length".
[0183] Effect of Temperature Cooling Rate
[0184] The FBRM was used to monitor the crystal growth profile as a
function of temperature cooling rate. The cooling rate effects the
time required for crystallization and the final size distribution.
A slow cooling rate supersaturates the solution slowly and the
crystal nucleation and growth becomes slow. Quick cooling induces
high supersaturation, and many small crystals form.
[0185] A linear temperature ramp from 22.degree. C. to 2.degree. C.
over various time periods was investigated. The results of the
equilibrium crystal distributions over 1, 4, 8 and 24 hour cooling
periods are shown in FIG. 4 and set forth in Table 2.
TABLE-US-00002 TABLE 2 Cooling Time # of particles (hour) Average
Size (.mu.m) (1-32 .mu.m) Solubility (g/L) 1 38 .+-. 20 810 0.81 4
43 .+-. 22 560 0.74 8 39 .+-. 18 550 1.0 24 44 .+-. 22 500 0.82
[0186] A 4-hour cooling rate provided acceptable results.
[0187] Effect of Agitation on Crystal Size
[0188] Approximately 0.4 L of SP elution buffer was crystallized at
three agitation rates. The three rates studies were the minimum
required to suspend most of the crystals (100 RPM), the maximum
agitation rate before drawings in air bubbles (250 RPM), and an
agitation rate in the middle (175 RPM). It was found that
crystallization was fastest with the highest agitation rate (250
RMP). The crystal size distribution was very similar for the
experiments run at 175 RPM and 250 RPM, and there was no noticeable
difference in microscopic images. 100 RPM did not provide enough
agitation to completely suspend all the particles. In addition,
some aggregation of the crystals was observed. In all cases, the
impeller was close to the air surface, and it was easy to drawn in
air. In large-scale applications this geometry might change, and
higher agitation rates can be used without damaging the protein by
exposure to the air-liquid interface.
[0189] Anti-Solvent Studies
[0190] Anti-solvents used in the crystallization process improve
crystallization efficiency by lowering the solubility of the
protein. Since any protein remaining in solution is lost during
filtration, it is important to drive solubility as low as possible
during the crystallization reaction.
[0191] Anti-solvents were screened by filling 5 mL syringes with
APO2L/TRAIL crystals or SP elution pool, and then adding an
appropriate amount of anti-solvent. The samples were agitated
slowly, over a span of two weeks at both room temperature and at
2-8.degree. C. 1 mL samples were passed through a 0.22 .mu.m filter
to remove all protein crystals and then run on an HPLC IEX to
determine APO2L/TRAIL concentration in solution.
[0192] Polyethylene glycol (PEG) in 400, 3350 and 10000 Da
molecular weights (PEG 440, PEG 3350 and PEG 10000, respectively)
was tested as an anti-solvent. The APO2L/TRAIL crystals were
dissolved in the SP elution buffer (200 mM NaCl, 50 mM HEPES, 0.05%
Triton X-100, 1 mM DTT, pH 7.5), and PEG was added. The mixtures
were agitated for 5 days at a temperature of 2-8.degree. C. The
results shown in FIG. 5 indicate that PEG 3350 and PEG 10000 are
superior over PEG 400, and are almost identical in terms of yield
improvement. Addition of 5% PEG 3350 improved the theoretical yield
from about 85% to about 96% relative to crystallization without the
addition of PEG or any other anti-solvent.
[0193] Next, the effect of ethanol and isopropyl alcohol on
APO2L/TRAIL solubility was examined. Both were found to provide
significant yield increases with concentrations between 5% and 10%.
The equilibrium APO2L/TRAIL solubility in using these solvents was
approximately equivalent to those for PEG.
[0194] Other commonly used organic anti-solvents, namely
2-methyl-2,4-pentanedol (MPD), ethylene glycol, and dioxane, were
also tested, but offered little or no benefit in terms of reducing
APO2L/TRAIL solubility.
[0195] Based on these studies, it has been determined that good
crystallization results and yields can be achieved by cooling the
SP elution pool with a linear temperature ramp between 22.degree.
C. and 4.degree. C., using a 4 hour cooling period, and PEG 3350 as
an anti-solvent.
Example 5
Two-Column Purification Process using Anti-Solvent in the
Crystallization Step
[0196] APO2L/TRAIL was purified essentially as described in Example
3, but adding 5% PEG 3350 during crystallization. After
crystallization, the material was split into 6 pools. 3 pools were
run on a CM-Sepharose column, and 3 pools were run on a
Phenyl-Sepharose column. The yield and purity results are give in
Table 4 below.
TABLE-US-00003 TABLE 4 Step Step Yield ECP (ppm) LAL (EU/mg)
homogenization 1.6 .times. 10.sup.6 85.3 SP-Sepharose 89% 172.90
1.9 Column Crystallization 96% 9.1 1.9 CM-Sepharose 86% 1.3 1.02
Column (Option #1) Phenyl-Sepharose 98% <0.35 0.23 Column
(Option #2)
[0197] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
Sequence CWU 1
1
21281PRTHomo Sapiens 1Met Ala Met Met Glu Val Gln Gly Gly Pro Ser
Leu Gly Gln Thr Cys1 5 10 15Val Leu Ile Val Ile Phe Thr Val Leu Leu
Gln Ser Leu Cys Val Ala 20 25 30Val Thr Tyr Val Tyr Phe Thr Asn Glu
Leu Lys Gln Met Gln Asp Lys 35 40 45Tyr Ser Lys Ser Gly Ile Ala Cys
Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60Trp Asp Pro Asn Asp Glu Glu
Ser Met Asn Ser Pro Cys Trp Gln Val65 70 75 80Lys Trp Gln Leu Arg
Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser 85 90 95Glu Glu Thr Ile
Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro 100 105 110Leu Val
Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly 115 120
125Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu
130 135 140Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg
Ser Gly145 150 155 160His Ser Phe Leu Ser Asn Leu His Leu Arg Asn
Gly Glu Leu Val Ile 165 170 175His Glu Lys Gly Phe Tyr Tyr Ile Tyr
Ser Gln Thr Tyr Phe Arg Phe 180 185 190Gln Glu Glu Ile Lys Glu Asn
Thr Lys Asn Asp Lys Gln Met Val Gln 195 200 205Tyr Ile Tyr Lys Tyr
Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys 210 215 220Ser Ala Arg
Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr225 230 235
240Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile
245 250 255Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His
Glu Ala 260 265 270Ser Phe Phe Gly Ala Phe Leu Val Gly 275
28021042DNAHomo Sapiensmisc_feature447n = T or G 2tttcctcact
gactataaaa gaatagagaa ggaagggctt cagtgaccgg ctgcctggct 60gacttacagc
agtcagactc tgacaggatc atggctatga tggaggtcca ggggggaccc
120agcctgggac agacctgcgt gctgatcgtg atcttcacag tgctcctgca
gtctctctgt 180gtggctgtaa cttacgtgta ctttaccaac gagctgaagc
agatgcagga caagtactcc 240aaaagtggca ttgcttgttt cttaaaagaa
gatgacagtt attgggaccc caatgacgaa 300gagagtatga acagcccctg
ctggcaagtc aagtggcaac tccgtcagct cgttagaaag 360atgattttga
gaacctctga ggaaaccatt tctacagttc aagaaaagca acaaaatatt
420tctcccctag tgagagaaag aggtccncag agagtagcag ctcacataac
tgggaccaga 480ggaagaagca acacattgtc ttctccaaac tccaagaatg
aaaaggctct gggccgcaaa 540ataaactcct gggaatcatc aaggagtggg
cattcattcc tgagcaactt gcacttgagg 600aatggtgaac tggtcatcca
tgaaaaaggg ttttactaca tctattccca aacatacttt 660cgatttcagg
aggaaataaa agaaaacaca aagaacgaca aacaaatggt ccaatatatt
720tacaaataca caagttatcc tgaccctata ttgttgatga aaagtgctag
aaatagttgt 780tggtctaaag atgcagaata tggactctat tccatctatc
aagggggaat atttgagctt 840aaggaaaatg acagaatttt tgtttctgta
acaaatgagc acttgataga catggaccat 900gaagccagtt ttttcggggc
ctttttagtt ggctaactga cctggaaaga aaaagcaata 960acctcaaagt
gactattcag ttttcaggat gatacactat gaagatgttt caaaaaatct
1020gaccaaaaca aacaaacaga aa 1042
* * * * *