U.S. patent application number 11/603584 was filed with the patent office on 2007-07-12 for methods for making apo-2 ligand using divalent metal ions.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Avi J. Ashkenazi, Sarah Hymowitz, Robert F. Kelley, Iphigenia Koumenis, Woon-Lam Susan Leung, Mark O'Connell, Roger Pai, Zahra Shahrokh, Laura Simmons.
Application Number | 20070161564 11/603584 |
Document ID | / |
Family ID | 22495287 |
Filed Date | 2007-07-12 |
United States Patent
Application |
20070161564 |
Kind Code |
A1 |
Ashkenazi; Avi J. ; et
al. |
July 12, 2007 |
Methods for making Apo-2 ligand using divalent metal ions
Abstract
Methods of making Apo-2 ligand and formulations of Apo-2 ligand
using divalent metal ions are provided. Such divalent metal ions
include zinc and cobalt which improve Apo-2 ligand trimer formation
and stability. The crystal structure of Apo-2 ligand is also
provided, along with Apo-2 ligand variant polypeptides identified
using oligonucleotide-directed mutagenesis.
Inventors: |
Ashkenazi; Avi J.; (San
Mateo, CA) ; Hymowitz; Sarah; (San Francisco, CA)
; Kelley; Robert F.; (San Bruno, CA) ; Koumenis;
Iphigenia; (Winston-Salem, NC) ; Leung; Woon-Lam
Susan; (San Mateo, CA) ; O'Connell; Mark;
(Montara, CA) ; Pai; Roger; (Los Altos, CA)
; Shahrokh; Zahra; (Weston, MA) ; Simmons;
Laura; (Burlingame, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
22495287 |
Appl. No.: |
11/603584 |
Filed: |
November 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09603866 |
Jun 26, 2000 |
|
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11603584 |
Nov 22, 2006 |
|
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60141342 |
Jun 28, 1999 |
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Current U.S.
Class: |
514/21.2 ;
514/18.9; 514/19.3 |
Current CPC
Class: |
C07K 14/70575 20130101;
A61K 9/0019 20130101; C07K 14/525 20130101; A61K 38/00 20130101;
A61P 35/00 20180101; A61P 43/00 20180101; A61K 9/19 20130101; A61K
47/02 20130101; A61P 31/12 20180101; Y02A 50/473 20180101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/17 20060101
A61K038/17 |
Claims
1. A formulation comprising Apo-2 ligand and one or more divalent
metal ions, wherein the concentration of said one or more divalent
metal ions present in the formulation is at a <2.times. molar
ratio to said Apo-2 ligand.
2. The formulation of claim 1 wherein said one or more divalent
metal ions comprises zinc or cobalt.
3. The formulation of claim 2 wherein said one or more divalent
ions comprises zinc.
4. The formulation of claim 3 wherein said zinc is selected from
the group consisting of zinc chloride, zinc acetate, zinc sulfate,
zinc carbonate and zinc citrate.
5. The formulation of claim 1 wherein said formulation is a
pharmaceutically acceptable formulation.
6. The formulation of claim 1 wherein said Apo-2 ligand comprises
amino acids 114 to 281 of FIG. 1 (SEQ ID NO:1).
7. The formulation of claim 1 wherein said Apo-2 ligand comprises
amino acids 1 to 281 of FIG. 1 (SEQ ID NO:1) or a biologically
active fragment or variant thereof.
8. The formulation of claim 1 wherein said formulation has a pH of
about 6 to about 9.
9. The formulation of claim 8 wherein said formulation has a pH of
about 7 to about 7.5.
10. The formulation of claim 1 wherein said formulation is an
aqueous formulation.
11. The formulation of claim 1 wherein said formulation is a
lyophilized formulation.
12. A formulation comprising Apo-2 ligand and one or more divalent
metal ions, wherein the concentration of said one or more divalent
metal ions present in the formulation is at a .gtoreq.2.times.
molar ratio to said Apo-2 ligand.
13. A method of enhancing formation of Apo-2 ligand trimers,
comprising exposing Apo-2 ligand polypeptides to an effective
amount of one or more divalent metal ions.
14. A method of making a pharmaceutically acceptable formulation of
Apo-2 ligand, comprising admixing Apo-2 ligand, an effective amount
of one or more divalent metal ions, and a pharmaceutically
acceptable carrier.
15. A method of reducing formation of disulfide-linked Apo-2 ligand
dimers, comprising exposing Apo-2 ligand polypeptides to an
effective amount of one or more divalent metal ions.
16. A method of making Apo-2 ligand, comprising the steps of: (a)
providing a host cell comprising a replicable vector containing a
nucleotide sequence encoding Apo-2 ligand polypeptide; (b)
providing culture media containing an effective amount of one or
more divalent metal ions; (c) culturing the host cell in the
culture media under conditions sufficient to express the Apo-2
ligand; and (d) recovering the Apo-2 ligand from the host cell or
culture media.
17. The method of claim 16 wherein said host cell is E. coli.
18. The method of claim 16 wherein said one or more divalent metal
ions comprises zinc.
19. The method of claim 18 wherein said zinc comprises zinc
sulfate.
20. The method of claim 16 wherein said one or more divalent metal
ions comprises cobalt.
21. The method of claim 20 wherein said cobalt comprises cobalt
chloride.
22. The method of claim 18 wherein said zinc is present in the
culture media at a concentration of about 50 micromolar to about
250 micromolar.
23. The method of claim 16 wherein said replicable vector comprises
a nucleotide sequence encoding one or more tRNA molecules.
24. The method of claim 23 wherein said replicable vector is the
pAPApo2-P2RU vector.
25. The method of claim 16 wherein said Apo-2 ligand comprises
amino acids 114 to 281 of FIG. 1 (SEQ ID NO:1).
26. The method of claim 16 wherein said Apo-2 ligand comprises
amino acids 1 to 281 of FIG. 1 (SEQ ID NO:1) or a biologically
active fragment or variant thereof.
27. A method of making Apo-2 ligand, comprising the steps of:
providing a host cell comprising a replicable vector containing a
nucleotide sequence encoding Apo-2 ligand; (b) providing culture
media; (c) culturing the host cell in the culture media under
conditions sufficient to express the Apo-2 ligand; (d) recovering
the Apo-2 ligand from the host cell or culture media; and (e)
purifying the Apo-2 ligand in the presence of an effective amount
of one of more divalent metal ions.
28. The method of claim 27 wherein in step (e), said Apo-2 ligand
is purified in the presence of one or more divalent metal ions and
a reducing agent.
29. The method of claim 27 wherein said Apo-2 ligand comprises
amino acids 114 to 281 of FIG. 1 (SEQ ID NO:1).
30. The method of claim 27 wherein said Apo-2 ligand comprises
amino acids 1 to 281 of FIG. 1 (SEQ ID NO:1) or a biologically
active fragment or variant thereof.
31. A method for recovering Apo-2 ligand from a prokaryotic cell
culture comprising the steps of (a) isolating Apo-2 ligand which
has been expressed in cultured prokaryote host cells; (b) exposing
said isolated Apo-2 ligand to a buffered solution containing one or
more divalent metal ions and reducing agent; and (c) recovering
said isolated Apo-2 ligand.
32. The method of claim 31 wherein in step (b), said one or more
divalent metal ions is selected from the group consisting of zinc
and cobalt and said reducing agent is selected from the group
consisting of DTT and BME.
33. The method of claim 31 wherein in step (c) said Apo-2 ligand is
recovered by sequentially contacting said Apo-2 ligand to a
cationic chromatography support, hydroxyapatite support, and
hydrophobic chromatographic support.
34. The method of claim 33 wherein said cationic chromatography
support is selected from the group consisting of SP-Sepharose,
CM-Sepharose, and Macro-Prep ceramic HS resin.
35. The method of claim 33 wherein said hydrophobic chromatographic
support is selected from the group consisting of phenyl agarose,
butyl agarose, TSK resin, and Toyopearl resin.
36. An isolated Apo-2 ligand variant polypeptide comprising an
amino acid sequence which differs from native sequence Apo-2 ligand
and has one or more of the following amino acid substitutions at
the residue position(s) in FIG. 1 (SEQ ID NO:1): R130A; N134A;
L136A; S138A; N140A; N143A; S153A; E155A; R158A; R170A; K179A;
R191A; Q193A; E195A; K197D; K201A; N.sub.2O.sub.2A; D203A; Y213A;
D218A; Y240A; K251A; S259A; D267A; D269A.
37. An isolated Apo-2 ligand variant polypeptide comprising an
amino acid sequence which differs from native sequence Apo-2 ligand
and has one or more of the following amino acid substitutions at
the residue position(s) in FIG. 1 (SEQ ID NO:1): S141A, K142A,
S159A, H264A.
38. An isolated Apo-2 ligand variant polypeptide comprising an
amino acid sequence which differs from native sequence Apo-2 ligand
and has one or more of the following amino acid substitutions at
the residue position(s) in FIG. 1 (SEQ ID NO:1): R149A; C230S;
C230A; Q205A; V207A; Y216A; E236A; Y237A.
39. An isolated nucleic acid comprising a nucleotide sequence
encoding the Apo-2 ligand variant of claim 36.
40. A vector comprising the nucleic acid of claim 39.
41. A host cell comprising the vector of claim 40.
42. The pAPApo2-P2RU vector.
43. A host cell comprising the vector of claim 42.
44. The host cell of claim 31 wherein said host cell is E.
coli.
45. An isolated Apo-2 ligand comprising amino acids 114 to 281 of
FIG. 1 (SEQ ID NO:1) and made according to the method of claim
16.
46. An isolated Apo-2 ligand comprising amino acids 1 to 281 of
FIG. 1 (SEQ ID NO:1) or a biologically active fragment or variant
thereof, and made according to the method of claim 16.
47. An isolated Apo-2 ligand comprising amino acids 114 to 281 of
FIG. 1 (SEQ ID NO:1) and made according to the method of claim
27.
48. An isolated Apo-2 ligand comprising amino acids 1 to 281 of
FIG. 1 (SEQ ID NO:1) or a biologically active fragment or variant
thereof, and made according to the method of claim 27.
Description
RELATED APPLICATIONS
[0001] This is a non-provisional application claiming priority
under Section 119(e) to provisional application No. 60/141,342
filed Jun. 28, 1999, the contents of which are incorporated herein
by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to making Apo-2
ligand and Apo-2 ligand formulations using divalent metal ions,
such as zinc or cobalt. The use of such Apo-2 ligand and Apo-2
ligand formulations having improved Apo-2L trimer formation and
stability is also provided. The present invention also relates to
Apo-2 ligand variants, particularly alanine substitution
variants.
BACKGROUND OF THE INVENTION
[0003] Control of cell numbers in mammals is believed to be
determined, in part, by a balance between cell proliferation and
cell death. One form of cell death, sometimes referred to as
necrotic cell death, is typically characterized as a pathologic
form of cell death resulting from some trauma or cellular injury.
In contrast, there is another, "physiologic" form of cell death
which usually proceeds in an orderly or controlled manner. This
orderly or controlled form of cell death is often referred to as
"apoptosis" [see, e.g., Barr et al., Bio/Technology, 12:487-493
(1994); Steller et al., Science, 267:1445-1449 (1995)]. Apoptotic
cell death naturally occurs in many physiological processes,
including embryonic development and clonal selection in the immune
system [Itoh et al., Cell, 66:233-243 (1991)].
[0004] 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, 4-1BB ligand, Apo-1
ligand (also referred to as Fas ligand or CD95 ligand), Apo-2
ligand (also referred to as TRAIL, AIM-1 or AGP-1), and Apo-3
ligand (also referred to as TWEAK) 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); 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; WO 97/46686
published Dec. 11, 1997; WO 97/33899 published Sep. 18, 1997;
Marsters et al., Curr. Biol., 8:525-528 (1998); Chicheportiche et
al., Biol. Chem., 272:32401-32410 (1997)]. Among these molecules,
TNF-.alpha., TNF-.beta., CD30 ligand, 4-1BB ligand, Apo-1 ligand,
Apo-2 ligand (TRAIL) and Apo-3 ligand (TWEAK) have been reported to
be involved in apoptotic cell death.
[0005] Induction of various cellular responses mediated by such TNF
family cytokines is believed to be initiated by their binding to
specific cell receptors. Two distinct TNF receptors of
approximately 55-kDa (TNFR1) and 75-kDa (TNFR2) have been
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] and human and mouse cDNAs
corresponding to both receptor types have been isolated and
characterized [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)]. Extensive
polymorphisms have been associated with both TNF receptor genes
[see, e.g., Takao et al., Immunogenetics, 37:199-203 (1993)]. Both
TNFRs share the typical structure of cell surface receptors
including extracellular, transmembrane and intracellular regions.
The extracellular portions of both receptors are 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)]. The cloning of recombinant soluble TNF receptors
was reported by Hale et al. [J. Cell. Biochem. Supplement 15F,
1991, p. 113 (P424)].
[0006] 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. Each CRD is about 40 amino acids long
and contains 4 to 6 cysteine residues at positions which are well
conserved [Schall et al., supra; Loetscher et al., supra; Smith et
al., supra; Nophar et al., supra; Kohno et al., supra]. In TNFR1,
the approximate boundaries of the four CRDs are as follows: CRD1-
amino acids 14 to about 53; CRD2- amino acids from about 54 to
about 97; CRD3- amino acids from about 98 to about 138; CRD4- amino
acids from about 139 to about 167. In TNFR2, CRD1 includes amino
acids 17 to about 54; CRD2- amino acids from about 55 to about 97;
CRD3- amino acids from about 98 to about 140; and CRD4- amino acids
from about 141 to about 179 [Banner et al., Cell, 73:431-435
(1993)]. The potential role of the CRDs in ligand binding is also
described by Banner et al., supra.
[0007] 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.,
J. Exp. Med., 169:1747-1756 (1989) 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. Recent studies on p75NGFR showed that
the deletion of CRD1 [Welcher, A. A. et al., Proc. Natl. Acad. Sci.
USA, 88:159-163 (1991)] or a 5-amino acid insertion in this domain
[Yan, H. and Chao, M. V., J. Biol. Chem., 266:12099-12104 (1991)]
had little or no effect on NGF binding [Yan, H. and Chao, M. V.,
supra]. p75 NGFR contains a proline-rich stretch of about 60 amino
acids, between its CRD4 and transmembrane region, which is not
involved in NGF binding [Peetre, C. et al., Eur. J. Hematol.,
41:414-419 (1988); Seckinger, P. et al., J. Biol. Chem.,
264:11966-11973 (1989); Yan, H. and Chao, M. V., supra]. A similar
proline-rich region is found in TNFR2 but not in TNFR1.
[0008] The TNF family ligands identified to date, with the
exception of lymphotoxin-.alpha., are type II transmembrane
proteins, whose C-terminus is extracellular. In contrast, most
receptors in the TNF receptor (TNFR) family identified to date are
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] Recently, other members of the TNFR family have been
identified. Such newly identified members of the TNFR family
include CAR1, HVEM and osteoprotegerin (OPG) [Brojatsch et al.,
Cell, 87:845-855 (1996); Montgomery et al., Cell, 87:427-436
(1996); Marsters et al., J. Biol. Chem., 272:14029-14032 (1997);
Simonet et al., Cell, 89:309-319 (1997)]. Unlike other known
TNFR-like molecules, Simonet et al., supra, report that OPG
contains no hydrophobic transmembrane-spanning sequence. OPG is
believed to act as a decoy receptor, as discussed below.
[0010] Another new member of the TNF/NGF receptor family has been
identified in mouse, a receptor referred to as GITR for
"glucocorticoid-induced tumor necrosis factor receptor
family-related gene" [Nocentini et al., Proc. Natl. Acad. Sci. USA
94:6216-6221 (1997)]. The mouse GITR receptor is a 228 amino acid
type I transmembrane protein that is expressed in normal mouse T
lymphocytes from thymus, spleen and lymph nodes. Expression of the
mouse GITR receptor was induced in T lymphocytes upon activation
with anti-CD3 antibodies, Con A or phorbol 12-myristate
13-acetate.
[0011] In Marsters et al., Curr. Biol., 6:750 (1996), investigators
describe a full length native sequence human polypeptide, called
Apo-3, which exhibits similarity to the TNFR family in its
extracellular cysteine-rich repeats and resembles TNFR1 and CD95 in
that it contains a cytoplasmic death domain sequence [see also
Marsters et al., Curr. Biol., 6:1669 (1996)]. Apo-3 has also been
referred to by other investigators as DR3, wsl-1, TRAMP, and LARD
[Chinnaiyan et al., Science, 274:990 (1996); Kitson et al., Nature,
384:372 (1996); Bodmer et al., Immunity, 6:79 (1997); Screaton et
al., Proc. Natl. Acad. Sci., 94:4615-4619 (1997)].
[0012] Pan et al. have disclosed another TNF receptor family member
referred to as "DR4" [Pan et al., Science, 276:111-113 (1997)]. 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 Apo-2 ligand
or TRAIL.
[0013] 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 the Apo-2 ligand (TRAIL) is described. That molecule
is referred to as DR5 (it has also been alternatively referred to
as Apo-2; TRAIL-R2, 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)]. Like DR4, DR5 is
reported to contain a cytoplasmic death domain and be capable of
signaling apoptosis.
[0014] Yet another death domain-containing receptor, DR6, was
recently identified [Pan et al., FEBS Letters, 431:351-356 (1998)].
Aside from containing four putative extracellular domains and a
cytoplasmic death domain, DR6 is believed to contain a putative
leucine-zipper sequence that overlaps with a proline-rich motif in
the cytoplasmic region. The proline-rich motif resembles sequences
that bind to src-homology-3 domains, which are found in many
intracellular signal-transducing molecules.
[0015] A further group of recently identified TNFR family members
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] and DCR3
[Pitti et al., Nature, 396:699-703 (1998)], both of which are
secreted, soluble proteins.
[0016] For a review of the TNF family of cytokines and their
receptors, see Ashkenazi et al., Science, 281:1305-1308 (1998);
Golstein, Curr. Biol., 7:750-753 (1997); and Gruss and Dower,
supra.
[0017] While zinc binding sites have been shown to play structural
roles in protein-protein interactions in certain proteins involving
diverse interfaces [Feese et al., Proc. Natl. Acad. Sci.,
91:3544-3548 (1994); Somers et al., Nature, 372:478-481 (1994);
Raman et al., Cell, 95:939-950 (1998)], none of the previously
structurally-characterized members of the TNF family (CD40 ligand,
TNF-alpha, or TNF-beta) bind metals. The use of metal ions, such as
zinc, in formulations of various hormones, such as human growth
hormone (hGH), has been described in the literature. [See, e.g., WO
92/17200 published Oct. 15, 1992). Zinc involvement in hGH binding
to receptors was likewise described in WO 92/03478 published Mar.
5, 1992. The roles of zinc binding in interferon-alpha dimers and
interferon-beta dimers were reported in Walter et al., Structure,
4:1453-1463 (1996) and Karpusas et al., Proc. Natl. Acad. Sci.,
94:11813-11818 (1997), respectively.
[0018] The structures and biological roles of various metal ions
such as zinc have been reviewed in the art, see, e.g., Christianson
et al., Advances in Protein Chemistry, 42:281-355 (1991).
SUMMARY OF THE INVENTION
[0019] The present invention is based on the discovery that the
inclusion of one or more divalent metal ions in methods or
processes for making Apo-2 ligand, or formulations containing Apo-2
ligand, results in increased yield and stability of Apo-2 ligand
trimers. It is presently believed that such inclusion of one or
more divalent metal ions may also improve folding of Apo-2 ligand
or Apo-2L trimer assembly upon expression in recombinant cell
culture. In oxidative environments, free cysteines on Apo-2L
monomers may form intermolecular disulfide bridges, giving rise to
free-standing Apo-2L dimers as well as disulfide-linked Apo-2L
dimer species within-trimeric forms of Apo-2L. Such formation of
Apo-2L dimers may lead to aggregation, precipitation, and/or
inactivation of Apo-2L. The presence of divalent metal ions in the
methods and formulations described herein may protect against such
disulfide bond formation. It appears that inclusion of divalent
metal ions during the process of synthesis and assembly of Apo-2L
trimers may further improve accumulation and recovery of properly
folded, homotrimeric Apo-2L. Applicants have found that Apo-2
ligand trimers are approximately 10-fold more active (in cytotoxic
activity in mammalian cancer cells) as compared to disulfide-linked
Apo-2L dimers.
[0020] While the description of the invention herein is primarily
directed to Apo-2 ligand, the use of divalent metal ions to make or
stabilize trimers of various other proteins is contemplated. Such
other proteins particularly include those proteins which require
trimer formation for biological activity, for instance, various
members of the TNF family.
[0021] In one embodiment, the invention provides a method of making
Apo-2 ligand using one or more divalent ions. The methods include
the steps of providing a host cell comprising a replicable vector
containing a nucleic acid encoding Apo-2 ligand, providing culture
media containing one or more divalent metal ions, culturing the
host cell in the culture media under conditions sufficient to
express the Apo-2 ligand, and recovering the Apo-2 ligand from the
host cells or the cell culture media. Optionally, one or more
divalent metal ions are used during the recovery or purification
process.
[0022] In another embodiment, the invention provides a formulation
comprising Apo-2 ligand and one or more divalent metal ions. The
composition may be a pharmaceutically acceptable formulation
useful, for instance, in inducing or stimulating apoptosis in
mammalian cancer cells.
[0023] A further embodiment of the invention provides articles of
manufacture and kits that include such Apo-2 ligand formulations
containing one or more divalent metal ions. The articles of
manufacture and kits include a container, a label on the container,
and a formulation contained within the container. The label on the
container indicates that the formulation can be used for certain
therapeutic or non-therapeutic applications. The formulation
contains Apo-2 ligand and one or more divalent ions.
[0024] In another embodiment, the invention provides Apo-2 ligand
polypeptides made in accordance with the methods described herein.
Such Apo-2 ligands may comprise amino acids 114-281 of FIG. 1 (SEQ
ID NO:1), amino acids 1-281 of FIG. 1 (SEQ ID NO:1), as well as
biologically active fragments or variants thereof.
[0025] In a still further embodiment, the invention provides Apo-2
ligand variants. Particularly, the invention provides Apo-2 ligand
variants comprising one or more amino acid substitutions in the
native sequence of Apo-2 ligand (FIG. 1; SEQ ID NO:1). Apo-2 ligand
variants comprising alanine substitutions are provided in Table I
below. The invention also provides nucleic acid molecules encoding
such Apo-2L variants and vectors and host cells containing nucleic
acid molecules encoding the Apo-2L variants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows the nucleotide sequence of human Apo-2 ligand
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".
[0027] FIG. 2 provides the crystal structure of Apo-2L. FIG. 2A
shows a view of the trimer along the three fold axis. Each monomer
is identical. The ordered protein structure commences at residue
120, residues 131-141 are disordered, as are residues 195-201
(marked as dashed lines). The zinc binding site including the three
symmetry related cysteines and the solvent ligand are shown as
space filling diagrams. FIG. 2B provides cross-eyed stereo close up
view of the zinc binding site; the angles between Sy-zinc-Sy are
112.degree. and the Sy-zinc-solvent angles are 107.degree. with 2.3
Angstrom zinc-sulfur and 2.3 Angstrom zinc-solvent bond distances.
FIGS. 2 (and 5) were made with the programs Molscript [Kraulis et
al., J. Appl. Cryst., 24:946-950 (1991)] and Raster3D [Merrit et
al., Acta Cryst., D50:869-873 (1994)]. FIG. 2C provides a summary
of the crystallographic data from the experiment described in
Example 2.
[0028] FIG. 3 shows a sequence alignment of selected TNF family
members: Apo2L (SEQ ID NO:1); TNF-beta (SEQ ID NO:3); TNF-alpha
(SEQ ID NO:4); CD40L (SEQ ID NO:5); FasL (SEQ ID NO:6); RANKL (SEQ
ID NO:7). Arrows over the sequence indicate beta-strands in Apo2L.
The numbering over the aligned sequences corresponds to the Apo2L
sequence numbering provided in FIG. 1 (SEQ ID NO:1).
[0029] FIG. 4 provides bioassay data showing the functional
importance of the zinc binding site. SK-MES-1 cell viability was
determined by a fluorescence assay of metabolic activity after
overnight incubation with various concentrations of Apo-2L (form
114-281), or Apo-2L (form 114-281) treated with chelating agents to
remove the zinc.
[0030] FIG. 5 shows mutational analysis mapped onto a space-filling
model of Apo-2L. The trimer is oriented as in FIG. 2. Residues with
a greater than 5-fold decrease in bioactivity when mutated to
alanine are labeled and darkly shaded. Other residues that have
been mutated are shown in medium shading and a few of these
residues are also labeled.
[0031] FIG. 6 shows circular dichroic spectra of Apo-2L (form
114-281) before and after treatment to remove the bound zinc.
[0032] FIG. 7 shows thermal denaturation of Apo-2L before and after
zinc removal monitored by circular dichroism at 225 nm. The dynode
voltage is reported for 2 micromolar solutions of Apo-2L.
[0033] FIG. 8 shows the effect (time course) of ZnSO.sub.4
additions on soluble Apo-2L product accumulation (gm/L) in an E.
coli expression system using an AP promoter.
[0034] FIG. 9 shows the elution profiles from MPHS chromatography
of cell lysates from the E. coli expression system (see Example 8)
conducted in the presence or absence of ZnSO.sub.4.
[0035] FIG. 10 shows the effect (time course) of ZnSO.sub.4
addition on soluble Apo-2L product accumulation (gm/L) in an E.
coli expression system using a trp promoter.
[0036] FIG. 11 shows the effect (time course) of CoCl.sub.2
addition on soluble Apo-2L product accumulation (gm/L) in an E.
coli expression system using an AP promoter.
[0037] FIG. 12 shows the pAPApo2-P2RU plasmid construct.
[0038] FIG. 13 shows the pAPOK5 plasmid construct.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039] I. Definitions
[0040] The terms "Apo-2 ligand", "Apo-2L", 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). 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 Pro119 (FIG. 1; SEQ ID NO:2) may be "CCT" or "CCG". In
another preferred embodiment, 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 sequences. The definition
encompasses substitutional variants of Apo-2 ligand in which at
least one of its native amino acids are substituted by an alanine
residue. Preferred substitutional variants include one or more of
the residue substitutions identified in Table I below. The
definition also encompasses a native sequence Apo-2 ligand isolated
from an Apo-2 ligand source or prepared by recombinant or synthetic
methods. The Apo-2 ligand of the invention includes the
polypeptides referred to as Apo-2 ligand or TRAIL disclosed in
WO97/01633 published Jan. 16, 1997 and WO97/25428 published Jul.
17, 1997. The terms "Apo-2 ligand" or "Apo-2L" are used to refer
generally to forms of the Apo-2 ligand which include monomer, dimer
or trimer forms of the polypeptide. All numbering of amino acid
residues referred to in the Apo-2L 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).
[0041] The term "Apo-2 ligand extracellular domain" or "Apo-2
ligand ECD" refers to a form of Apo-2 ligand 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.
[0042] The term "Apo-2 ligand monomer" or "Apo-2L monomer" refers
to a covalent chain of an extracellular domain sequence of
Apo-2L.
[0043] The term "Apo-2 ligand dimer" or "Apo-2L dimer" refers to
two Apo-2L monomers joined in a covalent linkage via a disulfide
bond. The term as used herein includes free standing Apo-2L dimers
and Apo-2L dimers that are within trimeric forms of Apo-2L (i.e.,
associated with another Apo-2L monomer).
[0044] The term "Apo-2 ligand trimer" or "Apo-2L trimer" refers to
three Apo-2L monomers that are non-covalently associated.
[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-2 ligand (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, 85:3378-3404 (1995); 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)].
[0046] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising Apo-2 ligand, 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, yet is short enough such that it does not interfere with
activity of the Apo-2 ligand. The tag polypeptide preferably also
is fairly unique so that the 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).
[0047] The term "divalent metal ion" refers to a metal ion having
two positive charges. Examples of divalent metal ions for use in
the present invention 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. A preferred divalent metal ion for use in the
present invention is zinc, and more preferably, the salt form, zinc
sulfate. Divalent metal ions, as described herein, are preferably
employed in concentrations or amounts (e.g., effective amounts)
which are sufficient to, for example, (1) enhance storage stability
of Apo-2L trimers over a desired period of time, (2) enhance
production or yield of Apo-2L trimers in a recombinant cell culture
or purification method, (3) enhance solubility (or reduce
aggregation) of Apo-2L trimers, or (4) enhance Apo-2L trimer
formation.
[0048] "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. Isolated protein
includes protein in situ within recombinant cells, since at least
one component of the Apo-2 ligand natural environment will not be
present. Ordinarily, however, isolated protein will be prepared by
at least one purification step.
[0049] An "isolated" Apo-2 ligand 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 Apo-2 ligand
nucleic acid. An isolated Apo-2 ligand nucleic acid molecule is
other than in the form or setting in which it is found in nature.
Isolated Apo-2 ligand nucleic acid molecules therefore are
distinguished from the Apo-2 ligand nucleic acid molecule as it
exists in natural cells. However, an isolated Apo-2 ligand nucleic
acid molecule includes Apo-2 ligand nucleic acid molecules
contained in cells that ordinarily express Apo-2 ligand where, for
example, the nucleic acid molecule is in a chromosomal location
different from that of natural cells.
[0050] "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 Apo-2 ligand 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.
[0051] 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.
[0052] 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.
[0053] "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; (b) capable of raising an
antibody, i.e., immunogenic; (c) capable of binding and/or
stimulating a receptor for Apo-2L; or (d) retaining the activity of
a native or naturally-occurring Apo-2L polypeptide.
[0054] 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, FACS analysis or DNA electrophoresis.
[0055] 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, small-cell lung cancer, non-small
cell lung cancer, glioma, gastrointestinal cancer, renal cancer,
ovarian cancer, liver cancer, 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.
[0056] The terms "treating", "treatment" and "therapy" as used
herein refer to curative therapy, prophylactic therapy, and
preventative therapy.
[0057] 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.
[0058] II. Compositions and Methods of the Invention
[0059] A novel cytokine related to the TNF ligand family, the
cytokine identified herein as "Apo-2 ligand" has been described.
The predicted mature amino acid sequence of human Apo-2 ligand
contains 281 amino acids, and has a calculated molecular weight of
approximately 32.5 kDa. The absence of a signal sequence and the
presence of an internal hydrophobic region suggests that Apo-2
ligand is a type II transmembrane protein. Soluble extracellular
domain Apo-2 ligand polypeptides have also been described. See,
e.g., WO97/25428 published Jul. 17, 1997. Apo-2L substitutional
variants have further been described. Alanine scanning techniques
have been utilized to identify various substitutional variant
molecules having biological activity. Particular substitutional
variants of the Apo-2 ligand include those in which at least one
amino acid is substituted by an alanine residue. These
substitutional variants are identified, for example, as "D203A";
"D218A" and "D269A." This nomenclature is used to identify Apo-2
ligand 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 Apo-2L
variants may comprise one or more of the alanine substitutions
which are recited in Table I below.
[0060] The x-ray crystal structure of the extracellular domain of
Apo-2 ligand is now provided in the present invention, and
alanine-scanning mutagenesis has been performed to provide the
mapping of its receptor contact regions. The structure obtained for
Apo-2 ligand reveals a homotrimeric protein which contains a novel
divalent metal ion (zinc) binding site that coordinates the
interaction of the Apo-2 ligand trimer molecule's three
subunits.
[0061] The x-ray structure of Apo-2L was determined by molecular
replacement using a model of TNF-alpha [Eck et al., J. Biol. Chem.,
264:17595-17605 (1989)] and refined to 3.9 Angstrom (for the
114-281 residue form) and 1.3 Angstrom (for the D218A variant;
91-281 form). Like other members of the TNF family, Apo-2L 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 (See FIG. 2A). The
position of the core beta-strands was well conserved compared to
the other structurally characterized members of the TNF family,
TNF-alpha [Eck et al., supra; Jones et al., Nature, 338:225-228
(1989)], TNF-beta [Eck et al., J. Biol. Chem., 267:2119-2122
(1992)], and CD40L [Karpusas et al., Structure, 3:1031-1039
(1995)], with a r.m.s.d. of 0.8 Angstrom when compared to the core
strands of TNF-alpha or TNF-beta. None of the residues in the
Apo-2L trimer interface appear to be absolutely conserved across
the sequences of the all the presently known human TNF family
members; however, the hydrophobic chemical nature of these residues
is preserved. (See FIG. 3). The conserved residues in the Apo-2L
trimer interface cluster near the base (the widest part of the
trimer) and along the three-fold axis. Near the top of the Apo-2L
trimer interface in the vicinity of Cys230, the structures appear
to diverge, and the conformation of the 190's and 230's loops are
variable in each structure.
[0062] In contrast to the beta-scaffold core, the structure of the
loops and receptor binding surfaces varies considerably among the
TNF family members. One difference between the structure of Apo-2
ligand and the structures of TNF-alpha, TNF-beta, and CD40L is the
connections between strands A and A'. In TNF-alpha, TNF-beta, and
CD40L, strand A is followed by a compact loop. In Apo-2 ligand, a
15-residue insertion lengthens this loop and alters its
conformation. The first part of the loop (residues 131 to 141) is
disordered while the second part of the loop (residues 142 to 154)
crosses the surface of the molecule from one monomer-monomer
interface to the next (see FIG. 2A) with a conformation that
resembles CD40L in its C-terminal portion.
[0063] Applicants surprisingly found a novel divalent metal ion
(zinc) binding site buried near the top of the trimerization
interface. The TNF family members can be divided by sequence
analysis into three groups with respect to Cys230: (1) proteins
such as TNF-alpha and Fas ligand in which a cysteine residue at the
position corresponding to Cys.sup.230 is accompanied by another
cysteine in the adjacent loop (the 194-203 loop in Apo-2L) with
which it can form a disulfide bridge precluding it from interacting
with a metal ion, (2) proteins without a cysteine corresponding to
Cys230 (such as TNF-beta and OPGL), and (3) proteins which have
only one cysteine residue corresponding to Cys230. Apo-2L and its
orthologs in other species meet the latter criteria (i.e., proteins
which have only Cys230) and are expected to bind divalent metal
ions at the trimer surface. The conformation of the main chain
immediately prior to Cys230 in Apo-2L differs from the disulfide
containing TNF family members such as TNF-alpha and CD40L. In
Apo-2L, the side chain of Cys230 is oriented towards the interface
instead of away from it.
[0064] The Cys230 residue in each Apo-2L monomer point inward
toward the trimer axis and coordinate a divalent metal ion in
conjunction with an interior solvent molecule. This divalent metal
ion binding site exhibits slightly distorted tetrahedral geometry
with bonds and angles appropriate for a zinc binding site and is
completely inaccessible to solvent (see FIG. 2B). The identity of
the bound metal was confirmed using inductively coupled plasma
atomic emission spectrometry (ICP-AES) (see Example 5). In a
quantitative analysis for Cd, Co, Zn, Ni, and Cu using ICP-AES,
0.79 moles of Zn and 0.06 moles of Co per molecule of Apo-2L trimer
were detected demonstrating that the bound ion in the structure was
zinc at approximately a one to one molar ratio (see Example 5). The
importance of this site was demonstrated by the observation that
alanine substitution of Cys230 resulted in a >8-fold decreased
apoptotic activity (See Example 7). Furthermore, removal of the
bound metal from Apo-2L by dialysis against chelating agents
resulted in a 7-fold decrease in DR5 affinity and a >90-fold
decrease in apoptotic activity (see Example 6). Upon removal of the
Zn, the cysteines became prone to oxidation and disulfide-linked
Apo-2L dimers were formed which had decreased apoptotic activity.
Since the metal binding site appears to be buried in the Apo-2L
trimer structure and is not expected to contact receptor, the data
suggests that divalent metal ion binding may be important to
maintain the trimer structure and stability of Apo-2L.
[0065] In order to map Apo-2 ligand's receptor binding site, amino
acid residues important for receptor binding and biological
activity were identified by alanine-scanning mutagenesis.
[Cunningham et al., Science, 244:1081-1085 (1989)]. Single alanine
substitutions at residues Arg149, Gln20.5, Val207, Tyr216, Glu236,
or Tyr237 resulted in a greater than 5-fold decrease in apoptotic
activity in a bioassay and showed decreased affinity for the
receptors (See Examples 3 and 4). Apo-2L binding to DR4, DR5 and
DcR2 was most affected by alanine substitutions at residues Gln205,
Tyr216, Glu236, or Tyr237, which resulted in at least a 5-fold
decreased affinity against all three receptors. All of these
variants with reduced apoptotic activity also exhibited impaired
binding to either DR4 or DR5 (or both) suggesting that receptor
binding is required for apoptotic activity.
[0066] Alanine substitutions at residues Asp218 and Asp269 resulted
in Apo-2L variants having increased apoptotic activity. (See
Example 4). Residue Asp2.18 is located near Tyr216, which is one of
the required residues for apoptotic activity. A comparison to the
low resolution Apo-2L structure (114-281 form) suggests that the
conformation of the 216-220 loop does not appear to be
significantly altered by the presence of the D218A mutation.
[0067] When the results of the mutagenesis analysis were mapped to
the Apo-2L trimer structure, the functional epitope on Apo-2L for
receptor binding and biological activity was found to be located on
the surface formed by the junction of two monomers (see FIG. 5),
similar to TNF-beta. A shallow groove at the monomer-monomer
interface forms the receptor binding site with both monomers
contributing to the binding site. Residues Arg32, Tyr87, and Asp143
in TNF-alpha (corresponding to Apo-2L residues Arg158, Tyr216, and
Asp267) also make contributions to TNF receptor binding. [Goh et
al., Protein Engineering, 4:785-791 (1991)]. In contrast, residues
of TNF-alpha (corresponding to residues Gln205, Glu236, and Tyr237
of Apo-2L) play only a minor role in TNFR binding. Thus, while for
TNF-alpha the base of the trimer structure makes the most important
contribution to receptor binding, in Apo-2L, important receptor
binding residues are also presented on the top of the trimer
structure. Apo-2L appears to be unique among the TNF family members
of known structure in having a larger and more extended contact
surface for interaction with its target receptors. It is believed
that preferred Apo-2L variants will comprise native residues (i.e.,
will not be mutated) at positions corresponding to Arg149, Gln205,
Val207, Tyr216, Glu236, and/or Tyr237.
[0068] The description below relates to methods of producing Apo-2
ligand by culturing host cells transformed or transfected with a
vector containing Apo-2 ligand encoding nucleic acid and recovering
the polypeptide from the cell culture.
[0069] The DNA encoding Apo-2 ligand may be obtained from any cDNA
library prepared from tissue believed to possess the Apo-2 ligand
mRNA and to express it at a detectable level. Accordingly, human
Apo-2 ligand 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 WO97/25428. The Apo-2
ligand-encoding gene may also be obtained from a genomic library or
by oligonucleotide synthesis.
[0070] Libraries can be screened with probes (such as antibodies to
the Apo-2 ligand 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, such as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual (New York:
Cold Spring Harbor Laboratory Press, 1989). An alternative means to
isolate the gene encoding Apo-2 ligand is to use PCR methodology
[Sambrook et al., supra; Dieffenbach et al., PCR Primer: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
[0071] Amino acid sequence fragments or variants of Apo-2 ligand
can be prepared by introducing appropriate nucleotide changes into
the Apo-2 ligand DNA, or by synthesis of the desired Apo-2 ligand
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 Apo-2 ligand 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 the sequences identified herein for the
intracellular, transmembrane, or extracellular domains of Apo-2
ligand, or the full-length sequence for Apo-2 ligand. The amino
acid changes also may alter post-translational processes of the
Apo-2 ligand, such as changing the number or position of
glycosylation sites or altering the membrane anchoring
characteristics.
[0072] Variations in the Apo-2 ligand 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.
[0073] 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, 244:1081 (1989)].
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., 150:1 (1976)].
[0074] Particular Apo-2L variants of the present invention include
those Apo-2L polypeptides which include one or more of the recited
alanine substitutions provided in TABLE I below. Such Apo-2L
variants will typically comprise a non-naturally occurring amino
acid sequence which differs from a native Apo-2L amino acid
sequence (such as provided in FIG. 1; SEQ ID NO:1, for a full
length or mature form of Apo-2L 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 Apo-2L variant as compared
to a native Apo-2L will comprise amino acid substitution(s) such as
those indicated in Table I. Apo-2L variants of the invention
include soluble Apo-2L 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 recited in TABLE I. Preferred Apo-2L
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 recited in TABLE I which enhance
biological activity, such as receptor binding.
[0075] Variations in the Apo-2 ligand sequence also included within
the scope of the invention relate to amino-terminal derivatives or
modified forms. Such Apo-2 ligand sequences include any of the
Apo-2 ligand 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.
[0076] The nucleic acid (e.g., cDNA or genomic DNA) encoding native
or variant Apo-2 ligand 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
WO97/25428.
[0077] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the Apo-2 ligand 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 Apo-2 ligand 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 Apo-2 ligand 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 Apo-2 ligand promoter sequence and many
heterologous promoters may be used to direct amplification and/or
expression of the Apo-2 ligand DNA.
[0078] Promoters suitable for use with prokaryotic and eukaryotic
hosts are known in the art, and are described in further detail in
WO97/25428.
[0079] A preferred method for the production of soluble Apo-2L in
E. coli employs 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.
[0080] Three inducible promoter systems (T7 polymerase, trp and
alkaline phosphatase (AP)) have been evaluated by Applicants for
the expression of Apo-2L (form 114-281). The use of each of these
three promoters resulted in significant amounts of soluble,
biologically active Apo-2L trimer being recovered from the
harvested cell paste. The AP promoter is preferred among these
three inducible promoter systems tested because of tighter promoter
control and the higher cell density and titers reached in harvested
cell paste.
[0081] 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
re-ligated in the form desired to generate the plasmids required.
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.,
9:309 (1981); Maxam et al., Methods in Enzymology, 65:499
(1980)].
[0082] Expression vectors that provide for the transient expression
in mammalian cells of DNA encoding Apo-2 ligand 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 Apo-2 ligand that are biologically active
Apo-2 ligand.
[0083] Other methods, vectors, and host cells suitable for
adaptation to the synthesis of Apo-2 ligand in recombinant
vertebrate cell culture are described in Gething et al., Nature,
293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP
117,060; and EP 117,058.
[0084] 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. licheniformis (e.g., B. licheniformis 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.
[0085] E. coli is the preferred host cell for use in the present
invention. E. coli is particularly well suited for the expression
of Apo-2 ligand (form 114-281), 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.
[0086] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for Apo-2 ligand-encoding vectors. Suitable host cells for the
expression of glycosylated Apo-2 ligand are derived from
multicellular organisms. Examples of all such host cells, including
CHO cells, are described further in WO97/25428.
[0087] Host cells are transfected and preferably transformed with
the above-described expression or cloning vectors for Apo-2 ligand
production and cultured in nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0088] 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.
[0089] 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 by Shaw et al., Gene, 23:315 (1983) and WO 89/05859
published 29 Jun. 1989. In addition, plants may be transfected
using ultrasound treatment as described in WO 91/00358 published 10
Jan. 1991.
[0090] For mammalian cells without such cell walls, the calcium
phosphate precipitation method of Graham and van der Eb, Virology,
52:456-457 (1978) 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., 130:946
(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829
(1979). 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, 185:527-537 (1990) and Mansour et al., Nature,
336:348-352 (1988).
[0091] Prokaryotic cells used to produce Apo-2 ligand 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 the
Examples below. Mammalian host cells used to produce Apo-2 ligand
may be cultured in a variety of culture media.
[0092] 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.
[0093] 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).
[0094] In accordance with the present invention, one or more
divalent metal ions will typically be added to or included in the
culture media for culturing or fermenting the host cells. The
divalent metal ions are preferably present in or added to the
culture media at a concentration level sufficient to enhance
storage stability, enhance solubility, or assist in forming stable
Apo-2L trimers coordinated by one or more zinc ions. The amount of
divalent metal ions which may be added will be dependent, in part,
on the host cell density in the culture or potential host cell
sensitivity to such divalent metal ions. At higher host cell
densities in the culture, it may be beneficial to increase the
concentration of divalent metal ions. If the divalent metal ions
are added during or after product expression by the host cells, it
may be desirable to adjust or increase the divalent metal ion
concentration as product expression by the host cells increases. It
is generally believed that trace levels of divalent metal ions
which may be present in typical commonly available cell culture
media may not be sufficient for stable trimer formation. Thus,
addition of further quantities of divalent metal ions, as described
herein, is preferred.
[0095] The divalent metal ions are preferably added to the culture
media at a concentration which does not adversely or negatively
affect host cell growth, if the divalent metal ions are being added
during the growth phase of the host cells in the culture. In shake
flask cultures, it was observed that ZnSO.sub.4 added at
concentrations of greater than 1 mM can result in lower host cell
density. Those skilled in the art appreciate that bacterial cells
can sequester metal ions effectively by forming metal ion complexes
with cellular matrices. Thus, in the cell cultures, it is
preferable to add the selected divalent metal ions to the culture
media after the growth phase (after the desired host cell density
is achieved) or just prior to product expression by the host cells.
To ensure that sufficient amounts of divalent metal ions are
present, additional divalent metal ions may be added or fed to the
cell culture media during the product expression phase.
[0096] The divalent metal ion concentration in the culture media
should not exceed the concentration which may be detrimental or
toxic to the host cells. In the methods of the invention employing
the host cell, E. coli, it is preferred that the concentration of
the divalent metal ion concentration in the culture media does not
exceed about 1 mM (preferably, .ltoreq.1 mM). Even more preferably,
the divalent metal ion concentration in the culture media is about
50 micromolar to about 250 micromolar. Most preferably, the
divalent metal ion used in such methods is zinc sulfate. It is
desirable to add the divalent metal ions to the cell culture in an
amount wherein the metal ions and Apo-2 ligand trimer can be
present at a one to one molar ratio.
[0097] The divalent metal ions can be added to the cell culture in
any acceptable form. For instance, a solution of the metal ion can
be made using water, and the divalent metal ion solution can then
be added or fed to the culture media.
[0098] In one embodiment of the invention, the selected Apo-2L
(form 114-281) is expressed in E. coli, and during the culturing or
fermentation of the cell culture, the process parameters are set
such that cellular activities are conducted at oxygen uptake rates
of approximately 1.0 to 3.0 mmoles/L-min for cultures at
approximately 40-50 gm/L dry cell weight. It is preferred that the
newly synthesized nascent Apo-2L polypeptides have sufficient time
for proper protein folding and trimerization of Apo-2L monomers.
The growth phase of the fermentation process is preferably
conducted at 30.degree. C. Just prior to the commencement of
product expression, the process temperature control set-point may
remain at 30.degree. C. or be down-shifted to 25.degree. C. for the
rest of the fermentation. Optionally, it may be desired to increase
cell density in the cell culture, and the above-mentioned
parameters may be adjusted (or increased) accordingly. For
instance, it may be advantageous to increase cell density in the
cell culture to increase volumetric yield. One skilled in the art
can, by using routine techniques known in the art, incrementally
increase the cell density and incrementally increase the
above-mentioned parameters, if desired.
[0099] Expression of the Apo-2L 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, 77:5201-5205 (1980)], 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. 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.
[0100] 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 Apo-2 ligand polypeptide or against a
synthetic peptide based on the DNA sequences provided herein or
against exogenous sequence fused to Apo-2 ligand DNA and encoding a
specific antibody epitope.
[0101] Apo-2 ligand preferably is recovered from the culture medium
as a secreted polypeptide, although it also may be recovered from
host cell lysates when directly produced without a secretory
signal. If the Apo-2 ligand is membrane-bound, it can be released
from the membrane using a suitable detergent solution (e.g.
Triton-X 100) or its extracellular region may be released by
enzymatic cleavage.
[0102] When Apo-2 ligand is produced in a recombinant cell other
than one of human origin, the Apo-2 ligand is free of proteins or
polypeptides of human origin. However, it is usually necessary to
recover or purify Apo-2 ligand from recombinant cell proteins or
polypeptides to obtain preparations that are substantially
homogeneous as to Apo-2 ligand. As a first step, the culture medium
or lysate may be centrifuged to remove particulate cell debris.
Apo-2 ligand thereafter is purified from contaminant soluble
proteins and polypeptides, with the following procedures being
exemplary of suitable purification procedures: by fractionation on
an ion-exchange column; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation-exchange resin such as DEAE
or CM; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
gel filtration using, for example, Sephadex G-75; diafiltration and
protein A Sepharose columns to remove contaminants such as IgG.
[0103] In a preferred embodiment, the Apo-2 ligand can be isolated
by affinity chromatography. Apo-2 ligand fragments or variants in
which residues have been deleted, inserted, or substituted are
recovered in the same fashion as native Apo-2 ligand, taking
account of any substantial changes in properties occasioned by the
variation. For example, preparation of an Apo-2 ligand fusion with
another protein or polypeptide, e.g., a bacterial or viral antigen,
facilitates purification; an immunoaffinity column containing
antibody to the antigen can be used to adsorb the fusion
polypeptide.
[0104] A protease inhibitor such as phenyl methyl sulfonyl fluoride
(PMSF) also may be useful to inhibit proteolytic degradation during
purification, and antibiotics may be included to prevent the growth
of adventitious contaminants. One skilled in the art will
appreciate that purification methods suitable for native Apo-2
ligand may require modification to account for changes in the
character of Apo-2 ligand or its variants upon expression in
recombinant cell culture.
[0105] During any such purification steps, it may be desirable to
expose the recovered Apo-2L to a divalent metal ion-containing
solution or to purification material (such as a chromatography
medium or support) containing one or more divalent metal ions. In a
preferred embodiment, the divalent metal ions and/or reducing agent
is used during recovery or purification of the Apo-2L. Optionally,
both divalent metal ions and reducing agent, such as DTT or BME,
may be used during recovery or purification of the Apo-2L. It is
believed that use of divalent metal ions during recovery or
purification will provide for stability of Apo-2L trimer or
preserve Apo-2L trimer formed during the cell culturing step.
[0106] A preferred method of recovering and purifying the expressed
Apo-2L from prokaryotic host cells (most preferably from bacterial
host cells) comprises the following steps: (a) extracting Apo-2L
(intracellular) from E. coli cells; (b) stabilizing the properly
folded Apo-2L in a buffer solution comprising divalent metal ions
and/or reducing agent; (c) purifying the Apo-2L by chromatography
using, sequentially, a cationic exchanger, a hydroxyapatite and a
hydrophobic interaction chromatograph, and (d) selectively eluting
Apo-2L in a buffer solution comprising divalent metal ions and/or
reducing agent from each such chromatographic support. The divalent
metal ions and the reducing agent utilized in such methods may
include a Zn sulfate, Zn chloride, Co sulfate, DTT and BME, and
more preferably, a Zn sulfate or DTT. An even more detailed
description of this recovery and purification process is provided
in Example 8 below.
[0107] As discussed above, such methods of the invention are
applicable and useful for various other proteins, besides Apo-2L,
which have improved activity when in a trimerized form or which
require trimerization of the protein for activity.
[0108] Formulations comprising Apo-2 ligand and one or more
divalent metal ions are also provided by the present invention. It
is believed that such formulations will be particularly suitable
for storage (and maintain Apo-2L trimerization), as well as for
therapeutic administration. Preferred formulations will comprise
Apo-2L and zinc or cobalt. More preferably, the formulation will
comprise an Apo-2L and zinc or cobalt solution in which the metal
is at a <2.times. molar ratio to the protein. If an aqueous
suspension is desired, the divalent metal ion in the formulation
may be at a >2.times. molar ratio to the protein. Using zinc
sulfate, Applicants have found Apo-2L (form 114-281) precipitates
and forms an aqueous suspension at about a 100 mM concentration of
zinc sulfate in the formulation. Those skilled in the art will
appreciate that at a >2.times. molar ratio, there may be an
upper range of concentration of the divalent metal ion in the
formulation at which the metal can become deleterious to the
formulation or would be undesirable as a therapeutic
formulation.
[0109] The formulations may be prepared by known techniques. For
instance, the Apo-2L formulation may be prepared by buffer exchange
on a gel filtration column.
[0110] Typically, an appropriate amount of a
pharmaceutically-acceptable salt is used in the formulation to
render the formulation isotonic. Examples of
pharmaceutically-acceptable carriers include saline, Ringer's
solution and dextrose solution. The pH of the formulation is
preferably from about 6 to about 9, and more preferably from about
7 to about 7.5. Preferably, the pH is selected so as to ensure that
the zinc remains bound to the Apo-2L. If the pH is too high or too
low, the zinc does not remain bound to the Apo-2L and as a result,
dimers of Apo-2L will tend to form. It will be apparent to those
persons skilled in the art that certain carriers may be more
preferable depending upon, for instance, the route of
administration and concentrations of Apo-2 ligand and divalent
metal ions.
[0111] Therapeutic compositions of the Apo-2L can be prepared by
mixing the desired Apo-2L molecule having the appropriate degree of
purity with optional pharmaceutically acceptable carriers,
excipients, or stabilizers (Remington's Pharmaceutical Sciences,
16th edition, Oslo, A. ed. (1980)), in the form of lyophilized
formulations, aqueous solutions or aqueous suspensions. Acceptable
carriers, excipients, or stabilizers are preferably nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as Tris, HEPES, PIPES, phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0112] Additional examples of such carriers include ion exchangers,
alumina, aluminum stearate, lecithin, serum proteins, such as human
serum albumin, buffer substances such as glycine, sorbic acid,
potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts, or electrolytes such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, and cellulose-based substances.
Carriers for topical or gel-based forms include polysaccharides
such as sodium carboxymethylcellulose or methylcellulose,
polyvinylpyrrolidone, polyacrylates,
polyoxyethylene-polyoxypropylene-block polymers, polyethylene
glycol, and wood wax alcohols. For all administrations,
conventional depot forms are suitably used. Such forms include, for
example, microcapsules, nano-capsules, liposomes, plasters,
inhalation forms, nose sprays, sublingual tablets, and
sustained-release preparations.
[0113] Effective dosages of Apo-2 ligand in the formulations may be
determined empirically, and making such determinations is within
the skill in the art. It is presently believed that an effective
dosage or amount of Apo-2 ligand may range from about 1
microgram/kg to about 100 mg/kg of body weight or more per day.
Interspecies scaling of dosages can be performed in a manner known
in the art, e.g., as disclosed in Mordenti et al., Pharmaceut.
Res., 8:1351 (1991). Those skilled in the art will understand that
the dosage of Apo-2 ligand that must be administered will vary
depending on, for example, the mammal which will receive the Apo-2
ligand, the route of administration, and other drugs or therapies
being administered to the mammal.
[0114] Apo-2L to be used for in vivo administration should be
sterile. This is readily accomplished by filtration through sterile
filtration membranes, prior to or following lyophilization and
reconstitution. Apo-2L ordinarily will be stored in lyophilized
form or in solution if administered systemically. If in lyophilized
form, Apo-2L is typically formulated in combination with other
ingredients for reconstitution with an appropriate diluent at the
time for use. An example of a liquid formulation of Apo-2L is a
sterile, clear, colorless unpreserved solution filled in a
single-dose vial for subcutaneous injection.
[0115] Therapeutic Apo-2L formulations generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle. The formulations are preferably administered as
repeated intravenous (i.v.), subcutaneous (s.c.), intramuscular
(i.m.) injections or infusions, or as aerosol formulations suitable
for intranasal or intrapulmonary delivery (for intrapulmonary
delivery see, e.g., EP 257,956).
[0116] Apo-2L 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 polyesters,
hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) as described by
Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and
Langer, Chem. Tech., 12: 98-105 (1'982) 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, 22: 547-556 (1983)), 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).
[0117] The Apo-2L and its formulations described herein can be
employed in a variety of therapeutic and non-therapeutic
applications. Among these applications are methods of treating
various cancers (provided above) and viral conditions. Such
therapeutic and non-therapeutic applications are described, for
instance, in WO97/25428 and WO97/01633.
[0118] An article of manufacture such as a kit containing Apo-2L
useful for the diagnosis or treatment of the disorders described
herein comprises at least a container and a label. 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 Apo-2L formulation
that is effective for diagnosing or treating the condition 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 condition of choice. The article of manufacture may
further comprise a second container comprising a
pharmaceutically-acceptable buffer, such as phosphate-buffered
saline, Ringer's solution, and 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. The
article of manufacture may also comprise a second or third
container with another active agent as described above.
[0119] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0120] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0121] 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
Expression and Purification of Apo-2L Variants
[0122] Alanine substitution variants of Apo-2L were constructed by
oligonucleotide-directed mutagenesis (Kunkel et al., Proc. Natl.
Acad. Sci., 82:488-492 (1985); Kunkel, Methods in Enzymology,
154:367-382 (1987)) of a plasmid (pAPOK5) (see FIG. 13), designed
for the intracellular E. coli expression of the 91-281 amino acid
form of Apo-2L under control of the trp promoter. pAPOK5 was
constructed by using PCR to clone the Apo-2L cDNA (encoding
residues 91-281) into plasmid pS1162 which carries the trp
promoter. E. coli strain 294 transformed with the mutated plasmids
were grown to mid-log phase at 37.degree. C. in 250 mL M9 media
plus 100 .mu.M ZnSO.sub.4, expression was induced by addition of 25
.mu.g/mL beta-indole acrylic acid, and the cultures were grown
overnight at 30.degree. C. Cells were harvested by centrifugation
and frozen.
[0123] The cell pellet was homogenized in 6 volumes 0.1 M Tris-HCl
pH 8, 0.2 M NaCl, 5 mM DTT, 1 mM EDTA, and Apo-2L was isolated from
the soluble fraction by IMAC on a chelating hiTRAP column
(Pharmacia) charged with nickel. The Apo-2L had a weak affinity for
immobilized metal and could be eluted with low concentrations of
imidazole. A final purification was obtained by cation exchange
chromatography on a SP hiTRAP column (Pharmacia). Concentrations of
purified Apo-2L variants were determined by absorbance measurements
using an e.sub.280 of 1.4 mg.sup.-1 ml cm.sup.-1.
[0124] The Apo-2L variants identified by the
oligonucleotide-directed mutagenesis are listed in Table I.
TABLE-US-00001 TABLE I Receptor binding and apoptotic activity of
Apo2L variants.sup.a Ratio (variant/wild-type) DR4-IgG DR5-IgG
DcR2-IgG Apoptosis Variant K.sub.D K.sub.D K.sub.D ED.sub.50
.DELTA.zinc 6.3 6.6 11.2 90.0 R130A 3 2.7 1.3 1.9 N134A 1.0 0.8 1.0
1.5 L136A 3.3 1.5 1.4 0.8 S138A 2.1 1.3 2.2 1.2 N140A 1.4 1.9 0.9
1.1 S141A 2.3 1.3 2.4 1.3 K142A 2.6 1.9 2.7 2.0 N143A 2.1 2.0 1.3
1.5 R149A 1.8 2.2 1.6 3.5 S153A 2.3 1.2 2.1 0.9 E155A 1.6 2 1.4 2.5
R158A 2.4 1.3 6.5 1.4 S159A 4.7 2.2 3.4 0.9 R170A 1.1 2.2 0.6 0.9
K179A 0.9 0.9 1.1 2.0 R191A 7.8 3.9 3.2 2.2 Q193A 1.7 1.1 1.2 2.2
E195A 4.6 1.4 2.6 0.8 K197D 2 2.1 2.9 1.1 K201A 4.3 2.7 10 2.5
N202A 2.5 2.5 1.9 3.2 D203A 1.5 1.1 0.6 0.5 Q205A 13.1 6.3 10.8 690
V207A 2.2 2.8 2.1 5.6 Y213A 1.3 1 1.5 1.2 Y216A 14.5 8.9 9.0 320
D218A 1.3 1.9 1.1 0.3 C230S 4.1 7.1 6.7 8.0 E236A 6.0 9.8 8.4 10.8
Y237A 7.3 5.0 48 8.3 Y240A 1.8 0.8 1.8 1.1 K251A 1.9 2 2.4 0.8
S259A 4.3 2.0 1.4 3.3 H264A 1.9 2.0 1.4 3.1 D267A 5.7 1.9 5.5 1.11
D269A 1.7 0.5 0.9 0.2 .sup.aValues shown represent the ratio of
variant to wild-type. For wild-type Apo-2L (residues 91-281), the
Kd values for DR4-IgG, DR5-IgG and DcR2-IgG are 0.8 .+-. 0.3 nM,
0.9 .+-. 0.4 nM, and 0.3 .+-. 0.2 nM. Wild-type Apo-2L (residues
91-281) gave an # ED50 of 24 .+-. 3.1 ng/mL in the apoptosis assay
while the 114-281 form of Apo-2L was slightly more active and gave
an ED.sub.50 of 16.0 .+-. 3.6 ng/ml. Only 2-fold changes from
wild-type values are considered to be significant.
Example 2
Crystallography Analysis of Apo-2L
[0125] Crystals of Apo-2L (amino acid residues 114-281) were grown
in 70 uL sitting drops containing 40 uL protein (at 2.6 mg/mL in 20
mM Tris, pH8.0), 20 uL 50 mM Tris pH 8.0, and 10 uL 8% peg 2K MME
over a well solution of 50% peg 2K MME at 20.degree. C. and were
members of the spacegroup P63 with two monomer in the asymmetric
unit and unit cell constants a=72.5, c=140 Angstrom and diffract to
3.9 Angstrom at room temperature. Crystals of D218A variant (see.
Example 1) grew in 14 uL sitting drops containing 4 uL of 4% MPD
and 10 uL protein (1.7 mg/ml in 20 mM Tris pH 7.5) over a well
solution of 32% MPD at 4.degree. C. and were members of the
spacegroup R32 with one monomer per asymmetric unit and unit cell
parameters 66.4, c=197.7 Angstrom and diffracted to 1.3 Angstrom at
-180.degree. C. with synchroton radiation. Data sets diffracting to
3.9 Angstrom for the Apo-2L (residues 114-281) crystals and 1.9
Angstrom for the D218A variant were measured on a Rigaku rotating
anode x-ray generator equipped with a MAR detector and processed
with DENZO/SCALEPACK [Otwinowski et al., Proceedings of the CCP4
Study Weekend:Data Collection and Processing (eds. Sawyer et al.)
pp. 56-62 Daresbury Laboratory, Daresbury, England, 1993]. A 1.3
Angstrom data set for the D218A variant was measured at the
Advanced Photon Source at Argonne National Labs and was processed
with HKL2000/SCALEPACK and had a Rsym of 6.4% (34% in the 1.35-1.30
shell), with 100% completeness and a redundancy of 12-fold, and
I/<I>=12.4.
[0126] The native Apo-2L structure was solved by molecular
placement using a model based on TNF-alpha (pdb code 1TNF) with the
program Amore [Acta Cryst., D50:760-763 (1994)] and was refined
[Brunger, X-PLOR:version 3.1, Yale Press, New Haven 1987] with
strict 2-fold non-crystallographic restraints until a R.sub.free of
35%. This structure refined against the 1.9 Angstrom dataset until
a R.sub.free of 25% and finally was refined against 1.3 Angstrom
data with Refmac and SHELXL [Sheldrick et al., Methods in
Enzymology, pp. 319-343, Academic Press, San Diego 1997] of
R.sub.free=22% and R.sub.factor of 20% with good geometry (rmsd
bonds 0.011 Angstrom, rmsd angle 1.7.degree.). All residues fall
within the allowed regions of a Ramachandran plot. During
refinement, a 28 sigma peak of electron density was observed
between symmetry related Cys230 on the trimer axis. This density
was modeled as a zinc ion and refined with B-factor of 10. It is
believed that a chlorine molecule on the trimer axis is present as
the fourth ligand to the zinc. The final model consists of residues
120-130, 142-194, 203-281 with 170 solvent molecules and one zinc
ion and one chloride ion. Residues 91-119, 131-141, and 195-202 are
disordered. N-terminal sequencing of several crystals confirmed
that the N-terminus is intact while mass spectrometry of the
starting material shows that it is full length.
[0127] A summary of the crystallographic data is provided in FIG.
2C.
Example 3
[0128] Determination of Receptor Binding Affinity of Apo-2L
Variants
[0129] Dissociation constants (Kd) for binding of Apo-2L variants
(see Table I) to immobilized receptor immunoadhesins were
determined from surface plasmon resonance (SPR) measurements on a
Pharmacia BIAcore 1000.
[0130] DR5-IgG (also referred to as Apo-2-IgG) and DcR2-IgG
receptor immunoadhesins were prepared as described in WO98/51793
published Nov. 19, 1998 and WO 99/10484 published Mar. 9, 1999,
respectively. DR4-IgG was prepared as follows. A mature DR4 ECD
sequence (amino acids 1-218; Pan et al., supra) was cloned into a
pCMV-1 Flag vector (Kodak) downstream of the Flag signal sequence
and fused to the CH1, hinge and Fc region of human immunoglobulin
G.sub.1 heavy chain as described previously [Aruffo et al., Cell,
61:1303-1313 (1990)]. The immunoadhesin was expressed by transient
transfection into human 293 cells and purified from the cell
supernatants by protein A affinity chromatography, as described by
Ashkenazi et al., Proc. Natl. Acad. Sci., 88:10535-10539
(1991)].
[0131] The receptor immunoadhesin proteins were coupled to the
sensor chip surface at a level of 1000-2000 resonance units using
amine coupling chemistry (Pharmacia Biosensor). Sensorgrams were
recorded for Apo-2L binding at concentrations ranging from 15.6 nM
to 500 nM in 2-fold increments. The kinetics constants were
determined by non-linear regression analysis and used to calculate
the binding constants.
[0132] The results are shown in Table I.
Example 4
[0133] Apoptotic Activity of Apo-2L Variants In Vitro
[0134] A bioassay which measures cell viability from the metabolic
conversion of a fluorescent dye was used to determine the apoptotic
activity of Apo-2L variants. Serial 2-fold dilutions of Apo-2L
(form 114-281) or Apo-2L variants (see Table I) were made in
RPMI-1640 media (Gibco) containing 0.1% BSA, and 50 .mu.L of each
dilution was transferred to individual wells of 96-well Falcon
tissue culture microplates. 50 .mu.L of SK-MES-1 human lung
carcinoma cells (ATCC HTB58) (in RMPI-1640, 0.1%. BSA) were added
at a density of 2.times.10.sup.4 cells/well. These mixtures were
incubated at 37.degree. C. for 24 hours. At 20 hours, 25 .mu.L of
alamar Blue (AccuMed, Inc., Westlake, Ohio) was added. Cell number
was determined by measuring the relative fluorescence at 590 nm
upon excitation at 530 nm. These data were analyzed by using a 4
parameter fit to calculate ED.sub.50, the concentration of Apo-2L
giving a 50% reduction in cell viability.
[0135] Single alanine substitutions at residues Arg149, Gln205,
Val207, Tyr216, Glu236 or Tyr237 resulted in a greater than 5-fold
decrease in apoptotic activity in the bioassay and showed decreased
affinity for DR4, DR5 and DcR2 (Table I). Apo-2L binding to these
receptors was most affected by alanine substitution of Gln205,
Tyr216, Glu236 and Tyr237, all of which resulted in at least a
5-fold decreased affinity for all three receptors. All of the
Apo-2L variants with reduced apoptotic activity also exhibited
impaired binding to either DR4 or DR5 (or both) suggesting that
receptor-binding is required for the biological effect. Alanine
substitution of Asp218 or Asp269 resulted in a greater than 2-fold
increase in apoptotic activity. It is noteworthy that most alanine
substitutions have similar effects on both DR4 and DR5 binding, the
only exceptions being mutation of Gln193, Glu195, Ser259, His264,
and Asp267, all of which had a greater than 5-fold effect on DR4
binding (decreasing affinity) but only a small or negligible effect
on DR5 binding.
[0136] Changes in DcR2-binding tended to parallel the effects
observed for DR4-binding. Diminished apoptotic activity appears to
be most closely linked with decreased DR5-binding suggesting that
DR5 is required for death signaling in SK-MES in response to Apo-2L
administration.
Example 5
[0137] Elemental and Quantitative Analysis to Determine Metal
Content of Apo-2L
[0138] Elemental analysis of Apo-2L was performed by using
inductively coupled plasma atomic emission spectrometry (ICP-AES).
For this determination, a 2 mg/mL solution of Apo-2L (residues
114-281 produced in E. coli using methods disclosed in WO97/25428;
additional quantities of divalent metal ions were not added during
fermentation or purification in accordance with the methods
described herein) formulated in 20 mM Tris pH 7.5 was used. Levels
of Cd, Co, Zn, Ni, and Cu in this sample and in a portion of the
formulation buffer were determined. TABLE-US-00002 TABLE II Sample
Cd Co Zn Ni Cu Buffer -0.058 -0.090 -0.098 -0.098 -0.082 Apo-2L
-0.058 0.199 1.712 -0.108 -0.075
[0139] The metals bound to the Apo-2L were Zn and Co (Table II).
The calculated molar ratios were 0.79 moles of Zn per mole of
Apo-2L trimer and 0.06 moles of Co per mole of Apo-2L trimer. These
data indicate Apo-2L has one zinc binding site per trimer. The site
is 85% occupied with metal in this preparation of Apo-2L.
Example 6
[0140] Effects of Removal of Bound Zinc from Apo-2L Using Chelating
Agents
[0141] A sample of Apo-2L (form 114-281) was treated with chelating
agents to remove the bound zinc. The sample was first dialyzed
against 2 changes of 1000 volumes each of 50 mM EDTA, then against
2 changes of 1000 volumes of 2 mM 1,10-phenanthroline, and finally
against 1000 volumes of metal-free 20 mM Tris pH 7.5. The sample,
before and after the chelating treatment, was assayed for
receptor-binding, metal content and apoptotic activity. Receptor
binding was measured as described in Example 3, metal content was
determined by ICP-AES and apoptotic activity was assayed as
described in Example 4. ICP-AES showed that the dialysis procedure
removed the bound zinc. After this treatment, the receptor affinity
was significantly reduced (Table I) and the apoptotic activity was
decreased 90-fold (FIG. 4).
[0142] Circular dichroic spectra were recorded on an AVIV
instrument (Lakewood, N.J.) model 202 spectropolarimeter. The
spectrum was scanned from 250 to 200 nm using a step size of 0.5
nm, an averaging time of 5 seconds, and quartz rectangular cuvettes
having a pathlength of 1 cm. The protein concentration was 2 .mu.M
in solutions containing PBS. As shown in FIG. 6, Apo-2L (form
114-281) gives a CD spectrum typical of a protein having a high
beta-sheet content. Removal of the bound zinc results in a
decreased intensity of the dichroic peaks suggesting that the
beta-sheet content has been diminished.
[0143] Circular dichroism was used to monitor the effect of zinc
removal on the thermal stability of Apo-2L (form 114-281). These
experiments also used 1 cm quartz rectangular cuvettes and a
protein concentration of 2 .mu.M. Circular dichroism at 225 nm was
monitored as the sample was heated from 30 to 100.degree. C.
Measurements were taken at 2.degree. C. increments after allowing
the sample to equilibrate at that temperature for 1 minute. Both
the ellipticity (CD) and dynode voltage were recorded and the
temperature dependence of the dynode voltage is plotted in FIG. 7.
The dynode voltage is proportional to the absorbance of the sample.
An increase in dynode voltage upon heating of the sample reflects
protein aggregation. The increase in dynode voltage was concomitant
with a loss of secondary structure as indicated by a decrease in
the ellipticity at 225 nm. These data suggest that Apo-2L (form
114-281) aggregates upon thermal denaturation with the midpoint for
this transition (Tm) occurring at about 75.degree. C. Removal of
the bound zinc results in a large decrease in the Tm for thermal
denaturation to about 54.degree. C. These data show that the bound
zinc is necessary for maintenance of the structure and stability of
homotrimeric Apo-2L.
Example 7
[0144] Effects of Removal of Zinc Binding Site from Apo-2L by
Mutation
[0145] Cys230 of Apo-2L (form 91-281) was replaced with Ala or Ser
by using oligonucleotide-directed mutagenesis as described in
Example 1. The variant proteins were then expressed and purified as
described in Example 1. As shown in Table I, both the C230A and
C230S mutants of Apo-2L (form 91-281) had reduced receptor-binding
affinity and greatly diminished-apoptotic activity. Since the
Apo-2L x-ray structure shows that Cys230 is a buried residue, and
thus is unlikely to directly contact receptor upon complex
formation, these data suggest that mutation of Cys230 indirectly
affects activity by changes in the structure or stability of
homotrimeric Apo-2L.
Example 8
Additions of Zn Improves Soluble Apo-2 Ligand Product Accumulation
and Recovery
A. Apo-2L (Amino Acid Residues 114-281) Expression Regulated by the
Alkaline Phosphatase Promoter
[0146] pAPApo2-P2RU (see FIG. 12) encodes for the co-expression of
Apo-2L (amino acid residues 114-281) and the tRNA's encoded by pro2
and argU. The pBR322-based plasmid [Sutcliffe, Cold Spring Harbor
Symp. Quant. Biol., 43:77-90 (1978)] pAPApo2-P2RU was used to
produce the Apo-2L in E. coli. The transcriptional and
translational sequences required for the expression of Apo-2L are
provided by the alkaline phosphatase promoter and the trp
Shine-Dalgarno, as described for the plasmid phGH1 [Chang et al.,
Gene, 55:189-196 (1987)]. The coding sequence for Apo-2L (form
114-281) is located downstream of the promoter and Shine-Dalgarno
sequences and is preceded by an initiation methionine. The coding
sequence includes nucleotides (shown in FIG. 1) encoding residues
114-281 of Apo-2L (FIG. 1) except that the codon encoding residue
Pro119 is changed to "CCG" instead of "CCT" in order to eliminate
potential secondary structure. The sequence encoding the lambda to
transcriptional terminator [Scholtissek et al., Nucleic Acids Res.,
15:3185 (1987)] follows the Apo-2L coding sequence. Additionally,
this plasmid also includes sequences for the expression of the
tRNA's pro2 [Komine et al., J. Mol. Biol., 212:579-598 (1990)] and
argU/dnaY [Garcia et al., Cell, 45:453-459 (1986)]. These genes
were cloned by PCR from E. coli w3110 and placed downstream of the
lambda to transcriptional terminator sequence. This plasmid confers
both tetracycline and ampicillin resistance upon the production
host.
[0147] Strain 43E7 (E. coli W3110 fhuA(tonA) phoA .DELTA.(argF-lac)
ptr3 degP kanS ompT ilvG+) was used as the production host for the
co-expression of the Apo-2 ligand and the tRNA's. Competent cells
of 43E7 were prepared and transformed with pAPApo2-P2RU using
standard procedures. Transformants were picked from LB plates
containing 20 .mu.g/ml tetracycline (LB+Tet20), streak-purified,
and grown in LB broth with 20 .mu.g/ml tetracycline in a 30.degree.
C. shaker/incubator before being stored in DMSO at -80.degree.
C.
[0148] A shake flask inoculum was prepared by inoculating sterile
medium using a freshly thawed stock culture vial. Appropriate
antibiotics were included in the medium to provide selective
pressure to ensure retention of the plasmid. The shake flask medium
composition is given in Table III. Flask cultures were incubated
with shaking at about 30.degree. C. (28.degree. C.-32.degree. C.)
for 14-18 hours. This culture was then used to inoculate the
production fermentation vessel. The inoculation volume was between
0.1% and 10% of the initial volume of medium. TABLE-US-00003 TABLE
III Shake Flask Medium Composition Ingredient Quantity/Liter
Tetracycline 4-20 mg Tryptone 8-12 g Yeast extract 4-6 g Sodium
chloride 8-12 g Sodium phosphate, added as pH 7 solution 4-6
mmol
[0149] Production of the Apo-2L was carried out in the production
medium given in Table IV. The fermentation process was conducted at
about 30.degree. C. (28-32.degree. C.) and pH controlled at
approximately 7.0 (6.5-7.5). The aeration rate and the agitation
rate were set to provide adequate transfer of oxygen to the
culture. At the onset of product expression, induced by phosphate
depletion, the process temperature was shifted from 30.degree. C.
to 25.degree. C. Throughout the fermentation process, the cell
culture was fed glucose based on a computer algorithm to meet its
carbon requirement while ensuring aerobic condition.
[0150] Two batch additions of ZnSO.sub.4 were made during the
fermentation process. One addition was made just prior to the
induction of product expression. The second addition was made at
approximately the mid-point of the production period. In this
example, the additions occurred at a culture optical density of
about 80-120 OD.sub.550 and at about 28 hours post-inoculation.
Sufficient amounts of 100 mM ZnSO.sub.4 were added to achieve
approximately 50-100 micromolar (final concentration) with each
batch addition of the metal ions.
[0151] The fermentation was allowed to proceed for about 34-45
hours, after which the cell paste was harvested from the broth for
subsequent product recovery evaluation. TABLE-US-00004 TABLE IV
Production Medium Composition for AP Promoter Expression System
Ingredient Quantity/Liter Tetracycline 4-20 mg Glucose.sup.a 10-250
g Ammonium sulfate.sup.a 2-8 g Sodium phosphate, monobasic,
dihydrate.sup.a 1-5 g Potassium phosphate, dibasic.sup.a 1-5 g
Potassium phosphate, monobasic.sup.a 0-5 g Sodium citrate,
dihydrate.sup.a 0.5-5 g Potassium chloride 0-5 g Magnesium sulfate,
heptahydrate.sup.a 1.0-10 g Antifoam 0-5 ml Ferric chloride,
hexahydrate.sup.a 20-200 mg Zinc sulfate, heptahydrate.sup.a 0.2-20
mg Cobalt chloride, hexahydrate.sup.a 0.2-20 mg Sodium molybdate,
dihydrate.sup.a 0.2-20 mg Cupric sulfate, pentahydrate.sup.a 0.2-20
mg Boric acid.sup.a 0.2-20 mg Manganese sulfate, monohydrate.sup.a
0.2-20 mg Casein hydrolysate.sup.a 5-25 g Yeast extract.sup.a 5-25
g .sup.aA portion of these ingredients may be fed to the culture
during the fermentation. Ammonium hydroxide was added as required
to control pH.
[0152] Broth samples were taken over the time course of the
fermentation process. Cells from 1 ml of broth samples diluted to a
cell density of 20 OD.sub.550 were collected by centrifugation and
the resultant cell pellets were stored at -20.degree. C. until
analysis. The cell pellets were thawed and resuspended in 0.5 ml of
extraction buffer (50 mM HEPES, pH 8.0, 50 mM EDTA and 0.2 mg/ml
hen egg-white lysozyme) and mechanically disrupted to release the
product from the cytoplasm. Solids were removed from the cell
lysates by centrifugation before the clarified lysates were loaded
onto a Dionex ProPac IEX HPLC column for trimer quantitation. The
HPLC assay method resolves the product away from the contaminating
E. coli proteins by use of a 5%-22% gradient of 1M NaCl in a 25 mM
phosphate (pH 7.5) buffer over 25 minutes at a flow rate of 0.5
ml/min.
[0153] Frozen cell paste was thawed and resuspended in extraction
buffer (100 mM Hepes buffer, pH 8.0, 50 mM EDTA, 5 mM DTT). After
multiple passes of the cell suspension through a mechanical
homogenizer to release the Apo-2L product from the cytoplasmic
compartment, 0.2% PEI (final concentration) was added and the
solids were removed by centrifugation. The clarified lysate was
diluted 1:1 (v/v) with H.sub.20 and pH adjusted to 7.2 prior to
loading onto the MPHS column (BioRad) pre-equilibrated with 3-4
column volumes of 50 mM Hepes/0.05% Triton/1 mM DTT pH 7.2. After a
wash step with the 2-3 column volumes of equilibration buffer
followed by a second wash step with 0.1M NaCl in the equilibration
buffer, Apo-2L proteins were eluted off the MPHS column with 6
column volumes of a 0.1M to 0.8M NaCl gradient in the equilibration
buffer. Column eluant fractions were collected, analyzed and the
relevant fractions were pooled and stored @ 4-8.degree. C.
[0154] Analysis of the Apo-2L accumulation during fermentation
showed that the ZnSO.sub.4 additions did not significantly affect
cell growth. The production of Apo-2 ligand began when the
phosphate in the medium was depleted, typically about 15-25 hours
after inoculation. FIG. 8 shows the time course in the accumulation
of soluble Apo-2L trimers detected by the IEX HPLC method. Cultures
with ZnSO.sub.4 additions had higher product concentration in the
cell lysate samples than the minus-ZnSO.sub.4 controls.
[0155] Analysis of the product recovery at the initial capture step
(involving IEX Chromatography) (FIG. 9) shows the elution profiles
of the MPHS chromatography of cell lysates from fermentations
conducted in the absence and presence of ZnSO.sub.4 additions.
After the initial flow-through and wash steps, two main peaks, Peak
A and Peak B, were resolved. By SDS-PAGE analysis, both peaks
consisted of mainly Apo-2L.
[0156] Purified material from both peaks was prepared and analyzed
for biological activity and stability. Results obtained from these
studies suggested that Peak A was a more stable pool of Apo-2L
product while Peak B had a greater tendency to aggregate over time.
To minimize instability, Peak B was excluded for further recovery.
The ratio of Peak A to Peak B was estimated by weighing the cut out
traces representing the integrated area under each of the peaks.
Results tabulated in Table V show a shift of the percent of Apo-2L
as Peak A from approximately 45% on the average for the
minus-ZnSO.sub.4 case to approximately 80% for the plus-ZnSO.sub.4
case, a significant increase in the amount of Apo-2L product in the
recoverable pool. TABLE-US-00005 TABLE V ZnSO4 Chromatography Run
ID Additions Scale Peak A % Peak B % SAPO2-113 No 0.66 .times. 15.5
cm 41.6 58.4 LAPO2-4 No 4.4 .times. 41.5 cm 50.4 49.6 SAPO2-138 Yes
0.66 .times. 17.0 cm 78.8 21.2
B. Apo-2L (Amino Acid Residues 114-281) Expression Regulated by the
trp Promoter:
[0157] PS1346.Apo2L.0 Plasmid Construction: DNA encoding residues
114-281 of Apo-2L (preceded by an initiating methionine codon) was
inserted into a pS1346 plasmid vector. The pS1346 plasmid is a
derivative of pHGH207-1 [DeBoer et al., Promoters: Structure and
Function, Praeger, New York, pp. 462-481 (1982)] and contains the
lambda- to transcriptional terminator [Scholtissek et al., Nucleic
Acids Res., 15:3185 (1987)] downstream of the Apo-2L encoding
sequence.
[0158] Strain 54C2 (E. coli W31-10 fhuA(tonA) lon galE
rpoHts(htpRts) clpP lacIq) was used as the production host for the
expression of Apo-2 ligand (amino acid residues 114-281) where the
ligand expression was regulated by the trp promoter. Competent
cells of 54C2 were prepared and transformed with pS1346.Apo2L.0
using standard procedures. Transformants were picked from LB plates
containing 20 .mu.g/ml tetracycline (LB+Tet20), streak purified,
and grown in LB broth with 20 .mu.g/ml tetracycline in a 30.degree.
C. shaker/incubator before being stored in DMSO at -80.degree.
C.
[0159] Experiments conducted with the production organism,
54C2/pS1346.Apo2L.0, were performed under similar fermentation
parameters as described above for the AP-promoter expression system
except for minor adjustments in the medium composition (Table VI),
the addition of an inducer, indole3-acrylic acid (IAA), and the
length of the process. Tryptophan was added to the initial medium
to repress promoter activity during the initial growth phase. The
temperature shift from 30.degree. C. to 25.degree. C. was made when
cell density of the broth reached approximately 30 OD.sub.550. The
inducer was added when the cell density of the broth reached
approximately 55 OD.sub.550. In the experiments where ZnSO.sub.4
additions were made, sufficient amounts of 100 mM ZnSO.sub.4
solution were added at cell density of 25 OD.sub.550 and at 24
hours post-inoculation to achieve a final concentration of
approximately 50-100 .mu.M. Cell pastes were harvested at 6 hours
post inducer addition and stored at -20.degree. C. to -80.degree.
C. TABLE-US-00006 TABLE VI Additions to the Production Medium for
AP Promoter Expression System necessary for the trp Promoter
Expression System Ingredient Quantity/Liter L-isoleucine 0.5-1 g
Tryptophan 0.1-5 g
[0160] Cell growth for the trp promoter system slowed after
reaching 40-60 OD.sub.550. There was significant leaky expression
of Apo-2 ligand prior to induction with IAA addition and was
probably responsible for the growth problem. Cell growth profiles
were comparable in the absence and the presence of ZnSO.sub.4
additions. FIG. 10 shows the time course in the accumulation of
soluble Apo-2L trimers detected by the IEX HPLC method. The
accumulation of soluble Apo-2L continued to increase in the run
with ZnSO.sub.4 additions and achieved a higher product
concentration in the cell lysate samples.
C. Apo-2L (Amino Acid Residues 114-281) Recovery and Purification
from E. coli Using Divalent Metal Ions/DTT:
[0161] The following protocol may be employed in recovery and
purification of Apo-2L from E. coli. First, the cells are
homogenized and extraction is performed as follows. Frozen
harvested Escherichia coli cells are suspended in 6 volumes of
extraction buffer (100 mM HEPES/5 mM DTT (or 5 mM Zn sulfate
instead of DTT), pH 8.0), or whole cell broth is conditioned with 5
mM DTT (or 5 mM Zn sulfate instead of DTT) @ pH 8.0. The suspension
is thoroughly mixed for 1 hour at 2-8.degree. C., then homogenized
in a homogenizer (Gaulin Corporation, Everett, Mass.). The broken
cell suspension is flocculated in 0.2% PEI for 1-2 hours and
centrifuged by a BTPX205 (Alfa Laval Separation AB, Sweden)
continuous feed centrifuge and clarified by depth filtration.
[0162] After extraction, the Apo-2L is purified as follows.
Macro-Prep ceramic High S (MP-HS) chromatography is performed by
conditioning the clarified cell suspension (extract) with an equal
volume of H.sub.2O/0.1% Triton-100 and adjusted pH to 7.2. The
conditioned extract is loaded onto a column of MP-HS cation
exchanger (Bio-Rad, Hercules, Calif.) that is equilibrated in 50 mM
HEPES/0.05% Triton-100/1 mM DTT (or 100 uM Zn sulfate), pH 7.2. (In
the preceding two steps, --SP-Sepharose Fast Flow (Amersham
Pharmacia, Sweden) may alternatively be employed). The non-binding
proteins are flowed through and removed by washing with
equilibration buffer to baseline @ A280. The column is washed with
3 column volumes of 0.1 M NaCl/equilibration buffer. The Apo-2L is
eluted using a linear, 8 column-volume gradient from 0.1 to 0.8M
sodium chloride in equilibration buffer. Fractions are collected
and those which contain properly-folded Apo-2L, as determined by
SDS-PAGE or SEC-HPLC assay, are pooled.
[0163] The pool of Apo-2L from the MP-HS column is loaded onto a
column of Macro-Prep Hydroxyapatite (Bio-Rad, Hercules, Calif.)
equilibrated in 50 mM HEPES/1 mM DTT (or 100 uM Zn sulfate), pH
7.2. (As an alternative to the Macro-Prep Hydroxyapatite,
SP-Sepharose Fast Flow may be employed). After the sample is
loaded, the column is washed with equilibration buffer to baseline
@ A280. The Apo-2L is eluted out of the column by using an
isocratic step of 0.15 M sodium phosphate in equilibration
buffer.
[0164] The pool of MP-HA is conditioned with an equal volume of 1.0
M Ammonium Sulfate/50 mM Tris/1 mM DTT (or 100 uM Zn sulfate), pH
7.5, and then loaded onto a column of Phenyl-Sepharose FF (Amersham
Pharmacia, Sweden) that is equilibrated in 0.5 M Ammonium
sulfate/50 mM Tris/1 mM DTT (or 100 uM Zn sulfate), pH 7.5. (As an
alternative to the 0.5M ammonium sulfate, 0.6M sodium sulfate may
be employed). The column is washed with equilibration buffer, and
the Apo-2L is collected in the column effluent.
[0165] The Apo-2L is then formulated by ultrafiltration and G-25
Gel Filtration (Amersham Pharmacia, Sweden) chromatography. The
pool of phenyl-Sepharose is concentrated with TFF Ultrafiltration
(Millipore, Bedford, Mass.) and formulated on a G-25 gel filtration
column with 20 mM Tris/8% Trehalose, pH 7.5. (As an alternative,
the material may be formulated by diafiltration). The final purity
of Apo-2L can be determined by SDS-PAGE, SEC-HPLC and Amino Acid
sequence analysis.
Example 9
Additions of Cobalt Chloride Improve Soluble Apo-2 Ligand Product
Accumulation and Recovery
[0166] Apo-2L (Amino Acid Residues 114-281) Expression Regulated by
the Alkaline Phosphatase Promoter
[0167] The same production organism, medium composition,
fermentation conditions and sample analysis described in Example 8A
were used in studying the effect of additions of metal ion other
than Zn, namely cobalt chloride, on Apo-2L (amino acid residues
114-281) product accumulation. A Solution of 100 mM CoCl.sub.2 in
H.sub.2O was used in place of the 100 mM ZnSO.sub.4 and sufficient
amounts were added to arrive at a final concentration of 50-100
.mu.M at each of the two additions.
[0168] FIG. 11 shows the benefits of additions of CoCl.sub.2 on
soluble Apo-2L accumulation during the fermentation process. Like
the ZnSO.sub.4 addition experiments, though not as significant, a
higher accumulation rate of soluble Apo-2L product was detected by
the IEX HPLC assay method. The data demonstrates that additions of
certain metal ions generally improve soluble Apo-2L accumulation,
likely as a result of their ability to stabilize the assembled
trimers of Apo-2L.
Example 10
[0169] Effects of Various Metal Ions on Apo-2L Formulations
[0170] In vitro assays were conducted by incubating 50 .mu.l Apo-2L
(form 114-281) at 5.degree. C. for 24 hours with 5 mM of metal salt
(each of which are recited in Table VII below; 100:1 molar ratio of
metal to protein) in a 20 mM Tris, 8% trehalose, 0.01% Tween 20, pH
7.5 formulation. The samples were then evaluated for apoptotic
activity using a SK-MES assay as described in Example 4.
[0171] The EC50 (or Apo-2L concentration that results in killing of
50% of the cells) is shown in Table VII (units of ng/ml). As shown
in Table VII, addition of Zn acetate and Zn sulfate to the culture
enhanced Apo-2L activity. TABLE-US-00007 TABLE VII Metal Added
Bioassay EC50 Control 19.0 .+-. 1.9 (n = 3) Mn acetate 19.8 Mn
chloride 19.9 Fe acetate 32.5 Co acetate 25.9 Co chloride 17.9 Co
sulfate 16.7 Ni acetate 18.7 Cu acetate 844 Cu chloride 984 Cu
sulfate 774 Ag acetate 20 Zn acetate 12.3 Zn chloride 17.2 Zn
sulfate (100:1) 9.3 .+-. 1.2 (n = 3) (10:1) 10.0 .+-. 0 (n = 2)
(1:1) 11.5 .+-. 0.7 (n = 2)
Example 11
[0172] Effects of Zinc Metal Ions on Apo-2 Ligand Formulation
Stability
[0173] As indicated in Example 8B, the first step in the
purification process using MPHS cation exchanger gives two peaks, A
and B. To investigate the storage stability of the two peaks,
Apo-2L (form 114-281) was extracted from the E. coli expressed
product (using the pS1346.ApoL.0 plasmid with trp promoter), and
purified by MPHS cation exchanger. The MPHS Peak A was further
purified by MP-Hydroxyapatite and Phenyl-Sepharose FF, and then
formulated using G-25 gel filtration. The MPHS Peak B was purified
by MP-Hydroxyapatite, Phenyl-Sepharose FF, and Ni-NTA Superflow and
then formulated on G-25 gel filtration into 20 mM Tris, 8%
trehalose, 0.01% Tween 20, pH 7.5. The samples were sterile filled
into 3 cc glass vials and sealed with teflon coated greybutyl
stoppers. The storage stability of the purified and formulated
Peaks A and B were evaluated at the time (weeks ("wk") or months
("mo")) and temperatures (.degree. C.) listed in Table VIII.
TABLE-US-00008 TABLE VIII SDS- SDS- SEC % SEC SEC % SEC % RP % Peak
A M % D recovery trimer main EC50 1 wk, -70 C. ND ND 98.60 78.10 1
wk, 37 C. ND ND 96.7 98.2 73.60 2 mo, -70 C. 88.85 7.50 93.93 74.52
2 mo, 30 C. 84.90 10.58 65.07 94.03 53.98 6 mo, -70 C. 93.00 5.80
ND 11.1 6 mo, 5 C. 87.00 9.44 100 ND 14.8 6 mo, 30 C. 80.00 8.68 76
ND 67.8 SDS- SDS- SEC % SEC SEC % SEC % RP % Peak B M % D recovery
trimer main 1 wk, -70 C. ND ND 98.70 75.3 1 wk, 37 C. ND ND 101.1
98.3 61.4 2 mo, -70 C. 94.03 3.79 90.00 65.77 2 mo, 30 C. 94.12
4.54 46.07 94.12 32.185 6 mo, -70 C. 82.00 11.25 ND 10.3 6 mo, 5 C.
73.50 17.00 92 ND 12.6 6 mo, 30 C. 47.00 23.20 38 ND Dead
[0174] Table VIII shows, among other things, the percent monomer (%
M) and percent dimer (% D); "ND" refers to those values not
determined. Peak A included slightly less dimer and other
impurities than Peak B at Day 0. As shown in Table VIII, Peak B was
found to be more thermally labile, as assessed by the more
extensive precipitation at 30.degree. C. (approximately 40% more at
the 2 month point). Even at 5.degree. C., Peak B was approximately
2-fold more prone to dimer formation than Peak A. Biochemical
analysis of Peaks A and B indicated that the material of each was
biochemically equivalent except for their respective stability
properties. It is believed that the lower stability of Peak B may
be related to improper (trimer) assembly or lower zinc content
(0.83 molar ratio of Zinc to protein for Peak A and 0.7 molar ratio
of Zinc to protein for Peak B).
Sequence CWU 1
1
7 1 281 PRT Homo sapiens 1 Met Ala Met Met Glu Val Gln Gly Gly Pro
Ser Leu Gly Gln Thr 1 5 10 15 Cys Val Leu Ile Val Ile Phe Thr Val
Leu Leu Gln Ser Leu Cys 20 25 30 Val Ala Val Thr Tyr Val Tyr Phe
Thr Asn Glu Leu Lys Gln Met 35 40 45 Gln Asp Lys Tyr Ser Lys Ser
Gly Ile Ala Cys Phe Leu Lys Glu 50 55 60 Asp Asp Ser Tyr Trp Asp
Pro Asn Asp Glu Glu Ser Met Asn Ser 65 70 75 Pro Cys Trp Gln Val
Lys Trp Gln Leu Arg Gln Leu Val Arg Lys 80 85 90 Met Ile Leu Arg
Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu 95 100 105 Lys Gln Gln
Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln 110 115 120 Arg Val
Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr 125 130 135 Leu
Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys 140 145 150
Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser 155 160
165 Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly 170
175 180 Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu
185 190 195 Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr
Ile 200 205 210 Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met
Lys Ser 215 220 225 Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr
Gly Leu Tyr 230 235 240 Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys
Glu Asn Asp Arg 245 250 255 Ile Phe Val Ser Val Thr Asn Glu His Leu
Ile Asp Met Asp His 260 265 270 Glu Ala Ser Phe Phe Gly Ala Phe Leu
Val Gly 275 280 2 1042 DNA Homo sapiens VARIATION 447 N CAN BE T OR
G 2 tttcctcact gactataaaa gaatagagaa ggaagggctt cagtgaccgg 50
ctgcctggct gacttacagc agtcagactc tgacaggatc atggctatga 100
tggaggtcca ggggggaccc agcctgggac agacctgcgt gctgatcgtg 150
atcttcacag tgctcctgca gtctctctgt gtggctgtaa cttacgtgta 200
ctttaccaac gagctgaagc agatgcagga caagtactcc aaaagtggca 250
ttgcttgttt cttaaaagaa gatgacagtt attgggaccc caatgacgaa 300
gagagtatga acagcccctg ctggcaagtc aagtggcaac tccgtcagct 350
cgttagaaag atgattttga gaacctctga ggaaaccatt tctacagttc 400
aagaaaagca acaaaatatt tctcccctag tgagagaaag aggtccncag 450
agagtagcag ctcacataac tgggaccaga ggaagaagca acacattgtc 500
ttctccaaac tccaagaatg aaaaggctct gggccgcaaa ataaactcct 550
gggaatcatc aaggagtggg cattcattcc tgagcaactt gcacttgagg 600
aatggtgaac tggtcatcca tgaaaaaggg ttttactaca tctattccca 650
aacatacttt cgatttcagg aggaaataaa agaaaacaca aagaacgaca 700
aacaaatggt ccaatatatt tacaaataca caagttatcc tgaccctata 750
ttgttgatga aaagtgctag aaatagttgt tggtctaaag atgcagaata 800
tggactctat tccatctatc aagggggaat atttgagctt aaggaaaatg 850
acagaatttt tgtttctgta acaaatgagc acttgataga catggaccat 900
gaagccagtt ttttcggggc ctttttagtt ggctaactga cctggaaaga 950
aaaagcaata acctcaaagt gactattcag ttttcaggat gatacactat 1000
gaagatgttt caaaaaatct gaccaaaaca aacaaacaga aa 1042 3 144 PRT Homo
sapiens 3 Lys Pro Ala Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn
Ser 1 5 10 15 Leu Leu Trp Arg Ala Asn Thr Asp Arg Ala Phe Leu Gln
Asp Gly 20 25 30 Phe Ser Leu Ser Asn Asn Ser Leu Leu Val Pro Thr
Ser Gly Ile 35 40 45 Tyr Phe Val Tyr Ser Gln Val Val Phe Ser Gly
Lys Ala Tyr Ser 50 55 60 Pro Lys Ala Thr Ser Ser Pro Leu Tyr Leu
Ala His Glu Val Gln 65 70 75 Leu Phe Ser Ser Gln Tyr Pro Phe His
Val Pro Leu Leu Ser Ser 80 85 90 Gln Lys Met Val Tyr Pro Gly Leu
Gln Glu Pro Trp Leu His Ser 95 100 105 Met Tyr His Gly Ala Ala Phe
Gln Leu Thr Gln Gly Asp Gln Leu 110 115 120 Ser Thr His Thr Asp Gly
Ile Pro His Leu Val Leu Ser Pro Ser 125 130 135 Thr Val Phe Phe Gly
Ala Phe Ala Leu 140 4 147 PRT Homo sapiens 4 Lys Pro Val Ala His
Val Val Ala Asn Pro Gln Ala Glu Gly Gln 1 5 10 15 Leu Gln Trp Leu
Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly 20 25 30 Val Glu Leu
Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu 35 40 45 Tyr Leu
Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro 50 55 60 Ser
Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val 65 70 75
Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 80 85
90 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr 95
100 105 Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp
110 115 120 Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe
Ala 125 130 135 Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 140
145 5 141 PRT Homo sapiens 5 Gln Ile Ala Ala His Val Ile Ser Glu
Ala Ser Ser Lys Thr Thr 1 5 10 15 Ser Val Leu Gln Trp Ala Glu Lys
Gly Tyr Tyr Thr Met Ser Asn 20 25 30 Asn Leu Val Thr Leu Glu Asn
Gly Lys Gln Leu Thr Val Lys Arg 35 40 45 Gln Gly Leu Tyr Tyr Ile
Tyr Ala Gln Val Thr Phe Cys Ser Asn 50 55 60 Arg Glu Ala Ser Ser
Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu 65 70 75 Lys Ser Pro Gly
Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn 80 85 90 Thr His Ser
Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu 95 100 105 Gly Gly
Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 110 115 120 Val
Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser 125 130 135
Phe Gly Leu Leu Lys Leu 140 6 137 PRT Homo sapiens 6 Arg Lys Val
Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met 1 5 10 15 Pro Leu
Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly 20 25 30 Val
Lys Tyr Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu 35 40 45
Tyr Phe Val Tyr Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn 50 55
60 Asn Leu Pro Leu Ser His Lys Val Tyr Met Arg Asn Ser Lys Tyr 65
70 75 Pro Gln Asp Leu Val Met Met Glu Gly Lys Met Met Ser Tyr Cys
80 85 90 Thr Thr Gly Gln Met Trp Ala Arg Ser Ser Tyr Leu Gly Ala
Val 95 100 105 Phe Asn Leu Thr Ser Ala Asp His Leu Tyr Val Asn Val
Ser Glu 110 115 120 Leu Ser Leu Val Asn Phe Glu Glu Ser Gln Thr Phe
Phe Gly Leu 125 130 135 Tyr Lys 7 152 PRT Homo sapiens 7 Gln Pro
Phe Ala His Leu Thr Ile Asn Ala Thr Asp Ile Pro Ser 1 5 10 15 Gly
Ser His Lys Val Ser Leu Ser Ser Trp Tyr His Asp Arg Gly 20 25 30
Trp Ala Lys Ile Ser Asn Met Thr Phe Ser Asn Gly Lys Leu Ile 35 40
45 Val Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe 50
55 60 Arg His His Glu Thr Ser Gly Asp Leu Ala Thr Glu Tyr Leu Gln
65 70 75 Leu Met Val Tyr Val Thr Lys Thr Ser Ile Lys Ile Pro Ser
Ser 80 85 90 His Thr Leu Met Lys Gly Gly Ser Thr Lys Tyr Trp Ser
Gly Asn 95 100 105 Ser Glu Phe His Phe Tyr Ser Ile Asn Val Gly Gly
Phe Phe Lys 110 115 120 Leu Arg Ser Gly Glu Glu Ile Ser Ile Glu Val
Ser Asn Pro Ser 125 130 135 Leu Leu Asp Pro Asp Gln Asp Ala Thr Tyr
Phe Gly Ala Phe Lys 140 145 150 Val Arg
* * * * *