U.S. patent application number 11/656174 was filed with the patent office on 2007-06-07 for al-2 neurotrophic factor.
Invention is credited to Ingrid W. Caras.
Application Number | 20070128259 11/656174 |
Document ID | / |
Family ID | 24546567 |
Filed Date | 2007-06-07 |
United States Patent
Application |
20070128259 |
Kind Code |
A1 |
Caras; Ingrid W. |
June 7, 2007 |
AL-2 neurotrophic factor
Abstract
The present invention provides nucleic acids encoding AL-2
protein, as well as AL-2 protein produced by recombinant DNA
methods. Such AL-2 protein and nucleic acid are useful in preparing
antibodies and antagonists and in diagnosing and treating various
neuronal disorders and disorders or conditions associated with
angiogenesis.
Inventors: |
Caras; Ingrid W.; (San
Francisco, CA) |
Correspondence
Address: |
HELLER EHRMAN LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Family ID: |
24546567 |
Appl. No.: |
11/656174 |
Filed: |
January 19, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10021121 |
Dec 6, 2001 |
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11656174 |
Jan 19, 2007 |
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08635130 |
Apr 19, 1996 |
6696557 |
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10021121 |
Dec 6, 2001 |
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Current U.S.
Class: |
424/445 ;
514/12.2; 514/13.2; 514/13.3; 514/13.7; 514/18.6; 514/19.3;
514/6.9; 514/8.4 |
Current CPC
Class: |
C07K 14/475 20130101;
A61L 2300/602 20130101; Y02A 50/30 20180101; C07K 2319/00 20130101;
A61L 2300/41 20130101; A61L 2300/42 20130101; A61L 17/005 20130101;
A61K 38/1709 20130101; A61L 2300/25 20130101; A61L 15/44 20130101;
A61L 27/54 20130101 |
Class at
Publication: |
424/445 ;
514/012 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61L 15/00 20060101 A61L015/00 |
Claims
1. A method for accelerating neovascularization of a wound,
comprising applying to the wound an angiogenically effective amount
of a pharmaceutical composition comprising an isolated polypeptide
comprising the amino acid sequence of (a) the mature human AL-2
amino acid sequence shown in FIGS. 1A-1C (SEQ ID NO: 2) or FIG.
2A-2D (SEQ ID NO: 4); or (b) a soluble AL-2 derived from SEQ ID NO:
2 or SEQ ID NO: 4; or (c) a mammalian homolog or a conservative
amino acid substitution variant of (a) having at least 95% sequence
identity with SEQ ID NO: 2 or SEQ ID NO: 4, and a physiologically
acceptable carrier.
2. The method of claim 1 wherein said wound is due to surgical
incision, burn, traumatized tissue, skin graft, or ulcer.
3. The method of claim 1 wherein normal healing of said wound is
retarded.
4. The method of claim 3 wherein the retardation is due to advanced
age, diabetes, cancer, or treatment with an anti-inflammatory drug
or an anticoagulant.
5. The method of claim 1 wherein said composition is a topical
composition.
6. The method of claim 5 wherein said topical composition is in the
form of an irrigant or salve.
7. The method of claim 1 wherein said composition is contained in a
suture, graft, or dressing.
8. The method of claim 1 wherein said composition is a sustained
release composition.
9. The method of claim 1 wherein a clustered soluble AL-2 of the
formula (soluble AL-2).sub.n is applied to the wound, wherein n is
2 or greater.
10. The method of claim 1 wherein an immunoadhesin comprising a
soluble AL-2 is applied to the wound.
11. The method of claim 1 wherein a compound of the formula
(AL-2).sub.nX is applied to the wound, wherein AL-2 is any of the
polypeptides defined in parts (a)-(c) of claim 1, and n is 2 or
greater, and X is an organic linker covalently binding each AL-2.
Description
TECHNICAL FIELD
[0001] This application relates to a receptor protein tyrosine
kinase ligand and its uses. In particular this application relates
to the production and use of purified forms of AL-2 and related
proteins.
BACKGROUND
[0002] Protein neurotrophic factors, or neurotrophins, which
influence growth and development of the vertebrate nervous system,
are believed to play an important role in promoting the
differentiation, survival, and function of diverse groups of
neurons in the brain and periphery. Neurotrophic factors are
believed to have important signaling functions in neural tissues,
based in part upon the precedent established with nerve growth
factor (NGF). NGF supports the survival of sympathetic, sensory,
and basal forebrain neurons both in vitro and in vivo.
Administration of exogenous NGF rescues neurons from cell death
during development. Conversely, removal or sequestration of
endogenous NGF by administration of anti-NGF antibodies promotes
such cell death (Heumann, J. Exp. Biol., 132:133-150 (1987); Hefti,
J. Neurosci., 6:2155-2162 (1986); Thoenen et al., Annu. Rev.
Physiol., 60:284-335 (1980)).
[0003] Additional neurotrophic factors related to NGF have since
been identified. These include brain-derived neurotrophic factor
(BDNF) (Leibrock, et al., Nature, 341:149-152 (1989)),
neurotrophin-3 (NT-3) (Kaisho, et al., FEBS Lett., 266:187 (1990);
Maisonpierre, et al., Science, 247:1446 (1990); Rosenthal, et al.,
Neuron, 4:767 (1990)), and neurotrophin 4/5 (NT-4/5) (Berkmeier, et
al., Neuron, 7:857-866 (1991)).
[0004] Neurotrophins, similar to other polypeptide growth factors,
affect their target cells through interactions with cell surface
receptors. According to our current understanding, two kinds of
transmembrane glycoproteins act as receptors for the known
neurotrophins. Equilibrium binding studies have shown that
neurotrophin-responsive neuronal cells possess a common low
molecular weight (65,000-80,000 Daltons), a low affinity receptor
typically referred to as p75.sup.LNGFR or p75, and a high molecular
weight (130,000-150,000 Dalton) receptor. The high and low affinity
receptors are members of the trk family of receptor tyrosine
kinases.
[0005] Receptor tyrosine kinases are known to serve as receptors
for a variety of protein factors that promote cellular
proliferation, differentiation, and survival. In addition to the
trk receptors, examples of other receptor tyrosine kinases include
the receptors for epidermal growth factor (EGF), fibroblast growth
factor (FGF), and platelet-derived growth factor (PDGF). Typically,
these receptors span the cell membrane, with one portion of the
receptor being intracellular and in contact with the cytoplasm, and
another portion of the receptor being extracellular. Binding of a
ligand to the extracellular portion of the receptor induces
tyrosine kinase activity in the intracellular portion of the
receptor, with ensuing phosphorylation of various intracellular
proteins involved in cellular signaling pathways.
[0006] Recently, a receptor tyrosine kinase subclass referred to as
the Eph receptor subclass or family has been identified. Eph was
the first member of this Eph subclass of receptor tyrosine kinases
to be identified and characterized by molecular cloning (Hirai et
al., Science, 238:1717-1720 (1987)). The name Eph is derived from
the name of the cell line from which the Eph cDNA was first
isolated, the erythropoietin-producing human hepatocellular
carcinoma cell line, ETL-1. The general structure of Eph is similar
to that of other receptor tyrosine kinases and consists of an
extracellular domain, a single membrane spanning region, and a
conserved tyrosine kinase catalytic domain. However, the structure
of the extracellular domain of Eph, which comprises an
immunoglobulin (Ig)-like domain at its amino terminus, followed by
a cysteine-rich region and two fibronectin type III repeats in
close proximity to the transmembrane domain, is completely distinct
from that of previously described receptor tyrosine kinases. The
juxtamembrane domain and carboxy-terminus regions of Eph also are
unrelated to the corresponding regions of other tyrosine kinase
receptors.
[0007] Newly discovered members of the Eph receptor family include
Elk, Cek5, Mek4, Cek4, Hek/Hek4 (Sajjadi et al., The New Biologist,
3:769-778 (1991)), Cek6 through Cek10 (Sajjadi et al., Oncogene,
8:1807-13 (1993), Sek, Hek2, and Ehk3 (Tuzi, et al., Br. J. Cancer,
69:417-421 (1994); Zhou, et al., J. Neurosci. Res., 37:129-143
(1994)). Other Eph-related receptor kinases that have been
identified include Sek (Gilardi-Hebenstreit et al, Oncogene,
7:2499-2506 (1992)), Eck (Lindberg et al., Mol. Cell. Biol.,
10:6316-6324 (1990)), Elk (Lhotak et al., Mol. Cell. Biol.,
11:2496-2502 (1991)), Eek (Chan et al., Oncogene, 6:1057-1061
(1991)), Rek7 (Winslow et al., Neuron, 14:973-981 (1995). Rek7 is a
rat homolog of chicken Cek7 and closely related to Ehk-1 (Davis et
al., Science, 266:816-819 (1994)) and bsk (Maisonpierre et al.
Oncogene, 8:3277-3288 (1993); Zhou et al., J. Neurosci. Res,
37:129-143 (1994)); the Rek7 cDNA corresponds to a splice variant
of Ehk-1, lacking the first of two tandem fibronectin type-III
domains. Human homologs of the chicken Cek receptors are referred
to as Hek receptors (See WO 95/28484, which is incorporated herein
by reference). For example, Hek5 (Fox et al., Oncogene,
10(5):897-905 (1995); WO 95/28484) is the human homolog of chicken
Cek5. The amino acid sequence of Hek5 is very closely related (96%
amino acid identity in the catalytic domain) to the chicken
receptor Cek5 (Pasquale et al., J. Neuroscience, 12:3956-3967
(1992); Pasquale, Cell Regulation, 2:523-534 (1991)). A portion of
the Hek5 sequence was previously disclosed as Erk, a human clone
encoding about sixty amino acids (Chan et al., Oncogene,
6:1057-1061 (1991)). Mature Erk showed high homology with Cek5
(92.5%) and mouse Nuk (99.1%) (Kiyokawa et al., Cancer Res., 54
(14):3645-50 (1994)). Other human Eph-family receptors include Hek
(Wicks et al., Proc. Natl. Acad. Sci. USA, 89(5):1611-1615 (1992);
also known as Hek4), Hek2 (Bohme et al., Oncogene, 8:2857-2862
(1993)), Heks 7, 8 and 11 (WO 95/28484), Hek3, which is a homolog
of rat Eek and murine Mdk-1, and Hek12, which is a homolog of rat
Ehk2.
[0008] Many of the Eph-receptor family members are "orphan
receptors." However, recently, ligands have been reported including
B61, an Eck receptor ligand (Bartley et al., Nature, 368:558-560
(1994) and Pandey et al., Science, (1995) 268:567-569), Elf-1, a
Mek4 and Sek receptor ligand (Cheng et al., Cell, (1995)
82:371-381; Cheng et al., Cell, 79:157-168 (1994)), Htk-L (Bennett
et al., Proc. Natl. Acad. Sci. USA, 92(6):1866-70 (1995)), AL-1
(Winslow et al., Neuron, 14:973-981 (1995)) and RAGS (Drescher et
al., Cell, (1995) 82:359-370), which are Rek7 ligands, Ehk-1-L
(Davis et al., Science, 266:816-819 (1994); see also efl-2 in WO
95/27060), Cek5-L, and Lerk2 (Beckmann et al., EMBO J.,
13:3757-3762 (1994)).
[0009] Aberrant expression of receptor tyrosine kinases correlates
with transforming ability. This relationship includes members of
the Eph subclass of receptor tyrosine kinases. For example,
carcinomas of the liver, lung, breast and colon show elevated
expression of Eph. Unlike many other tyrosine kinases, this
elevated expression can occur in the absence of gene amplification
or rearrangement. Such involvement of Eph in carcinogenesis also
has been shown by the formation of foci of NIH 3T3 cells in soft
agar and of tumors in nude mice following overexpression of Eph.
Moreover, Hek has been identified as a leukemia-specific marker
present on the surface of a pre-B cell leukemia cell line. As with
Eph, Hek also was overexpressed in the absence of gene
amplification or rearrangements in, for example, hemopoietic tumors
and lymphoid tumor cell lines. Over-expression of Myk-1 (a murine
homolog of human Htk (Bennett et al., J. Biol. Chem.,
269(19):14211-8 (1994)) was found in the undifferentiated and
invasive mammary tumors of transgenic mice expressing the Ha-ras
oncogene. (Andres et al., Oncogene, 9(5):1461-7 (1994) and Andres
et al., Oncogene, 9(8):2431 (1994)).
[0010] In addition to their roles in carcinogenesis, a number of
transmembrane tyrosine kinases have been reported to play key roles
during development. Some receptor tyrosine kinases are
developmentally regulated and predominantly expressed in embryonic
tissues. Examples include Cek1, which belongs to the FGF subclass,
and the Cek4 and Cek5 tyrosine kinases (Pasquale et al., Proc.
Natl. Acad Sci., USA, 86:5449-5453 (1989); Sajjadi et al., New
Biol., 3(8):769-78 (1991); and Pasquale, Cell Regulation, 2:523-534
(1991)).
[0011] Eph family members are expressed in many different adult
tissues, with several family members expressed in the nervous
system or specifically in neurons (Maisonpierre et al., Oncogene,
8:3277-3288 (1993); Lai et al., Neuron, 6:691-704 (1991)).
[0012] The aberrant expression or uncontrolled regulation of any
one of these receptor tyrosine kinases can result in different
malignancies and pathological disorders. Therefore, there exists a
need to identify means to regulate, control and manipulate receptor
tyrosine kinases and their ligands in order to provide new and
additional means for the diagnosis and therapy of Eph-pathway
related disorders and cellular processes. The present application
provides the clinician and researcher with such means by providing
new molecules that are specific for interacting with Eph-family
receptors. These compounds and their methods of use, as provided
herein, allow exquisite therapeutic control and specificity.
Additional advantages are provided as well.
SUMMARY
[0013] The present invention provides a novel cytokine, an
Eph-related tyrosine kinase receptor ligand referred to as
AL-2.
[0014] The present invention provides nucleic acid encoding AL-2,
particularly two forms referred to herein as AL-2s ("AL-2-short")
and AL-2l ("AL-2-long"), and methods to use the nucleic acid to
produce AL-2 in recombinant host cells for diagnostic or
therapeutic purposes. Also provided are uses of nucleic acids
encoding AL-2, and portions thereof, to identify related nucleic
acids in the cells or tissues of various animal species.
[0015] By providing the full nucleotide coding sequence for AL-2,
the invention enables the production of AL-2 by means of
recombinant DNA technology, thereby making available for the first
time sufficient quantities of substantially pure AL-2 protein or
AL-2 antagonists for diagnostic and therapeutic uses. For example,
method embodiments include treatment or prevention of a variety of
neurological disorders and diseases as well as conditions that are
angiogenesis-dependent such as solid tumors, diabetic retinopathy,
rheumatoid arthritis, and wound healing.
[0016] Also provided are derivatives and modified forms of AL-2,
including amino acid sequence variants and covalent derivatives
thereof, as well as antagonists of AL-2, that are preferably
biologically active (e.g., antigenically active. In one embodiment,
the invention provides a soluble form of the ligand with at least
the transmembrane region deleted. Usually, the cytoplasmic domain
will also be absent. Immunogens are provided for raising
antibodies, as well as to obtain antibodies, capable of binding to,
preferably neutralizing, AL-2 or derivatives or modified forms
thereof.
[0017] In a preferred embodiment, the invention provides AL-2 that
is free of other human proteins.
[0018] AL-2 and modified and variant forms of AL-2 are produced by
means of chemical or enzymatic treatment or by means of recombinant
DNA technology, including in vivo production. Variant polypeptides
can differ from native AL-2, for example, by virtue of one or more
amino acid substitutions, deletions or insertions, or in the extent
or pattern of glycosylation, but will substantially retain a
biological activity of native AL-2.
[0019] Chimeras comprising AL-2 (or a portion thereof) fused to
another polypeptide are provided. An example of such a chimera is
epitope-tagged AL-2. In another embodiment a soluble form of an
AL-2 chimera is provided, for example, as an immunoadhesin, which
is a fusion of the extracellular domain of AL-2 and an
immunoglobulin sequence.
[0020] Antibodies to AL-2 are produced by immunizing an animal with
AL-2 or a fragment thereof, optionally in conjunction with an
immunogenic polypeptide, and thereafter recovering antibodies from
the serum of the immunized animals. Alternatively, monoclonal
antibodies are prepared from cells of the immunized animal in
conventional fashion. Antibodies obtained by routine screening will
bind to AL-2 but, preferably, will not substantially bind to (i.e.,
cross react with) NGF, BDNF, NT-3, NT-4/5, GDNF, AL-1, Htk-L,
Lerk-2, or other neurotrophic factors or cytokines. Immobilized
anti-AL-2 antibodies are particularly useful in the detection of
AL-2 in clinical samples for diagnostic purposes, and in the
purification of AL-2.
[0021] AL-2, its derivatives, or its antibodies are formulated with
physiologically acceptable carriers, especially for therapeutic
use. Such carriers are used, for example, to provide
sustained-release formulations of AL-2.
[0022] In further aspects, the invention provides a method for
determining the presence of a nucleic acid molecule encoding AL-2
in test samples prepared from cells, tissues, or biological fluids,
comprising contacting the test sample with isolated DNA comprising
all or a portion of the nucleotide coding sequence for AL-2 and
determining whether the isolated DNA hybridizes to a nucleic acid
molecule in the test sample. DNA comprising all or a portion of the
nucleotide coding sequence for AL-2 is also used in hybridization
assays to identify and to isolate nucleic acids sharing substantial
sequence identity to the coding sequence for AL-2, such as nucleic
acids that encode allelic variants of AL-2.
[0023] Also provided is a method which involves contacting an AL-2
receptor with AL-2 in order to cause phosphorylation of the kinase
domain of the receptor.
[0024] Also provided is a method for amplifying a nucleic acid
molecule encoding AL-2 that is present in a test sample, comprising
the use of an oligonucleotide having a portion of the nucleotide
coding sequence for AL-2 as a primer in a polymerase chain
reaction.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1A-1B shows the AL-2l-encoding nucleotide sequence, its
complementary sequence, and the deduced amino acid sequence of AL-2
of the isolated AL-2l ("AL-2-long") cDNA. The deduced N-terminus of
the mature AL-2 protein begins with glycine-27 as numbered from the
initiation methionine. The C-terminal hydrophobic transmembrane
domain extends from amino acid Leu-220 to Ala-245. The deduced
extracellular domain sequence includes amino acids Gly-27 to
Pro-219.
[0026] FIG. 2A-2B shows the AL-2s-encoding nucleotide sequence, its
complementary sequence, and the deduced amino acid sequence of AL-2
of the isolated AL-2s ("AL-2-short") cDNA. The deduced N-terminus
of the mature AL-2 protein begins with glycine-27 as numbered from
the initiation methionine. The C-terminal hydrophobic transmembrane
domain extends from amino acid Leu-220 to Ala-245. The deduced
extracellular domain sequence includes amino acids Gly-27 to
Pro-219.
[0027] FIG. 3A-3B depicts an alignment of the AL-2l nucleotide
sequence with human EST sequence H10006.
[0028] FIG. 4 shows a comparison of the AL-2l and AL-2s amino acid
sequences with that of Lerk2 (Beckmann et al., EMBO J.,
13:3757-3762 (1994)) and human Htk-L (Bennett et al., Proc. Natl.
Acad. Sci. USA, 92:1866-70 (1995); WO 96/02645 published Feb. 1,
1996; both are incorporated by reference herein). Identical amino
acids are boxed, and gaps introduced for optimal alignment are
indicated by dashes. Conserved cysteine residues can be seen. The
deduced C-terminal amino acid for AL-2s is valine.
[0029] FIG. 5 shows a comparison of the AL-2l amino acid sequences
with that of Lerk2 and human Htk-L. Identical amino acids are
boxed, and gaps introduced for optimal alignment are indicated by
dashes. Conserved cysteine residues can be seen.
DETAILED DESCRIPTION
[0030] "AL-2" or "AL-2 protein" refers to a polypeptide or protein
encoded by the AL-2 nucleotide sequence set forth in FIGS. 1A-1B
(showing AL-2l) or 2 (showing AL-2s); a polypeptide that is the
translated amino acid sequence set forth in FIGS. 1A-1B or 2A-2B;
fragments thereof having greater than about 5 contiguous amino acid
residues and comprising an immune epitope or other biologically
active site of AL-2; amino acid sequence variants of the amino acid
sequence set forth in FIGS. 1A-1B or 2A-2B wherein one or more
amino acid residues are added at the N- or C-terminus of, or
within, said FIGS. 1A-1B or 2A-2B sequences or its fragments as
defined above; amino acid sequence variants of said FIGS. 1A-1B or
2A-2B sequences or its fragments as defined above wherein one or
more amino acid residues of said FIGS. 1A-2B or 2A-2B sequences or
fragment thereof are deleted, and optionally substituted by one or
more amino acid residues; and derivatives of the above proteins,
polypeptides, or fragments thereof, wherein an amino acid residue
has been covalently modified so that the resulting product is a
non-naturally occurring amino acid. Preferred embodiments retain a
biological property of AL-2. AL-2 amino acid sequence variants may
be made synthetically, for example, by site-directed or PCR
mutagenesis, or may exist naturally, as in the case of allelic
forms and other naturally occurring variants of the translated
amino acid sequence set forth in FIGS. 1A-1B or 2A-2B that occur in
human or other animal species. Accordingly, within the scope of the
present invention are AL-2 proteins derived from other animal
species, preferably mammalian, including about not limited to
murine, rat, bovine, porcine, or various primates. As used herein,
the term "AL-2" includes membrane-bound proteins (comprising a
cytoplasmic domain, a transmembrane region, and an extracellular
domain), including the long and short forms of AL-2, as well as
truncated proteins that retain Eph-family-receptor binding
property. Truncated AL-2 proteins include, for example, soluble
AL-2 comprising only the extracellular (receptor binding) domain.
Such fragments, variants, and derivatives exclude any polypeptide
heretofore identified, including any known neurotrophic factor,
such as nerve growth factor (NGF), brain derived neurotrophic
factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5
(NT-4/5), Eph family receptor ligand such as Erk-L or Lerk-2, as
well as statutorily obvious variants thereof. A preferred AL-2 is
one having a contiguous amino acid sequence of or derived from
mature AL-2 shown in FIGS. 1A-1B or 2A-2B.
[0031] By "Eph-related protein tyrosine kinase" or "Eph-related
kinase" or "Eph-family receptor" means herein a receptor tyrosine
kinase having an extracellular ligand binding domain, a
transmembrane domain, and a cytoplasmic catalytic domain, and
belonging to the Eph subclass of receptor tyrosine kinases.
Eph-family receptors include, for example, human receptor tyrosine
kinases, Eph, Erk/Nuk, Htk, Eck, and Heks (e.g., Hek, Hek2, Hek3,
Hek4, Hek5, Hek6, Hek7, Hek8, Hek9, Hek11, Hek12), and their
non-human counterparts including chicken Ceks (e.g., Cek5, Cek6,
Cek7, Cek8, Cek9, Cek10), murineNuk, Seks (e.g., Sek1, Sek2, Sek3,
Sek4; Gilardi-Hebenstreit et al, Oncogene 7:2499-2506 (1992)),
Myk-1 (Andres et al., Oncogene 9(5):1461-7 (1994)), Mek4, Mdk-1,
and rat Tyros (e.g., Tyro1, Tyro4, Tyro5, Tyro6, Tyro11), Rek7,
Ehk1, Ehk2, Ehk3, Bsk, Eek, and Elk. Natural ligands for these
receptors can be characterized by means of ligand attachment to a
cell membrane--either by a GPI-anchor (e.g., Lerk3 and Lerk4
(Kozlosky et al., Oncogene, 10(2):299-306 (1995)) or by a
transmembrane sequence. Preferred receptors for AL-2 are receptors
that are recognized by transmembrane-sequence type ligands.
Preferred receptors include rat Elk, Tyro5 (Marcelle et al.,
Oncogene, 7:2479-87 (1992)), and Tyro6, murine Nuk/Sek3 (Henkemeyer
et al., Oncogene, 9(4):1001-14 (1994); Becker et al., Mech. Dev.,
47(1):3-17 (1994)), Myk-1, and Sek4 (Becker et al., Mechanisms of
Development, 47:3-17 (1994)), chicken Cek5, Cek6 and Cek10, and
their human homologs. More preferred are human receptors Hek5,
Hek6, Hek3, Hek2, Keh9, Hek11, Hek12, Htk, Erk and Eph. Even more
preferred are human receptors Htk (WO 96/02645 published Feb. 1,
1996), Hek2 (Bohme et al., Oncogene, 8:2857-2862 (1993)), Hek5/Erk
(Fox et al., Oncogene, 10(5):897-905 (1995); (Kiyokawa et al.,
Cancer Res., 54 (14):3645-50 (1994), and Hek6. Of particularly
preferred interest as an AL-2 binding receptor candidate are
"orphan receptors," including human Eph, Hek3, Hek9, Hek11 and
Hek12, and less preferably their non-human homologs, as well as
Ehk3 for which a human homolog is not known.
[0032]
Eph-family-receptor-binding-transmembrane-sequence-containing
ligands include the human Erk-L or Lerk2 ligand (Fletcher et al.,
Oncogene, 9(11):3241-7 (1994)) and the human Htk-L or Lerk5 ligand
(Cerretti et al., Mol. Immunol., 32(16):1197-205 (1995)). Non-human
ligands include Cek5-L, Elf-2 and Elk-L.
[0033] Biologically active orantigenically active AL-2 polypeptides
embodiments of this invention include the polypeptide represented
by the entire translated nucleotide sequence of AL-2l and AL-2s
(including their signal sequence); mature AL-2, i.e., AL-2 without
the signal sequence; fragments consisting essentially of the
intracellular domain or transmembrane domain of AL-2; fragments of
the AL-2 having a contiguous sequence of at least 5, 10, 15, 20,
25, 30, or 40 consecutive amino acid residues from AL-2; amino acid
sequence variants of AL-2 wherein an amino acid residue has been
inserted N- or C-terminal to, or within, AL-2 or its fragment as
defined above; amino acid sequence variants of AL-2 or its fragment
as defined above wherein an amino acid residue of AL-2 or its
fragment as defined above has been substituted by another residue,
including predetermined mutations by, e.g., site-directed or PCR
mutagenesis, AL-2 of various animal species such as rabbit, rat,
porcine, non-human primate, equine, murine, and ovine AL-2 and
alleles or other naturally occurring variants of the foregoing and
human AL-2; derivatives of AL-2 or its fragments as defined above
wherein AL-2 or its fragments have been covalent modified, by
substitution, chemical, enzymatic, or other appropriate means, with
a moiety other than a naturally occurring amino acid; and
glycosylation variants of AL-2 (insertion of a glycosylation site
or alteration of any glycosylation site by deletion, insertion, or
substitution of suitable residues). The preferred AL-2 is human
AL-2, especially native human AL-2 having the sequence shown in
FIGS. 1A-1B or 2A-2B.
[0034] One embodiment of the present invention provides soluble
AL-2. By "soluble AL-2" is meant AL-2 which is essentially free of
at least a transmembrane sequence and, optionally, the
intracellular domain of native AL-2. By "essentially free" is meant
that the soluble AL-2 sequence has less than 2% of the
transmembrane domain, preferably less than 1% of the transmembrane
domain, and more preferably less than 0.5% of this domain. The
transmembrane domain of the native human mature amino acid
sequences are delineated in FIGS. 1A-1B and 2A-2B (for AL-2l and
AL-2s, respectively), i e., resides Gly-27 to Pro-219. Soluble
AL-2s have therapeutic advantages because they are generally
soluble in the patient's blood stream. Similarly, soluble ligands
may prove to be particularly useful as diagnostics since they are
expected to have a reduced tendency to incorporate in the cell
membrane. Soluble AL-2 polypeptides comprise all or part of the
extracellular domain of a native AL-2 but lack the tratismembrane
region that would cause retention of the polypeptide on a cell
membrane. Soluble AL-2 polypeptides advantageously comprise the
native (or a heterologous) signal peptide when initially
synthesized to promote secretion, but the signal peptide is cleaved
upon secretion. In preferred embodiments, the soluble AL-2
polypeptides retain the ability to bind an Eph-family receptor with
preferences as discussed herein. Soluble AL-2 can also include part
of the transmembrane region or part of the cytoplasmic domain or
other sequences, provided that the soluble AL-2 protein is capable
of being secreted or otherwise isolated.
[0035] In one embodiment a soluble AL-2 is an "immunoadhesin". The
term "immunoadhesin" is used interchangeably with the expression
"AL-2-immunoglobulin chimera" and refers to a chimeric molecule
that combines the extracellular domain ("ECD") of AL-2 with an
immunoglobulin sequence. The immunoglobulin sequence preferably,
but not necessarily, is an immunoglobulin constant domain. The
immunoglobulin moiety in the chimeras of the present invention may
be obtained from IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA, IgE,
IgD or IgM, but preferably IgG-1 or IgG-3. The expression
"extracellular domain" or "ECD" when used herein refers to any
polypeptide sequence that shares a receptor binding function of the
extracellular domain of the naturally occurring AL-2 disclosed
herein. Receptor binding function refers to the ability of the
polypeptide to bind the extracellular domain of a Eph-family
receptor, with preferences as discussed herein, and, optionally,
activate the receptor. Accordingly, it is not necessary to include
the entire extracellular domain since smaller segments are commonly
found to be adequate for receptor binding. The term ECD encompasses
polypeptide sequences in which the cytoplasmic domain and
hydrophobic transmembrane sequence (and, optionally, 1-20 amino
acids amino-terminal to the transmembrane domain) of the mature
AL-2 have been deleted. The extracellular domain sequence of AL-2
is provided in FIGS. 1A-1B and 2A-2B.
[0036] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising the entire AL-2, or a portion
thereof, fused to a "tag polypeptide." The tag polypeptide has
sufficient amino acids to provide an antibody-binding epitope but
not interfere with activity of the AL-2. The tag polypeptide
preferably also is fairly unique so that an antibody against it
does not substantially cross-react with other epitopes. Suitable
tag polypeptides generally have at least 6 amino acid residues and
usually between about 8-50 amino acid residues, preferably between
about 9-30 residues.
[0037] "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 interfere with diagnostic or therapeutic uses
for the protein, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous solutes.
[0038] "Essentially pure" protein means a composition comprising at
least about 90% by weight of the protein, based on total weight of
the composition, preferably at least about 95% by weight.
"Essentially homogeneous" protein means a composition comprising at
least about 99% by weight of protein, based on total weight of the
composition.
[0039] An AL-2 amino acid sequence variant is included within the
scope of the invention provided that it is functionally active. As
used herein, "functionally active" and "functional activity" in
reference to AL-2 for the purposes herein means an in vivo effector
or antigenic function or activity that is performed by AL-2 of the
sequences in FIGS. 1A-1B or 2A-2B (whether in its native or
denatured conformation). A principal effector function is the
ability of AL-2 to bind to, and/or activate, a receptor from the
Eph-receptor family, preferably a receptor for the
transmembrane-ligand family that is also, more preferably, a human
receptor. Less preferred are their non-human homologs.
[0040] Generally, the ligand will bind to the extracellular domain
of the receptor and thereby activate its intracellular tyrosine
kinase domain. Consequently, binding of the ligand to the receptor
can result in enhancement or inhibition of proliferation and/or
differentiation and/or activation of cells having a receptor for
AL-2 in vivo, ex vivo, or in vitro. Other effector functions
include signal transduction, any enzyme activity or enzyme
modulatory activity (e.g., tyrosine kinase activity), or any
structural role, for example. An antigenic function means
possession of an epitope or antigenic site that is capable of
cross-reacting with antibodies raised against the polypeptide
sequence of a naturally occurring polypeptide comprising the
polypeptide sequences of FIGS. 1A-1B and 2A-2B.
[0041] In preferred embodiments, antigenically active AL-2 is a
polypeptide that binds with an affinity of at least about 10.sup.6
l/mole to an antibody capable of binding AL-2. Ordinarily, the
polypeptide binds with an affinity of at least about 10.sup.7
l/mole. In particular, an AL-2 is able to promote or enhance the
growth, survival, function, activation, and/or differentiation of
neurons and glia, whether the neurons be central, peripheral,
motoneurons, or sensory neurons, e.g., photoreceptors, vestibular
ganglia, spinal ganglia, auditory hair cells, and the AL-2 is
immunologically cross-reactive with an antibody directed against an
epitope of naturally occurring AL-2. Therefore, AL-2 amino acid
sequence variants generally will share at least about 75%
(preferably at least 80%, more preferably at least 90%, even more
preferably at least 95%, with increasing preference to at least
99%, and finally 100%) sequence identity with the translated amino
acid sequence set forth in FIGS. 1A-1B and 2A-2B, after aligning
the sequences and introducing gaps, if necessary, to achieve
maximal percent identity. This is typically determined, for
example, by the Fitch, et al., Proc. Nat. Acad. Sci. USA,
80:1382-1386 (1983), version of the algorithm described by
Needleman, et al., J. Mol. Biol., 48:443-453 (1970). None of
N-terminal, C-terminal, or internal extensions, deletions, or
insertions into the AL-2 sequence shall be construed as affecting
sequence identity or homology. Preferably, the AL-2 nucleic acid
molecule that hybridizes to nucleic acid sequence encoding AL-2
contains at least 20, more preferably 40, even more preferably 70,
and most preferably 90 bases. For fragments, the percent identity
is calculated for that portion of a native sequence that is present
in the fragment.
[0042] In one embodiment an isolated AL-2 protein induces
phosphorylation of an Eph-family receptor and contains an amino
acid sequence selected from the group consisting of (a) the amino
acid sequence for mature AL-2l, (b) the amino acid sequence for
mature AL-2s, (c) the naturally occurring amino acid sequence for
mature AL-2 from a non-human animal species, (d) allelic variants
of the sequences of (a), (b), or (c), and (e) the sequences of (a),
(b), (c), or (d) having a single preferred conservative amino acid
substitution as defined in Table 1. In a preferred embodiment the
phosphorylation-inducing AL-2 has the amino acid sequence for
mature human AL-2 shown in FIGS. 1A-1B or 2A-2B. Generally the AL-2
will be a chimera, membrane or liposome bound, or epitope tagged
and "clustered" (see WO 95/27060, which is incorporated herein by
reference), thus mimicking its membrane-bound state and ability to
induce receptor phosphorylation. In another embodiment an isolated
AL-2 protein binds to the Eph-family receptor and contains an amino
acid sequence selected from the group consisting of (a) the amino
acid sequence for mature AL-2l, (b) the amino acid sequence for
mature AL-2s, (c) the naturally occurring amino acid sequence for
mature AL-2 from a non-human animal species, (d) allelic variants
of the sequences of (a), (b), or (c), and (e) the sequences of (a),
(b), (c), or (d) having a single preferred conservative amino acid
substitution as defined in Table 1. In a preferred embodiment the
AL-2 has the amino acid sequence for mature human AL-2 shown in
FIGS. 1A-1B or 2A-2B. In another embodiment isolated soluble AL-2
binds to a Eph-family receptor and contains an amino acid sequence
selected from the group consisting of (a) the amino acid sequence
for mature soluble AL-2l, (b) the amino acid sequence for mature
soluble AL-2s, (c) the naturally occurring amino acid sequence for
mature soluble AL-2 from a non-human animal species, (d) allelic
variants of the sequences of (a), (b), or (c), and (e) the
sequences of (a), (b), (c), or (d) having a single preferred
conservative amino acid substitution as defined in Table 1. In a
preferred embodiment the soluble AL-2 has the amino acid sequence
for mature soluble human AL-2 shown in FIGS. 1A-1B or 2A-2B. In
another preferred embodiment, the soluble AL-2 is a chimeric
polypeptide containing an amino acid sequence encoding mature
soluble AL-2 fused to an immunoglobulin sequence. In a more
preferred embodiment the chimeric polypeptide contains a fusion of
an AL-2 extracellular domain sequence to an immunoglobulin constant
domain sequence. Preferably the constant domain sequence is that of
an immunoglobulin heavy chain. Also preferred are chimeric
polypeptides containing a mature, soluble Al-2 amino acid sequence
fused to an epitope tag polypeptide sequence.
[0043] AL-2 can be recovered from culture of cells expressing AL-2,
preferably from the culture medium as a secreted polypeptide;
although, AL-2 can be recovered from host cell lysates when
directly produced without a secretory signal. When AL-2 is
membrane-bound, it can be released from the membrane using a
suitable detergent solution (e.g., Triton-X 100.TM.). When AL-2 is
produced in a recombinant cell other than one of human origin, AL-2
is completely free of proteins or polypeptides of human origin.
However, it is necessary to purify AL-2 from recombinant cell
proteins or polypeptides to obtain preparations that are
substantially homogeneous in AL-2. As a first step, the culture
medium or lysate is centrifuged to remove particulate cell debris.
Then AL-2 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; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
gel filtration using, for example, Sephadex.TM. G-75; and protein A
Sepharose.TM. columns to remove contaminants such as IgG.
[0044] In a preferred embodiment, an AL-2-receptor-Fc fusion, using
the preferred AL-2-receptors in Fc constructs, for example as
disclosed by Bennett et al., J. Biol. Chem., 269(19):14211-8
(1994), is immobilized on a protein A Sepharose.TM. column and AL-2
can be isolated by affinity purification using this column.
[0045] AL-2 variants in which residues have been deleted, inserted,
or substituted are recovered in the same fashion as native AL-2,
taking account of any substantial changes in properties resulting
from the variation. For example, preparation of an AL-2 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. Immunoaffinity columns such as a rabbit polyclonal
anti-AL-2 column can be employed to absorb the AL-2 variant by
binding it to at least one remaining immune AL-2 epitope. 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 AL-2 can
require modification to account for changes in the character of
AL-2 or its variants upon expression in recombinant cell
culture.
[0046] Amino acid sequence variants of AL-2 are prepared by
introducing appropriate nucleotide changes into AL-2 DNA and
thereafter expressing the resulting modified DNA in a host cell, or
by in vitro synthesis. Such variants include, for example,
deletions from, or insertions or substitutions of, amino acid
residues within the AL-2 amino acid sequence set forth in FIGS.
1A-1B and 2A-2B. Any combination of deletion, insertion, and
substitution may be made to arrive at an amino acid sequence
variant of AL-2, provided that such variant possesses the desired
characteristics described herein. Changes that are made in the
amino acid sequence set forth in FIGS. 1A-1B and 2A-2B to arrive at
an amino acid sequence variant of AL-2 also may result in further
modifications of AL-2 upon its expression in host cells, for
example, by virtue of such changes introducing or moving sites of
glycosylation, or introducing membrane anchor sequences as
described, for example, in PCT Pat. Pub. No. WO 89/01041 (published
Feb. 9, 1989).
[0047] There are two principal variables in the construction of
amino acid sequence variants of AL-2: the location of the mutation
site and the nature of the mutation. These are variants from the
amino acid sequence set forth in FIGS. 1A-1B and 2A-2B, and may
represent naturally occurring allelic forms of AL-2, or
predetermined mutant forms of AL-2 made by mutating AL-2 DNA,
either to arrive at an allele or a variant not found in nature. In
general, the location and nature of the mutation chosen will depend
upon the AL-2 characteristic to be modified.
[0048] For example, due to the degeneracy of nucleotide coding
sequences, mutations can be made in the AL-2 nucleotide sequence
set forth in FIGS. 1A-1B and 2A-2B without affecting the amino acid
sequence of the AL-2 encoded thereby. Other mutations can be made
that will result in a AL-2 that has an amino acid sequence
different from that set forth in FIGS. 1A-1B and 2A-2B, but which
is functionally active. Such functionally active amino acid
sequence variants of AL-2 are selected, for example, by
substituting one or more amino acid residues in the amino acid
sequence set forth in FIGS. 1A-1B and 2A-2B with other amino acid
residues of a similar or different polarity or charge.
[0049] One useful approach is called "alanine scanning
mutagenesis." Here, a an amino acid residue or group of target
residues are identified (e.g., charged residues such as arginine,
aspartic acid, histidine, lysine, and glutamic acid) and, by means
of recombinant DNA technology, replaced by a neutral or negatively
charged amino acid (most preferably alanine or polyalanine) to
affect the interaction of the amino acids with the surrounding
aqueous environment in or outside the cell, (Cunningham, et al.,
Science, 244:1081-1085 (1989)). Those domains demonstrating
functional sensitivity to the substitutions then are refined by
introducing further or other variants at or for the sites of
substitution. Obviously, such variations that, for example, convert
the amino acid sequence set forth in FIGS. 1A-1B and 2A-2B to the
amino acid sequence of a known neurotrophic factor, such as NGF,
BDNF, NT-3, NT-4/5, Eph-family receptor ligand (e.g., see FIGS. 4
and 5), or another known polypeptide or protein are not included
within the scope of this invention, nor are any other fragments,
variants, and derivatives of the amino acid AL-2 that are not novel
and unobvious over the prior art. Thus, while the site for
introducing an amino acid sequence variation is predetermined, the
nature of the mutation per se need not be predetermined. For
example, to optimize the performance of a mutation at a given site,
ala scanning or random mutagenesis is conducted at the target codon
or region and the expressed AL-2 variants are screened for
functional activity.
[0050] Amino acid sequence deletions generally range from about 2
to 30 residues, more preferably about 1 to 10 residues, and
typically are contiguous. Deletions from regions of substantial
homology with other tyrosine kinase receptor ligands, for example,
are more likely to affect the functional activity of AL-2.
Generally, the number of consecutive deletions will be selected so
as to preserve the tertiary structure of AL-2 in the affected
domain, e.g., beta-pleated sheet or alpha helix.
[0051] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one amino acid
residue to polypeptides containing a hundred or more residues, as
well as intrasequence insertions of single or multiple amino acid
residues. Intrasequence insertions, i.e., insertions made within
the amino acid sequence set forth in FIGS. 1A-1B or 2A-2B, may
range generally from about 1 to 10 residues, more preferably 2 to
5, even more preferably 2 to 3, and most preferably 1 to 2.
Examples of terminal insertions include AL-2 with an N-terminal
methionyl residue (such as may result from the direct expression of
AL-2 in recombinant cell culture), and AL-2 with a heterologous
N-terminal signal sequence to improve the secretion of AL-2 from
recombinant host cells. Such signal sequences generally will be
homologous to the host cell used for expression of AL-2, and
include STII or Ipp for E. coli, alpha factor for yeast, and viral
signals such as herpes gD for mammalian cells. Other insertions
include the fusion to the N- or C-terminus of AL-2 of immunogenic
polypeptides (for example, bacterial polypeptides such as
beta-lactamase or an enzyme encoded by the E. coli trp locus, or
yeast protein), and C-terminal fusions with proteins having a long
half-life such as immunoglobulin constant regions, albumin, or
ferritin, as described in PCT Pat. Pub. No. WO 89/02922 published
Apr. 6, 1989.
[0052] The third group of variants are those in which at least one
amino acid residue in the amino acid sequence set forth in FIGS.
1A-1B or 2A-2B, preferably one to four, more preferably one to
three, even more preferably one to two, and most preferably only
one, has been removed and a different residue inserted in its
place. The sites of greatest interest for making such substitutions
are in the regions of the amino acid sequence set forth in FIGS.
1A-1B or 2A-2B that have the greatest homology with other tyrosine
kinase receptor ligands (for non-limiting examples, see comparisons
in FIGS. 4 and 5). Those sites are likely to be important to the
functional activity of the AL-2. Accordingly, to retain functional
activity, those sites, especially those falling within a sequence
of at least three other identically conserved sites, are
substituted in a relatively conservative manner. Such conservative
substitutions are shown in Table 1 under the heading of preferred
substitutions. If such substitutions do not result in a change in
functional activity, then more substantial changes, denominated
exemplary substitutions in Table 1, or as further described below
in reference to amino acid classes, may be introduced and the
resulting variant AL-2 analyzed for functional activity.
TABLE-US-00001 TABLE 1 Original Residue Exemplary Substitutions
Preferred Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln;
asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser
ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro pro His (H) asn;
gln; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leu
Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln;
asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala leu
Pro (P) gly gly Ser (S) thr thr Thr (T) ser ser Trp (W) ryr tyr Tyr
(Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala;
norleucine leu
[0053] Insertional, deletional, and substitutional changes in the
amino acid sequence set forth in FIGS. 1A-1B and 2A-2B may be made
to improve the stability of AL-2. For example, trypsin or other
protease cleavage sites are identified by inspection of the encoded
amino acid sequence for an arginyl or lysinyl residue. These are
rendered inactive to protease by substituting the residue with
another residue, preferably a basic residue such as glutamine or a
hydrophobic residue such as serine; by deleting the residue; or by
inserting a prolyl residue immediately after the residue. Also, any
cysteine residues not involved in maintaining the proper
conformation of AL-2 for functional activity may be substituted,
generally with serine, to improve the oxidative stability of the
molecule and prevent aberrant crosslinking.
[0054] Additional sites for mutation are those sites that are
conserved in AL-2 amongst species variants of AL-2 but are not
conserved between AL-2 and another ligand in the Eph ligand family,
preferably between AL-2 and at least two ligands, and more
preferably at least three ligands. Such sites, which are not
conserved between AL-2 and another transmembrane-ligand, are
candidates sites for modulating receptor specificity and
selectivity. Sites that are conserved between AL-2 and other
transmembrane-ligands are candidate sites for modulating activities
shared by transmembrane-ligands, such as stability, folding,
tertiary conformation, protease susceptibility, and amount of
ligand specific activity.
[0055] A comparison of AL-2 amino acid sequences with other
Eph-family receptor ligand sequences (see FIGS. 4 and 5) reveals
AL-2 as a new Eph-family receptor ligand. AL-2, having a
transmembrane sequence, is more closely related to other
transmembrane-containing ligands than to the GPI-anchored ligands,
of which AL-1 is an example. Transmembrane-containing ligands
include Lerk-2, a ligand for the Eph-related receptor Hek5, and
Htk-L, a ligand for the Htk receptor. Percent identities of ligand
comparisons are provided in Table 2, in which "ECD" indicates
extracellular domain. TABLE-US-00002 TABLE 2 % IDENTITY Ligand Full
Length ECD Cytoplasmic Domain Lerk2 vs. HtkL 56.0% 49.3% 74.7% AL-2
vs Lerk2 41.5% 42.1% 48.2% AL-2 vs HtkL 40.8% 39.5% 56.6% AL-2 vs
AL-1 28.0%
[0056] Covalent modifications of AL-2 molecules also are included
within the scope of this invention. For example, covalent
modifications are introduced into AL-2 by reacting targeted amino
acid residues of the AL-2 with an organic derivatizing agent that
is capable of reacting with selected amino acid side chains or the
N- or C-terminal residues.
[0057] Cysteinyl residues most commonly are reacted with
.alpha.-haloacetates (and corresponding amines), such as
chloroacetic acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole.
[0058] Histidyl residues are derivatized by reaction with
diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively
specific for the histidyl side chain. Para-bromophenacyl bromide
also is useful; the reaction is preferably performed in 0.1M sodium
cacodylate at pH 6.0.
[0059] Lysinyl and amino terminal residues are reacted with
succinic or other carboxylic acid anhydrides. Derivatization with
these agents has the effect of reversing the charge of the lysinyl
residues. Other suitable reagents for derivatizing
.alpha.-amino-containing residues include imidoesters such as
methyl picolinimidate; pyridoxal phosphate; pyridoxal;
chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea;
2,4-pentanedione; and transaminase-catalyzed reaction with
glyoxylate.
[0060] Arginyl residues are modified by reaction with one or
several conventional reagents, among them phenylglyoxal,
2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.
Derivatization of arginine residues requires that the reaction be
performed in alkaline conditions because of the high pK.sub.a of
the guanidine functional group. Furthermore, these reagents may
react with the groups of lysine as well as the arginine
epsilon-amino group.
[0061] The specific modification of tyrosyl residues may be made,
with particular interest in introducing spectral labels into
tyrosyl residues by reaction with aromatic diazonium compounds or
tetranitromethane. Most commonly, N-acetylimidizole and
tetranitromethane are used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively. Tyrosyl residues are iodinated
using .sup.125I or .sup.131I to prepare labeled proteins for use in
radioimmunoassay, the chloramine T method described above being
suitable.
[0062] Carboxyl side groups (aspartyl or glutamyl) are selectively
modified by reaction with carbodiimides (R'--N.dbd.C.dbd.N--R'),
where R and R' are different alkyl groups, such as
1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,
aspartyl and glutamyl residues are converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
[0063] Derivatization with bifunctional agents is useful for
crosslinking AL-2 to a water-insoluble support matrix or surface
for use in the method for purifying anti-AL-2 antibodies, or for
therapeutic use. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides
such as bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate yield
photoactivatable intermediates that are capable of forming
crosslinks in the presence of light. Alternatively, reactive
water-insoluble matrices such as cyanogen bromide-activated
carbohydrates and the reactive substrates described in U.S. Pat.
Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and
4,330,440 are employed for protein immobilization.
[0064] Glutaminyl and asparaginyl residues are frequently
deamidated to the corresponding glutamyl and aspartyl residues,
respectively. Alternatively, these residues are deamidated under
mildly acidic conditions. Either form of these residues falls
within the scope of this invention.
[0065] Other modifications include hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of seryl or threonyl
residues, methylation of the .alpha.-amino groups of lysine,
arginine, and histidine side chains, acetylation of the N-terminal
amine, and amidation of any C-terminal carboxyl group, (Creighton,
Proteins: Structure and Molecular Properties, pp. 79-86 (W.H.
Freeman & Co., 1983)). AL-2 also is covalently linked to
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat.
Nos. 4,179,337; 4,301,144; 4,496,689; 4,640,835; 4,670,417; or
4,791,192.
[0066] "AL-2 antagonist" or "antagonist" refers to a substance that
opposes or interferes with a functional activity of AL-2.
[0067] "Cell," "host cell," "cell line." and "cell culture" are
used interchangeably and all such terms should be understood to
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of times the cultures have
been passaged. It should also be understood that all progeny may
not be precisely identical in DNA content, due to deliberate or
inadvertent mutations.
[0068] "Plasmids" are DNA molecules that are capable of replicating
within a host cell, either extrachromosomally or as part of the
host cell chromosome(s), and are designated by a lower case "p"
preceded and/or followed by capital letters and/or numbers. The
starting plasmids herein are commercially available, are publicly
available on an unrestricted basis, or can be constructed from such
available plasmids as disclosed herein and/or in accordance with
published procedures. In certain instances, as will be apparent to
the ordinarily skilled artisan, other plasmids known in the art may
be used interchangeably with plasmids described herein.
[0069] "Control sequences" refers to DNA sequences necessary for
the expression of an operably linked nucleotide coding sequence in
a particular host cell. The control sequences that are suitable for
expression in prokaryotes, for example, include origins of
replication, promoters, ribosome binding sites, and transcription
termination sites. The control sequences that are suitable for
expression in eukaryotes, for example, include origins of
replication, promoters, ribosome binding sites, polyadenylation
signals, and enhancers.
[0070] An "exogenous" element is one that is foreign to the host
cell, or homologous to the host cell but in a position within the
host cell in which the element is ordinarily not found.
[0071] "Digestion" of DNA refers to the catalytic cleavage of DNA
with an enzyme that acts only at certain locations in the DNA. Such
enzymes are called restriction enzymes or restriction
endonucleases, and the sites within DNA where such enzymes cleave
are called restriction sites. If there are multiple restriction
sites within the DNA, digestion will produce two or more linearized
DNA fragments (restriction fragments). The various restriction
enzymes used herein are commercially available and their reaction
conditions, cofactors, and other requirements as established by the
enzyme manufacturers are used. Restriction enzymes commonly are
designated by abbreviations composed of a capital letter followed
by other letters representing the microorganism from which each
restriction enzyme originally was obtained and then a number
designating the particular enzyme. In general, about 1 .mu.g of DNA
is digested with about 1-2 units of enzyme in about 20 .mu.l of
buffer solution. Appropriate buffers and substrate amounts for
particular restriction enzymes are specified by the manufacturer,
and/or are well known in the art.
[0072] "Recovery" or "isolation" of a given fragment of DNA from a
restriction digest typically is accomplished by separating the
digestion products, which are referred to as "restriction
fragments," on a polyacrylamide or agarose gel by electrophoresis,
identifying the fragment of interest on the basis of its mobility
relative to that of marker DNA fragments of known molecular weight,
excising the portion of the gel that contains the desired fragment,
and separating the DNA from the gel, for example by
electroelution.
[0073] "Ligation" refers to the process of forming phosphodiester
bonds between two double-stranded DNA fragments. Unless otherwise
specified, ligation is accomplished using known buffers and
conditions with 10 units of T4 DNA ligase per 0.5 .mu.g of
approximately equimolar amounts of the DNA fragments to be
ligated.
[0074] "Oligonucleotides" are short-length, single- or
double-stranded polydeoxynucleotides that are chemically
synthesized by known methods (involving, for example, triester,
pbosphoramidite, or phosphonate chemistry), such as described by
Engels, et al., Agnew. Chem. Int. Ed. Engl, 28:716-734 (1989). They
are then purified, for example, by polyacrylamide gel
electrophoresis.
[0075] "Polymerase chain reaction," or "PCR," as used herein
generally refers to a method for amplification of a desired
nucleotide sequence in vitro, as described in U.S. Pat. No.
4,683,195. In general, the PCR method involves repeated cycles of
primer extension synthesis, using two oligonucleotide primers
capable of hybridizing preferentially to a template nucleic acid.
Typically, the primers used in the PCR method will be complementary
to nucleotide sequences within the template at both ends of or
flanking the nucleotide sequence to be amplified, although primers
complementary to the nucleotide sequence to be amplified also may
be used (Wang, et al., in PCR Protocols, pp. 70-75 (Academic Press,
1990); Ochman, et al., in PCR Protocols, pp. 219-227; Triglia, et
al., Nuc. Acids Res., 16:8186 (1988)).
[0076] "PCR cloning" refers to the use of the PCR method to amplify
a specific desired nucleotide sequence that is present amongst the
nucleic acids from a suitable cell or tissue source, including
total genomic DNA and cDNA transcribed from total cellular RNA
(Frohman, et al., Proc. Nat. Acad. Sci. USA, 85:8998-9002 (1988);
Saiki, et al., Science, 239:487-492 (1988); Mullis, et at., Meth.
Enzymol., 155:335-350 (1987)).
[0077] "Stringent conditions" for hybridization or annealing of
nucleic acid molecules are those that (1) employ low ionic strength
and high temperature for washing, for example, 0.015 M NaCl/0.0015
M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 50.degree.
C., or (2) employ during hybridization a denaturing agent such as
formamide, for example, 50% (vol/vol) formamide with 0.1% bovine
serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate
at 42.degree. C. Another example is use of 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times. Denhardt's
solution, sonicated salmon sperm DNA (50 .mu.g/mL), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree. C.
in 0.2.times.SSC and 0.1% SDS.
[0078] "AL-2 nucleic acid" is RNA or DNA that encodes AL-2. "AL-2
DNA" is DNA that encodes AL-2. AL-2 DNA is obtained from cDNA or
genomic DNA libraries, or by in vitro synthesis. Identification of
AL-2 DNA within a cDNA or a genomic DNA library, or in some other
mixture of various DNAs, is conveniently accomplished by the use of
an oligonucleotide hybridization probe that is labeled with a
detectable moiety, such as a radioisotope (Keller, et al., DNA
Probes, pp. 149-213 (Stockton Press, 1989)). To identify DNA
encoding AL-2, the nucleotide sequence of the hybridization probe
preferably is selected so that the hybridization probe is capable
of hybridizing preferentially to DNA encoding the AL-2 amino acid
sequence set forth in FIGS. 1A-1B or 2A-2B, or a variant or
derivative thereof as described herein, under the hybridization
conditions chosen. Another method for obtaining AL-2 nucleic acid
is to chemically synthesize it using one of the methods described,
for example, by Engels, et al., Agnew. Chem. Int. Ed. Engl.,
28:716-734 (1989). A preferred embodiment is an isolated nucleic
acid molecule that includes a nucleotide sequence encoding the
amino acid sequence shown in FIGS. 1A-1B or 2A-2B for mature AL-2,
and in which, more preferably, the AL-2 codons are contiguous. A
preferred nucleotide sequence encoding the amino acid sequence for
mature AL-2 can be found in FIGS. 1A-1B or 2A-2B. Also included are
AL-2-encoding nucleic acid sequences based on the codon degeneracy
of the genetic code.
[0079] If the entire nucleotide coding sequence for AL-2 is not
obtained in a single cDNA, genomic DNA, or other DNA, as
determined, for example, by DNA sequencing or restriction
endonuclease analysis, then appropriate DNA fragments (e.g.,
restriction fragments or PCR amplification products) may be
recovered from several DNAs and covalently joined to one another to
construct the entire coding sequence. The preferred means of
covalently joining DNA fragments is by ligation using a DNA ligase
enzyme, such as T4 DNA ligase.
[0080] "Isolated" AL-2 nucleic acid is AL-2 nucleic acid that is
identified and separated from (or otherwise substantially free
from) contaminant nucleic acid encoding another polypeptide or from
nucleic acid with which it is normally associated in the natural
source of AL-2 nucleic acid. Isolated AL-2 nucleic acid molecules
therefore are distinguished from the AL-2 nucleic acid molecule as
it occurs naturally in cells. However, an isolated AL-2 nucleic
acid molecule includes AL-2 nucleic acid molecules contained in
cells that ordinarily express AL-2 where, for example, the nucleic
acid molecule is in a chromosomal location different from that of
natural cells. The isolated AL-2 nucleic acid can be incorporated
into a plasmid or expression vector for in vitro, ex vivo or in
vivo use, or can be labeled for diagnostic and probe purposes,
using a label as described further herein in the discussion of
diagnostic assays and nucleic acid hybridization methods.
[0081] For example, isolated AL-2 DNA, or a fragment thereof
comprising at least about 15 nucleotides, is used as a
hybridization probe to detect, diagnose, or monitor disorders or
diseases that involve changes in AL-2 expression, such as may
result from neuron damage. In one embodiment of the invention,
total RNA in a tissue sample from a patient (that is, a human or
other mammal) can be assayed for the presence of AL-2 messenger
RNA, wherein the decrease in the amount of AL-2 messenger RNA is
indicative of neuronal degeneration.
[0082] The present invention further provides antisense or sense
oligonucleotides comprising a single-stranded nucleic acid sequence
(either RNA or DNA) capable of binding to target AL-2 mRNA (sense)
or AL-2 DNA (antisense) sequences. Antisense or sense
oligonucleotides, according to the present invention, comprise a
fragment of the coding region of AL-2 cDNA. Such a fragment
generally comprises at least about 14 nucleotides, preferably from
about 14 to about 30 nucleotides. The ability to create an
antisense or a sense oligonucleotide, based upon a cDNA sequence
for a given protein is described for example, in Stein et al.,
Cancer Res., 48:2659 (1988) and van der Krol et al., BioTechniques,
6:958,1988. Although not to be restricted by the following working
model, it is generally believed that binding of antisense or sense
oligonucleotides to target nucleic acid sequences results in the
formation of duplexes that block translation (RNA) or transcription
(DNA) by one of several means, including enhanced degradation of
the duplexes, premature termination of transcription or
translation, or by other means. The antisense oligonucleotides can
be used to block expression of AL-2 proteins. Antisense or sense
oligonucleotides further comprise oligonucleotides having modified
sugar-phosphodiester backbones (or other sugar linkages, such as
those described in WO91/06629) and wherein such sugar linkages are
resistant to endogenous nucleases. Such oligonucleotides with
resistant sugar linkages are stable in vivo (i.e., capable of
resisting enzymatic degradation) but retain sequence specificity to
be able to bind to target nucleotide sequences. Other examples of
sense or antisense oligonucleotides include those oligonucleotides
which are covalently linked to organic moieties, such as those
described in WO 90/10448, and other moieties that increase affinity
of the oligonucleotide for a target nucleic acid sequence, such as
poly-(L-lysine). Further still, intercalating agents, such as
ellipticine, and alkylating agents or metal complexes can be
attached to sense or antisense oligonucleotides to modify binding
specificities of the antisense or sense oligonucleotide for the
target nucleotide sequence.
[0083] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO4-mediated DNA
transfectibn, electroporation, or other gene transfer vectors such
as Epstein-Barr virus or adenovirus. Antisense or sense
oligonucleotides are preferably introduced into a cell containing
the target nucleic acid sequence by insertion of the antisense or
sense oligonucleotide into a suitable retroviral vector, then
contacting the cell with the retrovirus vector containing the
inserted sequence, either in vivo or ex vivo. Suitable retroviral
vectors include, but are not limited to, the murine retrovirus
M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy
vectors designated DCT5A. DCT5B and DCT5C (see PCT U.S. application
Ser. No. 90/02656). Alternatively, other promotor sequences may be
used to express the oligonucleotide. Most preferably, target-tissue
specific promoters (either constitutive or inducible) are used.
[0084] Sense or antisense oligonucleotides are also introduced into
a cell containing the target nucleotide sequence by formation of a
conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to specific cell surface
receptors. Alternatively, a sense or an antisense oligonucleotide
is introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by all endogenous lipase.
[0085] Isolated AL-2 nucleic acid also is used to produce AL-2 by
recombinant DNA and recombinant cell culture methods. In various
embodiments of the invention, host cells are transformed or
transfected with recombinant DNA molecules comprising an isolated
AL-2 DNA, to obtain expression of the AL-2 DNA and thus the
production of AL-2 in large quantities. DNA encoding amino acid
sequence variants of AL-2 is prepared by a variety of methods known
in the art. These methods include, but are not limited to,
isolation from a natural source (in the case of naturally occurring
amino acid sequence variants of AL-2) or preparation by
site-directed (or oligonucleotide-mediated) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared DNA
encoding a variant or a non-variant form of AL-2.
[0086] Site-directed mutagenesis is a preferred method for
preparing substitution, deletion, and insertion variants of AL-2
DNA. This technique is well known in the art (Zoller et al., Meth
Enz., 100:4668-500 (1983); Zoller et al., Meth. Enz.,
154:329-350(1987); Carter, Meth. Enz., 154:382-403 (1987); Horwitz
et al., Meth. Enz. 185:599-611 (1990)), and has been used, for
example, to produce amino acid sequence variants of trypsin and T4
lysozyme, which variants have certain desired functional properties
(Perry et al., Science, 226:555-557 (1984); Craik et al., Science,
228:291-297 (1985)).
[0087] Briefly, in carrying out site-directed mutagenesis of AL-2
DNA, the AL-2 DNA is altered by first hybridizing an
oligonucleotide encoding the desired mutation to a single strand of
such AL-2 DNA. After hybridization, a DNA polymerase is used to
synthesize an entire second strand, using the hybridized
oligonucleotide as a primer, and using the single strand of AL-2
DNA as a template. Thus, the oligonucleotide encoding the desired
mutation is incorporated in the resulting double-stranded DNA.
[0088] Oligonucleotides for use as hybridization probes or primers
may be prepared by any suitable method, such as by purification of
a naturally occurring DNA or by in vitro synthesis. For example,
oligonucleotides are readily synthesized using various techniques
in organic chemistry, such as described by Narang et al., Meth.
Enzymol., 68:90-98 (1979); Brown et al., Meth Enzymol., 68:109-151
(1979); and Caruther et al., Meth. Enzymol., 154:287-313 (1985).
The general approach to selecting a suitable hybridization probe or
primer is well known (Keller et al., DNA Probes, pp. 11-18
(Stockton Press, 1989)). Typically, the hybridization probe or
primer will contain 10-25 or more nucleotides, and will include at
least 5 nucleotides on either side of the sequence encoding the
desired mutation so as to ensure that the oligonucleotide will
hybridize preferentially to the single-stranded DNA template
molecule.
[0089] Multiple mutations are introduced into AL-2 DNA to produce
amino acid sequence variants of AL-2 comprising several or a
combination of insertions, deletions, or substitutions of amino
acid residues as compared to the amino acid sequence set forth in
FIGS. 1A-1B or 2A-2B. If the sites to be mutated are located close
together, the mutations may be introduced simultaneously using a
single oligonucleotide that encodes all of the desired mutations.
If, however, the sites to be mutated are located some distance from
each other (separated by more than about ten nucleotides), it is
more difficult to generate a single oligonucleotide that encodes
all of the desired changes. Instead, one of two alternative methods
may be employed.
[0090] In the first method, a separate oligonucleotide is generated
for each desired mutation. The oligonucleotides are then annealed
to the single-stranded template DNA simultaneously, and the second
strand of DNA that is synthesized from the template will encode all
of the desired amino acid substitutions.
[0091] The alternative method involves two or more rounds of
mutagenesis to produce the desired mutant. The first round is as
described for introducing a single mutation: a single strand of a
previously prepared AL-2 DNA is used as a template, an
oligonucleotide encoding the first desired mutation is annealed to
this template, and a heteroduplex DNA molecule is then generated.
The second round of mutagenesis utilizes the mutated DNA produced
in the first round of mutagenesis as the template. Thus, this
template already contains one or more mutations. The
oligonucleotide encoding the additional desired amino acid
substitution(s) is then annealed to this template, and the
resulting strand of DNA now encodes mutations from both the first
and second rounds of mutagenesis. This resultant DNA can be used as
a template in a third round of mutagenesis, and so on.
[0092] PCR mutagenesis is also suitable for making amino acid
sequence variants of AL-2 (Higuchi, in PCR Protocols, pp. 177-183
(Academic Press, 1990); Vallette et al., Nuc. Acids Res.,
17:723-733 (1989)). Briefly, when small amounts of template DNA are
used as starting material in a PCR, primers that differ slightly in
sequence from the corresponding region in a template DNA can be
used to generate relatively large quantities of a specific DNA
fragment that differs from the template sequence only at the
positions where the primers differ from the template. For
introduction of a mutation into a plasmid DNA, for example, one of
the primers is designed to overlap the position of the mutation and
to contain the mutation; the sequence of the other primer must be
identical to a nucleotide sequence within the opposite strand of
the plasmid DNA, but this sequence can be located anywhere along
the plasmid DNA. It is preferred, however, that the sequence of the
second primer is located within 200 nucleotides from that of the
first, such that in the end the entire amplified region of DNA
bounded by the primers can be easily sequenced. PCR amplification
using a primer pair like the one just described results in a
population of DNA fragments that differ at the position of the
mutation specified by the primer, and possibly at other positions,
as template copying is somewhat error-prone (Wagner et al., in PCR
Topics, pp. 69-71 (Springer-Verlag, 1991)).
[0093] If the ratio of template to product amplified DNA is
extremely low, the majority of product DNA fragments incorporate
the desired mutation(s). This product DNA is used to replace the
corresponding region in the plasmid that served as PCR template
using standard recombinant DNA methods. Mutations at separate
positions can be introduced simultaneously by either using a mutant
second primer, or performing a second PCR with different mutant
primers and ligating the two resulting PCR fragments simultaneously
to the plasmid fragment in a three (or more)-part ligation.
[0094] Another method for preparing variants, cassette mutagenesis,
is based on the technique described by Wells et al., Gene,
34:315-323 (1985). The starting material is the plasmid (or other
vector) comprising the AL-2 DNA to be mutated. The codon(s) in the
AL-2 DNA to be mutated are identified. There must be a unique
restriction endonuclease site on each side of the identified
mutation site(s). If no such restriction sites exist, they may be
generated using the above-described oligonucleotide-mediated
mutagenesis method to introduce them at appropriate locations in
the AL-2 DNA. The plasmid DNA is cut at these sites to linearize
it. A double-stranded oligonucleotide encoding the sequence of the
DNA between the restriction sites but containing the desired
mutation(s) is synthesized using standard procedures, wherein the
two strands of the oligonucleotide are synthesized separately and
then hybridized together using standard techniques. This
double-stranded oligonucleotide is referred to as the cassette.
This cassette is designed to have 5' and 3' ends that are
compatible with the ends of the linearized plasmid, such that it
can be directly ligated to the plasmid. This plasmid now contains
the mutated AL-2 DNA sequence.
[0095] In another embodiment, AL-2 suitable for therapy is AL-2
covalently joined to another protein, such as an immunoglobulin
domain (for example, to produce an AL2-IgG fusion protein).
Immunoglobulin fusions, immunoadhesins, are chimeric antibody-like
molecules that combine the functional domain(s) of a binding
protein (in this case AL-2 or its receptor) with the an
immunoglobulin sequence. The immunoglobulin sequence preferably
(but not necessarily) is an immunoglobulin constant domain.
Immunoglobulins (Ig) and certain variants thereof are known and
many have been prepared in recombinant cell culture. For example,
see U.S. Pat. No. 4,745,055; EP 256,654; Faulkner et al., Nature,
298:286 (1982); EP 120,694; EP 125,023; Morrison, J. Immun.,
123:793 (1979); Kohler et al., Proc. Nat'l. Acad. Sci. USA, 77:2197
(1980); Raso et al., Cancer Res., 41:2073 (1981); Morrison et al.,
Ann. Rev. Immunol., 2:239 (1984); Morrison, Science, 229:1202
(1985); Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851
(1984); EP 255,694; EP 266,663; and WO 88/03559. Reassorted
immunoglobulin chains also are known. See for example U.S. Pat. No.
4,444,878; WO 88/03565; and EP 68,763 and references cited therein.
The immunoglobulin moiety in the chimeras of the present invention
may be obtained from IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA,
IgE, IgD or IgM, but preferably IgG-1 or IgG-3.
[0096] Chimeras constructed from a receptor sequence linked to an
appropriate immunoglobulin constant domain sequence
(immunoadhesins) are known in the art. Immunoadhesins reported in
the literature include fusions of the T cell receptor* (Gascoigne
et al., Proc. Natl. Acad. Sci. USA, 84:2936-2940 (1987)); CD4*
(Capon et al., Nature, 337:525-531 (1989); Traunecker et al.,
Nature, 339:68-70 (1989); Zettmeissl et al, DNA Cell Biol. USA,
9:347-353 (1990); Byrn et al., Nature, 344:667-670 (1990));
L-selectin (homing receptor) (Watson et al., J. Cell. Biol.,
110:2221-2229 (1990); Watson et al., Nature, 349:164-167 (1991));
CD44* (Aruffo et al., Cell, 61:1303-1313 (1990)); CD28* and B7*
(Linsley et al., J. Exp. Med., 173721-730 (1991)); CTLA-4* (Lisley
et al., J. Exp. Med., 174:61-569 (1991)); CD22* (Stamenkovic et
al., Cell, 66:1133-1144 (1991)); where the asterisk (*) indicates
that the receptor is member of the immunoglobulin superfamily.
[0097] The simplest and most straightforward immunoadhesin design
combined the binding region(s) of the `adhesin` protein (in this
case AL-2) with the hinge and Fc regions of an immunoglobulin heavy
chain. Ordinarily, when preparing chimeras of the present
invention, nucleic acid encoding the extracellular domain or a
fragment thereof of AL-2 will be fused C-terminally to nucleic acid
encoding the N-terminus of an immunoglobulin constant domain
sequence, however N-terminal fusions are also possible. Typically,
in such fusions the encoded chimeric polypeptide will retain at
least functionally active hinge, CH2 and CH3 domains of the
constant region of an immunoglobulin heavy chain. Fusions are also
made to the C-terminus of the Fc portion of a constant domain, or
immediately N-terminal to the CH1 of the heavy chain or the
corresponding region of the light chain. The precise site at which
the fusion is made is not critical; particular sites are well known
and may be selected in order to optimize the biological activity,
secretion or binding characteristics of AL-2-immunoglobulin
chimeras.
[0098] In some embodiments, chimeras are assembled as monomers, or
hetero- or homo-multimers, and particularly as dimers or tetramers,
essentially as illustrated in WO 91/08298. In a preferred
embodiment, the AL-2 extracellular domain sequence is fused to the
N-terminus of the C-terminal portion of an antibody (in particular
the Fc domain), containing the effector functions of an
immunoglobulin, e.g., immunoglobulin G.sub.1 (IgG-1). It is
possible to fuse the entire heavy chain constant region to the AL-2
extracellular domain sequence. Preferably a sequence beginning in
the hinge region just upstream of the papain cleavage site (which
defines IgG Fc chemically; residue 216, taking the first residue of
heavy chain constant region to be 114, or analogous sites of other
immunoglobulins) is used in the fusion. In one embodiment, an AL-2
amino acid sequence is fused to the hinge region and CH2 and CH3 or
CH1, hinge, CH2 and CH3 domains of an IgG-1, IgG-2, or IgG-3 heavy
chain. The precise site at which the fusion is made is not
critical, and the optimal site can be determined by routine
experimentation. The immunoglobulin portion can genetically
engineered or chemically modified to inactivate a biological
activity of the immunoglobulin portion, such as T-cell binding,
while retaining desirable properties such as its scaffolding
property for presenting AL-2 function to an axon or target cell.
Chimeras can be assembled as multimers, particularly as homo-dimers
or -tetramers. Generally, these assembled immunoglobulins will have
known unit structures. A basic four chain structural unit is the
form in which IgG, IgD, and IgE exist. A four unit is repeated in
the higher molecular weight immunoglobulins; IgM generally exists
as a pentamer of basic four units held together by disulfide bonds.
IgA globulin, and occasionally IgG globulin, may also exist in
multimeric form in serum. In the case of multimer, each four unit
may be the same or different. Alternatively, the AL-2 extracellular
domain sequences can be inserted between immunoglobulin heavy chain
and light chain sequences such that an immunoglobulin comprising a
chimeric heavy chain is obtained. In this embodiment, the sequences
are fused to the 3' end of an immunoglobulin heavy chain in each
arm of an immunoglobulin, either between the hinge and the CH2
domain, or between the CH2 and CH3 domains (see Hoogenboom et al.,
Mol. Immunol., 28:1027-1037 (1991)). The presence of an
immunoglobulin light chain is not required in the immunoadhesins of
the present invention; an immunoglobulin light chain might be
present either covalently associated to a immunoglobulin heavy
chain fusion polypeptide, or directly fused to the AL-2
extracellular domain. In the former case, DNA encoding an
immunoglobulin light chain is typically coexpressed with the DNA
encoding the AL-2-immunoglobulin heavy chain fusion protein. Upon
secretion, the hybrid heavy chain and the light chain will be
covalently associated to provide an immunoglobulin-like structure
comprising two disulfide-linked immunoglobulin heavy chain-light
chain pairs. Preparation of such structures are, for example,
disclosed in U.S. Pat. No. 4,816,567 issued 28 Mar. 1989. The
immunoglobulin sequences used in the construction of the
immunoadhesins of the present invention can be from an IgG
immunoglobulin heavy chain constant domain. For human
immunoadhesins, the use of human IgG1 and IgG3 immunoglobulin
sequences is preferred. A major advantage of using IgG1 is that
IgG1 immunoadhesins can be purified efficiently on immobilized
protein A. In contrast, purification of IgG3 requires protein G, a
significantly less versatile medium. However, other structural and
functional properties of immunoglobulins should be considered when
choosing the Ig fusion partner for a particular immunoadhesin
construction. For example, the IgG3 hinge is longer and more
flexible, so it can accommodate larger `adhesin` domains that may
not fold or function properly when fused to IgG1. Another
consideration may be valency; IgG immunoadhesins are bivalent
homodimers, whereas Ig subtypes like IgA and IgM may give rise to
dimeric or pentameric structures, respectively, of the basic Ig
homodimer unit. For AL-2-Ig immunoadhesins designed for in vivo
application, the pharmacokinetic properties and the effector
functions specified by the Fc region are important as well.
Although IgG1, IgG2 and IgG4 all have in vivo half-lives of 21
days, their relative potencies at activating the complement system
are different. IgG4 does not activate complement, and IgG2 is
significantly weaker at complement activation than IgG1. Moreover,
unlike IgG1, IgG2 does not bind to Fc receptors on mononuclear
cells or neutrophils. While IgG3 is optimal for complement
activation, its in vivo half-life is approximately one third of the
other IgG isotypes. Another important consideration for
immunoadhesins designed to be used as human therapeutics is the
number of allotypic variants of the particular isotype. In general,
IgG isotypes with fewer serologically-defined allotypes are
preferred. For example, IgG1 has only four serologically-defined
allotypic sites, two of which (G1m1 and 2) are located in the Fc
region; and one of these sites G1m1, is non-immunogenic. In
contrast, there are 12 serologically-defined allotypes in IgG3, all
of which are in the Fc region; only three of these sites (G3m5, 11
and 21) have one allotype which is nonimmunogenic. Thus, the
potential immunogenicity of a .gamma.3 immunoadhesin is greater
than that of a .gamma.1 immunoadhesin.
[0099] In designing the chimeras of the present invention domains
that are not required for neurotrophin binding and/or biological
activity may be deleted. In such structures, it is important to
place the fusion junction at residues that are located between
domains, to avoid misfolding. With respect to the parental
immunoglobulin, a useful joining point is just upstream of the
cysteines of the hinge that form the disulfide bonds between the
two heavy chains. In a frequently used design, the codon for the
C-terminal residue of the `adhesin` part of the molecule is placed
directly upstream of the codons for the sequence DKTHTCPPCP of the
IgG1 hinge region.
[0100] The general methods suitable for the construction and
expression of immunoadhesins are the same those disclosed
hereinabove with regard to (native or variant) AL-2. For example,
AL-2-Ig immunoadhesins are most conveniently constructed by fusing
the cDNA sequence encoding the AL-2 portion in-frame to an Ig cDNA
sequence. However, fusion to genomic Ig fragments can also be used
(see, e.g., Gascoigne et al., Proc. Natl. Acad. Sci. USA,
84:2936-2940 (1987); Aruffo et al., Cell, 61:1303-1313 (1990);
Stamenkovic et al., Cell, 66:1133-1144 (1991)). The latter type of
fusion requires the presence of Ig regulatory sequences for
expression. cDNAs encoding IgG heavy-chain constant regions can be
isolated based on published sequence from cDNA libraries derived
from spleen or peripheral blood lymphocytes, by hybridization or by
polymerase chain reaction (PCR) techniques. The cDNAs encoding the
`adhesin` and the Ig parts of the immunoadhesin are inserted in
tandem into a plasmid vector that directs efficient expression in
the chosen host cells. For expression in mammalian cells pRK5-based
vectors (Schall et al., Cell, 61:361-370 (1990)) and CDM8-based
vectors (Seed, Nature, 329:840 (1989)). The exact junction can be
created by removing the extra sequences between the designed
junction codons using oligonucleotide-directed deletional
mutagenesis (Zoller et al., Nucleic Acids Res., 10:6487 (1982);
Capon et al., Nature, 337:525-531 (1989)). Synthetic
oligonucleotides can be used, in which each half is complementary
to the sequence on either side of the desired junction; ideally,
these are 36 to 48-mers. Alternatively, PCR techniques can be used
to join the two parts of the molecule in-frame with an appropriate
vector.
[0101] The choice of host cell line for the expression of AL-2-Ig
immunoadhesins depends mainly on the expression vector. Another
consideration is the amount of protein that is required. Milligram
quantities often can be produced by transient transfections. For
example, the adenovirus EIA-transformed 293 human embryonic kidney
cell line can be transfected transiently with pRK5-based vectors by
a modification of the calcium phosphate method to allow efficient
immunoadhesin expression. CDM8-based vectors can be used to
transfect COS cells by the DEAE-dextran method (Aruffo et al.,
Cell, 61:1303-1313 (1990); Zettmeissl et al., DNA Cell Biol., (US)
9:347-353 (1990)). If larger amounts of protein are desired, the
immunoadhesin can be expressed after stable transfection of a host
cell line. For example, a pRK5-based vector can be introduced into
Chinese hamster ovary (CHO) cells in the presence of an additional
plasmid encoding dihydrofolate reductase (DHFR) and conferring
resistance to G418. Clones resistant to G418 can be selected in
culture; these clones are grown in the presence of increasing
levels of DHFR inhibitor methotrexate; clones are selected, in
which the number of gene copies encoding the DHFR and immunoadhesin
sequences is co-amplified. If the immunoadhesin contains a
hydrophobic leader sequence at its N-terminus, it is likely to be
processed and secreted by the transfected cells. The expression of
immunoadhesins with more complex structures may require uniquely
suited host cells; for example, components such as light chain or J
chain may be provided by certain myeloma or hybridoma cell hosts
(Gascoigne et al., Proc. Natl. Acad. Sci. USA, 84:2936-2940 (1987);
Martin et al., J. Virol., 67:3561-3568 (1993)).
[0102] Immunoadhesins can be conveniently purified by affinity
chromatography. The suitability of protein A as an affinity ligand
depends on the species and isotype of the immunoglobulin Fc domain
that is used in the chimera. Protein A can be used to purify
immunoadhesins that are based on human .gamma.1, .gamma.2, or
.gamma.4 heavy chains (Lindmark et al., J. Immunol. Meth., 62:1-13
(1983)). Protein G is recommended for all mouse isotypes and for
human .gamma.3 (Guss et al., EMBO J., 5:15671575 (1986)). The
matrix to which the affinity ligand is attached is most often
agarose, but other matrices are available. Mechanically stable
matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. The conditions
for binding an immunoadhesin to the protein A or G affinity column
are dictated entirely by the characteristics of the Fc domain; that
is, its species and isotype. Generally, when the proper ligand is
chosen, efficient binding occurs directly from unconditioned
culture fluid. One distinguishing feature of immunoadhesins is
that, for human .gamma.1 molecules, the binding capacity for
protein A is somewhat diminished relative to an antibody of the
same Fc type. Bound immunoadhesin can be efficiently eluted either
at acidic pH (at or above 3.0), or in a neutral pH buffer
containing a mildly chaotropic salt. This affinity chromatography
step can result in an immunoadhesin preparation that is >95%
pure.
[0103] Other methods known in the art can be used in place of, or
in addition to, affinity chromatography on protein A or G to purify
immunoadhesins. Immunoadhesins behave similarly to antibodies in
thiophilic gel chromatography (Hutchens et al., Anal. Biochem.,
159:217-226 (1986)) and immobilized metal chelate chromatography
(Al-Mashikhi et al., J. Dairy Sci., 71:1756-1763 (1988)). In
contrast to antibodies, however, their behavior on ion exchange
columns is dictated not only by their isoelectric points, but also
by a charge dipole that may exist in the molecules due to their
chimeric nature.
[0104] If desired, the immunoadhesins can be made bispecific, that
is, directed against two distinct ligands. Thus, the immunoadhesins
of the present invention can have binding specificities for AL-2,
or can specifically bind to a AL-2 and to an other determinant, for
example one specifically expressed on the cells expressing a
receptor to which the AL-2 portion of the immunoadhesin structure
binds. For bispecific molecules, trimeric molecules, composed of a
chimeric antibody heavy chain in one arm and a chimeric antibody
heavy chain-light chain pair in the other arm of their
antibody-like structure are advantageous, due to ease of
purification. In contrast to antibody-producing quadromas
traditionally used for the production of bispecific immunoadhesins,
which produce a mixture of ten tetramers, cells transfected with
nucleic acid encoding the three chains of a trimeric immunoadhesin
structure produce a mixture of only three molecules, and
purification of the desired product from this mixture is
correspondingly easier.
[0105] This application encompasses chimeric polypeptides
comprising AL-2 fused to another polypeptide (such as the
immunoadhesins mentioned above). In one preferred embodiment, the
chimeric polypeptide contains a fusion of the AL-2 (or a fragment
thereof, e.g., the ECD of the AL-2) with a tag polypeptide which
provides an epitope to which an anti-tag antibody can selectively
bind. The epitope tag is generally provided at the amino- or
carboxyl-terminus of the AL-2. Such epitope-tagged-AL-2 can be
detected using a labelled antibody against the tag polypeptide.
Also, the epitope tag allows AL-2 to be readily purified by
anti-tag antibody affinity purification. Affinity purification
techniques and diagnostic assays involving antibodies are
well-known.
[0106] Tag polypeptides and their respective antibodies are well
known in the art. Examples include the flu HA tag polypeptide and
its antibody 12CA5, (Field et al., Mol. Cell. Biol., 8:2159-2165
(1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10
antibodies thereto (Evan et al., Molecular and Cellular Biology,
5(12):3610-3616 (1985)); and the Herpes Simplex virus glycoprotein
D (gD) tag and its antibody (Paborsky et al., Protein Engineering,
3(6):547-553 (1990)). Other tag polypeptides have been disclosed.
Examples include the Flag-peptide (Hopp et al., BioTechnology,
6:1204-1210 (1988)); the KT3 epitope peptide (Martin et al.,
Science, 255:192-194 (1992)); an .alpha.-tubulin epitope peptide
(Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)); and the
T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990). Once the tag polypeptide has
been selected, an antibody thereto can be generated using the
techniques disclosed herein.
[0107] The general methods suitable for the construction and
production of epitope tagged AL-2 are the same as those disclosed
herein with regard to (native or variant) AL-2. AL-2-tag
polypeptide fusions are most conveniently constructed by fusing the
cDNA sequence encoding the AL-2 portion in-frame to the tag
polypeptide DNA sequence and expressing the resultant DNA fusion
construct in appropriate host cells. Ordinarily, nucleic acid
encoding the AL-2 (or a fragment thereof) will be fused at its 3'
end to nucleic acid encoding the N-terminus of the tag polypeptide,
however 5' fusions are also possible.
[0108] Epitope tagged AL-2 can be conveniently purified by affinity
chromatography using the anti-tag antibody. The matrix to which the
affinity antibody is attached is most often agarose, but other
matrices are available (e.g. controlled pore glass or
poly(styrenedivinyl)benzene). The epitope tagged AL-2 can be eluted
from the affinity column by varying the buffer pH or ionic strength
or adding chaotropic agents, for example.
[0109] In another embodiment of the invention, multimeric soluble
ligands are prepared by expression as chimeric molecules utilizing
flexible linker loops. A DNA construct encoding the chimeric
protein is designed such that it expresses two or more soluble or
extracellular domains fused together in tandem (e.g.,
"head-to-head") by a flexible loop. This loop may be entirely
artificial (e.g. polyglycine repeats interrupted by serine or
threonine at a certain interval) or "borrowed" from naturally
occurring proteins (e.g. the hinge region of hIgG). Molecules can
be engineered in which the length and composition of the loop is
varied, to allow for selection of molecules with desired
characteristics. Although not wishing to be limited by theory, it
is believed that membrane attachment of the ligands can facilitate
ligand clustering, which in turn can promote receptor
multimerization and activation. Thus, one means of obtaining
biological activity of the soluble AL-2 is mimicking, in solution,
membrane associated ligand clustering. Thus, a biologically active,
clustered soluble Eph-family ligand comprises (soluble AL-2).sub.n,
wherein the soluble AL-2 is the receptor-binding AL-2 extracellular
domain and n is 2 or greater. For example, despite the fact that
receptor phosphorylation is markedly induced by stimulating
receptor expressing reporter cells with mammalian cells
overexpressing membrane-linked forms of the ligands AL-1 or B61,
there is little or no observable phosphorylation using soluble
forms of these ligands. However, when secreted forms of B61 are
myc-tagged and antibodies are used to cluster the ligands, or when
AL-1-IgG chimera is used, the previously inactive soluble ligands
strongly induce receptor tyrosine phosphorylation in reporter cells
expressing Ehk-1 or Rek7 receptors, respectively. Dimerization of
the soluble ligand, e.g., utilizing Fc, can be sufficient for
achieving a biological response, however, further clustering of the
ligand according to the invention, for example using anti-Fc
antibodies, may achieve an increase in biological activity. Cells
of the present invention may transiently or, preferably,
constitutively and permanently express AL-2 in native form, or in
soluble form as chimeric tagged AL-2, AL-2 immunoadhesin, or
clustered AL-2 as described herein.
[0110] Accordingly, a method of enhancing the biological activity
of the soluble AL-2 or its ECD is provided that includes the steps
of (a) expressing the soluble domain of AL-2 with an epitope tag
and (b) exposing the tagged soluble domain to anti-tag antibodies.
The position of the tag with respect to AL-2 is not important so
long as the tag does not interfere with AL-2 function and, in tum,
AL-2 does not interfere with tag function. The tag is preferably
located at either termini of AL-2, more preferably at the
C-terminus of AL-2. However, the tag may be attached by covalent
means, including with oxime linkages as taught for example in WO
9425071 published Nov. 11, 1994.
[0111] In additional embodiments are compounds of the formula
(AL-2).sub.nX, where n is an integer greater than or equal to 2 and
X is an organic linker covalently binding each AL-2. For example,
AL-2 dimers and multimers can be made by attaching AL-2 peptides to
an organic linker or baseplate (designated as X in the formula)
using methods and linkers (e.g., baseplates or templates) described
in the art, for example in WO 94/25071, WO 95/19567, or WO
95/04543. Accordingly, a biologically active, soluble AL-2 is
provided that contains 2 or greater number of soluble AL-2 peptides
where the soluble AL-2 is the AL-2 extracellular domain that binds
an Eph-family receptor. Preferably n is 2 to 20, more preferably 2
to 10, even more preferably 2 to 4. In one preferred embodiment n
is 2. For example, multiple AL-2 are covalently attached to the
same baseplate, e.g., an organic molecule such as a penta-lysine,
where each AL-2 is attached site-specifically via a covalent
linkage, e.g., an oxime linkage, which can be formed by reaction of
a reactive group on AL-2 with its complementary reactive group on
the baseplate. Oxime linkages have superior hydrolysis stability
over a range of physiological conditions compared to hydrazones,
etc. Oxime linkages are not commonly subject to enzymatic
hydrolysis. Polyoximes are therefore suited to applications where
integrity and stability of the complex is desired. The linker (or
baseplate) should not interfere with AL-2 activity. Baseplates can
be designed to improve solubility of peptides, as well as to
present peptides to receptors. A chemically reactive group suitable
for oxime linkage formation can be site-specifically added to AL-2
through methods known in the art (see for example WO 90/02135, WO
94/25071, or EP 243929 B1 issued Sep. 27, 1995).
[0112] It is apparent that AL-2 antagonists can be prepared or used
applying the above guidelines appropriately. For example, a
AL-2-binding Eph-family-receptor-IgG chimera fusion (see Winslow et
al., Neuron, 14:973-981 (1995)), for a method of making
receptor-IgG fusions by recombinant means that is suitable for use
with other Eph-family receptors and their extracellular domains) or
anti-AL-2 antibody can be adsorbed onto a membrane, such as a
silastic membrane, which can be implanted in proximity to tumors or
arthritic tissue, or can be incorporated into liposomes (see for
example WO 91/04014 published Apr. 4, 1991).
[0113] It will be appreciated that some screening of the recovered
variant will be needed to select one having the desired activity. A
change in the immunological character of the AL-2 molecule, such as
affinity for a given antibody, can be measured by a
competitive-type immunoassay. The variant is assayed for changes in
the suppression or enhancement of its enzymatic activity by
comparison to the activity observed for native AL-2 in the same
assay. For example, one can screen for the ability of the variant
AL-2 to stimulate protein kinase activity of an Eph-family receptor
using the techniques set forth, for example, in Lokker et al., EMBO
J., 11:2503-2510 (1992), Winslow et al., Neuron, 14:973-981 (1995),
or Bennett et al., J. Biol. Chem., 269(19):14211-8 (1994). Other
potential modifications of protein or polypeptide properties such
as redox or thermal stability, hydrophobicity, susceptibility to
proteolytic degradation, or the tendency to aggregate with carriers
or into multimers are assayed by methods well known in the art.
[0114] AL-2 DNA, whether cDNA or genomic DNA or a product of in
vitro synthesis, is ligated into a replicable vector for further
cloning or for expression. "Vectors" are plasmids and other DNAs
that are capable of replicating autonomously within a host cell,
and as such, are useful for performing two functions in conjunction
with compatible host cells (a vector-host system). One function is
to facilitate the cloning of the nucleic acid that encodes the
AL-2, i.e., to produce usable quantities of the nucleic acid. The
other function is to direct the expression of AL-2. One or both of
these functions are performed by the vector-host system. The
vectors will contain different components depending upon the
function they are to perform as well as the host cell with which
they are to be used for cloning or expression.
[0115] To produce AL-2, an expression vector will contain nucleic
acid that encodes AL-2 as described above. The AL-2 of this
invention is expressed directly in recombinant cell culture, or as
a fusion with a heterologous polypeptide, preferably a signal
sequence or other polypeptide having a specific cleavage site at
the junction between the heterologous polypeptide and the AL-2.
[0116] In one example of recombinant host cell expression,
mammalian cells are transfected with an expression vector
comprising AL-2 DNA and the AL-2 encoded thereby is recovered from
the culture, preferably cell culture medium in which the
recombinant host cells are grown. But the expression vectors and
methods disclosed herein are suitable for use over a wide range of
prokaryotic and eukaryotic organisms.
[0117] Prokaryotes may be used for the initial cloning of DNAs and
the construction of the vectors useful in the invention. However,
prokaryotes may also be used for expression of DNA encoding AL-2.
Polypeptides that are produced in prokaryotic host cells typically
will be non-glycosylated.
[0118] Plasmid or viral vectors containing replication origins and
other control sequences that are derived from species compatible
with the host cell are used in connection with prokaryotic host
cells, for cloning or expression of an isolated DNA. For example,
E. coli typically is transformed using pBR322, a plasmid derived
from an E. coli species (Bolivar, et al., Gene, 2:95-113 (1987)).
PBR322 contains genes for ampicillin and tetracycline resistance so
that cells transformed by the plasmid can easily be identified or
selected. For it to serve as an expression vector, the pBR322
plasmid, or other plasmid or viral vector, must also contain, or be
modified to contain, a promoter that functions in the host cell to
provide messenger RNA (mRNA) transcripts of a DNA inserted
downstream of the promoter (Rangagwala, et al., Bio/Technology,
9:477-479 (1991)).
[0119] In addition to prokaryotes, eukaryotic microbes, such as
yeast, may also be used as hosts for the cloning or expression of
DNAs useful in the invention. Yeast, for example, Saccharomyces
cerevisiae, is a commonly used eukaryotic microorganism. Plasmids
useful for cloning or expression in yeast cells of a desired DNA
are well known, as are various promoters that function in yeast
cells to produce mRNA transcripts.
[0120] Furthermore, cells derived from multicellular organisms also
may be used as hosts for the cloning or expression of DNAs useful
in the invention. Mammalian cells are most commonly used, and the
procedures for maintaining or propagating such cells in vitro,
which procedures are commonly referred to as tissue culture, are
well known. Kruse and Patterson, eds., Tissue Culture (Academic
Press, 1977). Examples of useful mammalian cells are human cell
lines such as 293, HeLa, and WI-38, monkey cell lines such as COS-7
and VERO, and hamster cell lines such as BHK-21 and CHO, all of
which are publicly available from the American Type Culture
Collection (ATCC), Rockville, Md. 20852 USA.
[0121] Expression vectors, unlike cloning vectors, should contain a
promoter that is recognized by the host organism and is operably
linked to the AL-2 nucleic acid. Promoters are untranslated
sequences that are located upstream from the start codon of a gene
and that control transcription of the gene (that is, the synthesis
of mRNA). Promoters typically fall into two classes, inducible and
constitutive. Inducible promoters are promoters that initiate high
level transcription of the DNA under their control in response to
some change in culture conditions, for example, the presence or
absence of a nutrient or a change in temperature.
[0122] A large number of promoters are known, that may be operably
linked to AL-2 DNA to achieve expression of AL-2 in a host cell.
This is not to say that the promoter associated with naturally
occurring AL-2 DNA is not usable. However, heterologous promoters
generally will result in greater transcription and higher yields of
expressed AL-2.
[0123] Promoters suitable for use with prokaryotic hosts include
the .beta.-lactamase and lactose promoters, (Goeddel et al.,
Nature, 281:544-548 (1979)), tryptophan (trp) promoter, (Goeddel et
al., Nuc. Acids Res., 8:4057-4074 (1980)), and hybrid promoters
such as the tac promoter, (deBoer et al., Proc. Natl. Acad. Sci.
USA, 80:21-25 (1983)). However, other known bacterial promoters are
suitable. Their hucleotide sequences have been published,
(Siebenlist et al., Cell, 20:269-281 (1980)), thereby enabling a
skilled worker operably to ligate them to DNA encoding AL-2 using
linkers or adaptors to supply any required restriction sites (Wu et
al., Meth. Enz., 152:343-349 (1987)).
[0124] Suitable promoters for use with yeast hosts include the
promoters for 3-phosphoglycerate kinase, (Hitzeman et al., J. Biol.
Chem., 255:12073-12080 (1980); Kingsman et al., Meth. Enz.,
185:329-341 (1990)), or other glycolytic enzymes such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase (Dodson et
al., Nuc. Acids Res., 10:2625-2637 (1982); Emr, Meth. Enz.,
185:231-279 (1990)).
[0125] Expression vectors useful in mammalian cells typically
include a promoter derived from a virus. For example, promoters
derived from polyoma virus, adenovirus, cytomegalovirus (CMV), and
simian virus 40 (SV40) are commonly used. Further, it is also
possible, and often desirable, to utilize promoter or other control
sequences associated with a naturally occurring DNA that encodes
AL-2, provided that such control sequences are functional in the
particular host cell used for recombinant DNA expression.
[0126] Other control sequences that are desirable in an expression
vector in addition to a promoter are a ribosome binding site, and
in the case of an expression vector used with eukaryotic host
cells, an enhancer. Enhancers are cis-acting elements of DNA,
usually about from 10-300 bp, that act on a promoter to increase
the level of transcription. Many enhancer sequences are now known
from mammalian genes (for example, the genes for globin, elastase,
albumin, .alpha.-fetoprotein and insulin). Typically, however, the
enhancer used will be one from a eukaryotic cell virus. Examples
include the SV40 enhancer on the late side of the replication
origin (bD 100-270), the cytomegalovirus early promoter enhancer,
the polyoma enhancer on the late side of the replication origin,
and adenovirus enhancers (Kriegler, Meth. Enz., 185:512-527
(1990)).
[0127] Expression vectors may also contain sequences necessary for
the termination of transcription and for stabilizing the messenger
RNA (mRNA) (Balbas et al., Meth. Enz., 185:14-37 (1990)); Levinson,
Meth. Enz, 185:485-511 (1990)). In the case of expression vectors
used with eukaryotic host cells, such transcription termination
sequences may be obtained from the untranslated regions of
eukaryotic or viral DNAs or cDNAs. These regions contain
polyadenylation sites as well as transcription termination sites
(Birnsteil et al., Cell, 41:349-359 (1985)).
[0128] In general, control sequences are DNA sequences necessary
for the expression of an operably liked coding sequence in a
particular host cell. "Expression" refers to transcription and/or
translation. "Operably linked" refers to the covalent joining of
two or more DNA sequences, by means of enzymatic ligation or
otherwise, in a configuration relative to one another such that the
normal function of the sequences can be performed. 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. Linking is
accomplished by ligation at convenient restriction sites. If such
sites do not exist, then synthetic oligonucleotide adaptors or
linkers are used, in conjunction with standard recombinant DNA
methods.
[0129] Expression and cloning vectors also will contain a sequence
that enables the vector to replicate in one or more selected host
cells. Generally, in cloning vectors this sequence is one that
enables the vector to replicate independently of the host
chromosome(s), and includes origins of replication or autonomously
replicating sequences. Such sequences are well known for a variety
of bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most gram-negative bacteria, the
2.mu. plasmid origin is suitable for yeast, and various viral
origins (for example, from SV40, polyoma, or adenovirus) are useful
for cloning vectors in mammalian cells. Most expression vectors are
"shuttle" vectors, i.e., they are capable of replication in at
least one class of organisms but can be transfected into another
organism for expression. For example, a vector may be cloned in E.
coli and then the same vector is transfected into yeast or
mammalian cells for expression even though it is not capable of
replicating independently of the host cell chromosome.
[0130] The expression vector may also include an amplifiable gene,
such as that comprising the coding sequence for dihydrofolate
reductase (DHFR). Cells containing an expression vector that
includes a DHFR gene may be cultured in the presence of
methotrexate, a competitive antagonist of DHFR. This leads to the
synthesis of multiple copies of the DHFR gene and, concomitantly,
multiple copies of other DNA sequences comprising the expression
vector (Ringold et al., J. Mol. Apl. Genet., 1:165-175 (1981)),
such as a DNA sequence encoding AL-2. In that manner, the level of
AL-2 produced by the cells may be increased.
[0131] DHFR protein encoded by the expression vector also may be
used as a selectable marker of successful transfection. For
example, if the host cell prior to transformation is lacking in
DHFR activity, successful transformation by an expression vector
comprising DNA sequences encoding AL-2 and DHFR protein can be
determined by cell growth in medium containing methotrexate. Also,
mammalian cells transformed by an expression vector comprising DNA
sequences encoding AL-2, DHFR protein, and aminoglycoside 3'
phosphotransferase (APH) can be determined by cell growth in medium
containing an aminoglycoside antibiotic such as kanamycin or
neomycin. Because eukaryotic cells do not normally express an
endogenous APH activity, genes encoding APH protein, commonly
referred to as neo.sup.r genes, may be used as dominant selectable
markers in a wide range of eukaryotic host cells, by which cells
transfected by the vector can easily be identified or selected
(Jiminez et al., Nature, 287:869-871 (1980); Colbere-Garapin et
al., J. Mol. Biol., 150:1-14 (1981); Okayama et al., Mol. Cell.
Biol., 3:280-289 (1983)).
[0132] Many other selectable markers are known that may be used for
identifying and isolating recombinant host cells that express AL-2.
For example, a suitable selection marker for use in yeast is the
trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al.,
Nature, 282:39-43 (1979); Kingsman et al., Gene, 7:141-152 (1979);
Tschemper, et al., Gene, 10:157-166 (1980)). The trp1 gene provides
a selection marker for a mutant strain of yeast lacking the ability
to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1
(available from the American Type Culture Collection, Rockville,
Md. 20852 USA) (Jones, Genetics, 85:12 (1977)). The presence of the
trp1 lesion in the yeast host cell genome then provides an
effective environment for detecting transformation by growth in the
absence of tryptophan. Similarly, Leu2-deficient yeast strains
(ATCC Nos. 20622 or 38626) are complemented by known plasmids
bearing the Leu2 gene.
[0133] Accordingly, a method for producing AL-2 is provided that
includes the steps of transforming a cell containing an endogenous
AL-2 gene with a homologous DNA comprising an amplifiable gene and
a flanking sequence of at least about 150 base pairs that is
homologous with a DNA sequence within or in proximity to the
endogenous AL-2 gene, whereby the homologous DNA integrates into
the cell genome by recombination, then culturing the cells under
conditions that select for amplification of the amplifiable gene
whereby the AL-2 gene is also amplified, and thereafter recovering
AL-2 from the cells.
[0134] For diagnostic applications, anti-AL-2 antibodies typically
will be labeled with a detectable moiety. The detectable moiety can
be any one which is capable of producing, either directly or
indirectly, a detectable signal. For example, the detectable moiety
may be a radioisotope, such as .sup.3H, .sup.14C, .sup.32P,
.sup.35S, or .sup.125I, a fluorescent or chemiluminescent compound,
such as fluorescein isothiocyanate, rhodamine, or luciferin;
radioactive isotopic labels, such as, e.g., .sup.125I, .sup.32P,
.sup.14C, or .sup.3H, or an enzyme, such as alkaline phosphatase,
beta-galactosidase or horseradish peroxidase.
[0135] Any method known in the art for separately conjugating the
antibody to the detectable moiety may be employed, including those
methods described by David et al., Biochemistry, 13:1014-1021
(1974); Pain et al., J. Immunol. Meth., 40:219-231 (1981); and
Bayer et al., Meth. Enz., 184:138-163 (1990).
[0136] The anti-AL-2 antibodies may be employed in any known assay
method, such as competitive binding assays, direct and indirect
sandwich assays, and immunoprecipitation assays (Zola, Monoclonal
Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.,
1987)). The term "antibody" is used in the broadest sense and
specifically covers single anti-AL-2 monoclonal antibodies
(including agonist and antagonist antibodies) and anti-AL-2
antibody compositions with polyepitopic specificity.
[0137] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally-occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen.
[0138] The monoclonal antibodies herein include hybrid and
recombinant antibodies produced by splicing a variable (including
hypervariable) domain of an anti-AL-2 antibody with a constant
domain (e.g. "humanized" antibodies), or a light chain with a heavy
chain, or a chain from one species with a chain from another
species, or fusions with heterologous proteins, regardless of
species of origin or immunoglobulin class or subclass designation,
as well as antibody fragments (e.g., Fab, F(ab').sub.2, and Fv), so
long as they exhibit the desired biological activity. (See, e.g.,
U.S. Pat. No. 4,816,567 and Mage et al., Monoclonal Antibody
Production Techniques and Applications, pp. 79-97 (Marcel Dekker,
Inc., New York (1987)).
[0139] Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. Monoclonal
antibodies include hybrid and recombinant antibodies produced by
splicing a variable (including hypervariable) domain of an
anti-AL-2 antibody with a constant domain (e.g. "humanized"
antibodies), or a light chain with a heavy chain, or a chain from
one species with a chain from another species, or fusions with
heterologous proteins, regardless of species of origin or
immunoglobulin class or subclass designation, as well as antibody
fragments (e.g. Fab, F(ab').sub.2, and Fv), so long as they exhibit
the desired biological activity. (See, e.g., Mage et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
79-97 (Marcel Dekker, Inc., New York (1987)). The monoclonal
antibodies to be used in accordance with the present invention can
be made by hybridoma method known in the art, or may be made by
recombinant DNA methods (U.S. Pat. No. 4,816,567). The "monoclonal
antibodies" may also be isolated from phage libraries generated
using the techniques described in McCafferty et al., Nature,
348:552-554 (1990), for example. The individual antibodies
comprising the monoclonal antibody population are identical except
for possible naturally-occurring mutations that may be present in
minor amounts.
[0140] "Humanized" forms of non-human (e.g. murine) antibodies are
specific chimeric immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementary determining region (CDR) of
the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore, the
humanized antibody may comprise residues which are found neither in
the recipient antibody nor in the imported CDR or framework
sequences. These modifications are made to further refine and
optimize antibody performance. In general, the humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin.
[0141] DNA encoding the monoclonal antibodies of the invention is
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA.
[0142] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingiy, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567 by
Cabilly et al.) wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence
from a non-human species. In practice, humanized antibodies are
typically human antibodies in which some CDR residues, and possibly
some FR residues, are substituted by residues from analogous sites
in rodent antibodies.
[0143] It is important that antibodies be humanized with retention
of high affinity for the antigen and other favorable biological
properties. To achieve this goal, according to a preferred method,
humanized antibodies are prepared by a process of analysis of the
parental sequences and various conceptual humanized products using
three dimensional models of the parental and humanized sequences.
Three dimensional immunoglobulin models are familiar to those
skilled in the art. Computer programs are available which
illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role of
the residues in the functioning of the candidate immunoglobulin
sequence, i.e., the analysis of residues that influence the ability
of the candidate immunoglobulin to bind its antigen. In this way,
FR residues can be selected and combined from the consensus and
import sequence so that the desired antibody characteristic, such
as increased affinity for the target antigen(s), is achieved. In
general, the CDR residues are directly and most substantially
involved in influencing antigen binding. For further details see WO
92/22653, published Dec. 23, 1992.
[0144] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for a AL-2, the other one is for any other
antigen, and preferably f6r a receptor or receptor subunit. For
example, bispecific antibodies specifically binding a Htk receptor
and AL-2 are within the scope of the present invention.
[0145] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the coexpression of two immunoglobulin heavy
chain-light chain pairs, where the two heavy chains have different
specificities (Millstein et al., Nature, 305:537-539 (1983)).
Because of the random assortment of immunoglobulin heavy and light
chains, these hybridomas (quadromas) produce a potential mixture of
10 different antibody molecules, of which only one has the correct
bispecific structure. The purification of the correct molecule,
which is usually done by affinity chromatography steps, is rather
cumbersome, and the product yields are low. Similar procedures are
disclosed in WO 93/08829, published May 13, 1993, and in Traunecker
et al., EMBO J., 10:3655-3659 (1991).
[0146] According to a different and more preferred approach,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an
immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2 and CH3 regions. It is preferred to have the
first heavy chain constant region (CH1) containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are cotransfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance. In a preferred embodiment of this approach, the
bispecific antibodies are composed of a hybrid immunoglobulin heavy
chain with a first binding specificity in one arm, and a hybrid
immunoglobulin heavy chain-light chain pair (providing a second
binding specificity) in the other arm. It was found that this
asymmetric structure facilitates the separation of the desired
bispecific compound from unwanted immunoglobulin chain
combinations, as the presence of an immunoglobulin light chain in
only one half of the bispecific molecule provides for a facile way
of separation. This approach is disclosed in WO 94/04690 published
Mar. 3, 1994. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0147] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980), and for treatment of HIV infection (WO
91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies
may be made using any convenient cross-linking methods. Suitable
cross-linking agents are well known in the art, and are disclosed
in U.S. Pat. No. 4,676,980, along with a number of cross-linking
techniques.
[0148] Competitive binding assays rely on the ability of a labeled
standard (e.g., AL-2 or an immunologically reactive portion
thereof) to compete with the test sample analyte (AL-2) for binding
with a limited amount of antibody. The amount of AL-2 in the test
sample is inversely proportional to the amount of standard that
becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies generally are
insolubilized before or after the competition, so that the standard
and analyte that are bound to the antibodies may conveniently be
separated from the standard and analyte which remain unbound.
[0149] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
of the protein to be detected. In a sandwich assay, the test sample
analyte is bound by a first antibody which is immunobilized on a
solid support, and thereafter a second antibody binds to the
analyte, thus forming an insoluble three part complex (for example,
see U.S. Pat. No. 4,376,110). The second antibody may itself be
labeled with a detectable moiety (direct sandwich assays) or may be
measured using an anti-immunoglobulin antibody that is labeled with
a detectable moiety (indirect sandwich assay). For example, one
type of sandwich assay is an ELISA assay, in which case the
detectable moiety is an enzyme.
[0150] AL-2 antibodies may be useful in certain therapeutic
indications to block activity of the AL-2 (for example in
carcinogenesis).
[0151] Therapeutic AL-2 antibody formulations and modes for
administration will be similar to those described herein for AL-2.
A typical daily dosage of the antibody ranges from about 1 .mu.g/kg
to up to 5 mg/kg or more, depending on the factors mentioned herein
for AL-2 administration.
[0152] AL-2 antibodies may also be useful in diagnostic assays for
AL-2, e.g., detecting its expression in specific cells, tissues, or
serum. The antibodies are labeled in the same fashion as AL-2
described above and/or are immobilized on an insoluble matrix. AL-2
antibodies also are useful for the affinity purification of AL-2
from recombinant cell culture or natural sources. AL-2 antibodies
that do not detectably cross-react with other proteins can be used
to purify AL-2 free from these other known proteins. Suitable
diagnostic assays for AL-2 and its antibodies are described
herein.
[0153] The anti-AL-2 antibodies of the invention also are useful
for in vivo imaging, wherein an antibody labeled with a detectable
moiety is administered to a host, preferably into the bloodstream,
and the presence and location of the labeled antibody in the host
is assayed. This imaging technique is useful in the staging and
treatment of various neurological disorders. The antibody may be
labeled with any moiety that is detectable in a host, whether by
nuclear magnetic resonance, radiology, or other detection means
known in the art.
[0154] Neutralizing anti-AL-2 antibodies are useful as antagonists
of AL-2. The term "neutralizing anti-AL-2 antibody" as used herein
refers to an antibody that is capable of specifically binding to
AL-2, and which is capable of substantially inhibiting or
eliminating the functional activity of AL-2 in vivo or in vitro.
Typically a neutralizing antibody will inhibit the functional
activity of AL-2 at least about 50%, preferably greater than 80%,
and more preferably greater than 90% as determined, for example, by
an in vitro receptor binding assay, or in vitro cell-based receptor
activation assays (for example, see Winslow et al., Neuron,
14:973-981 (1995)).
[0155] Other AL-2 antagonists are prepared using AL-2 receptor
proteins. One example of an AL-2 antagonist is an
Eph-family-receptor-IgG chimeric protein that binds AL-2 as
described herein. Another example of an AL-2 antagonist is a
soluble form of an AL-2 receptor, which comprises the extracellular
domain or the receptor substantially free of the transmembrane
domain. The soluble form of the receptor can be used directly as an
antagonist, or it can be used to screen for small molecules that
would antagonize AL-2 activity.
[0156] As stated previously, receptor tyrosine kinases are involved
in many signal transduction events that regulate important cellular
processes. Such processes include, for example, cellular
differentiation and proliferation. Abnormal regulation or
expression of the signal transduction machinery can lead to
aberrant and malignant growth of the abnormally regulated cells.
Abnormal expression of Eph is known to be associated with
carcinomas of the liver, lung, breast and colon, for example.
Likewise, since some Eph-related tyrosine kinases are, at least,
found within the same tissues as Eph, their abnormal expression may
also lead to the development of the carcinomas described above as
well as other types of cancers. Additionally, cancers of the
neuronal linage are likely to be caused by the abnormal expression
or regulation of Cek5, since this Eph-related kinase is found
exclusively in neuronal tissues. Cek5 and the other Eph-related
kinases expressed in the nervous system also are likely to be
involved in nerve regeneration. A change in the amount or activity
of an Eph-related kinase in a sample, compared to a normal sample,
will be indicative of cancerous stages and of their level of
malignancy. Depending on whether the normal state is caused by the
presence or absence of an Eph-related kinase, the change can
involve either an increase or decrease in the amount or activity of
the Eph-related kinase. One skilled in the art can measure these
parameters and compare them to those obtained from a normal sample.
Methods for determining the levels or activity of Eph-related
kinases are known to one skilled in the art and, include, for
example, RNA and protein blot analysis, ELISA using specific
antibodies to each of the Eph-related kinases and direct
measurement of catalytic activity such as tyrosine kinase activity.
Such methods can be found in Harlow et al., Antibodies: A
Laboratory Manual Cold Spring Harbor Laboratory (1989), which is
incorporated herein by reference. The compositions and methods of
receptor modulation as taught herein can then be effectively
applied where AL-2-binding receptors are involved.
[0157] AL-2 is believed to find therapeutic use for treating
mammals via stimulation or inhibition of growth and/or
differentiation and/or activation and/or metabolism of cells having
a receptor for AL-2 as described herein, such as Htk, Hek2, or
Hek5. The interaction of ligand and receptor may result in
activation of the receptor and transduction of a signal which
modulates the physiological state of the receptor-bearing cells.
The ligand can act as a growth factor to stimulate the
proliferation of target cells. Alternatively, ligand binding may
not activate the receptor. In this instance, the ligand may act as
an antagonist for other molecules which activate the receptor and
induce signal transduction.
[0158] The invention provides a method of modulating the endogenous
enzymatic activity of an AL-2-binding Eph-family receptor. The
method includes the step of administering to a mammal an effective
amount of AL-2 to modulate the receptor enzymatic activity. In one
embodiment is provided a method for stimulating the proliferation,
differentiation, metabolism, regeneration, growth, process-out
growth, or cell migration of AL-2-receptor expressing cells in a
mammal by administering a therapeutically effective amount of
receptor-activating AL-2. Receptor-activating forms of AL-2, such
as AL-2-IgG, find use in alleviating cell damage or promoting
neurogenesis following disease or injury, such as cytotoxicity,
caused by chemotherapy. For example, a method for stimulating
proliferation of neurons innervating the liver includes the step of
administering a therapeutically effective amount of AL-2. Treatment
with AL-2 is useful for repairing liver damage resulting from
disease or injury.
[0159] Soluble Eph-family-receptor polypeptides can be used to
modulate the activation of the cell-associated receptors, typically
by competing with the cell-bound receptor for unbound AL-2.
Modulation of Eph-family receptor activation may in turn alter the
proliferation and/or differentiation of receptor-bearing cells.
[0160] Antibodies to Eph-like receptors are useful reagents for the
detection of receptors in different cell types using immunoassays
conventional to the art. Antibodies are also useful therapeutic
agents for modulating receptor activation. Antibodies may bind to
the receptor so as to directly or indirectly block ligand binding
and thereby act as an antagonist of receptor activation.
Alternatively, antibodies may act as an agonist by binding to
receptor so as to facilitate ligand binding and bring about
receptor activation at lower ligand concentrations. In addition,
antibodies can themselves act as a ligand by inducing receptor
activation. In this context the present invention provides
anti-idiotype antibodies, i.e., anti-AL-2-antibodies, that
recognize an AL-2-binding-Eph-family receptor.
[0161] Accordingly, a method for modulating the activation of an
AL-2-binding-Eph-family receptor by administering a
modulation-effective amount of AL-2 or soluble AL-2. The term
"modulation-effective amount" is that amount which effects an
increase or decrease in the activation of an
AL-2-binding-Eph-family receptor. Preferably the amount will range
from about 0.01 .mu.g to about 100 mg of polypeptide per kg body
weight. In general, for therapeutic purposes, therapy will be
appropriate for a patient having a condition in part related to the
state of proliferation and/or differentiation of receptor-bearing
cells. Based in part upon the tissue distribution of AL-2, and thus
presumably its receptors in some embodiments, treatment with the
pharmaceutical compositions of the invention may be particularly
indicated for disorders involving brain, heart, muscle, lung,
kidney, pancreas, skeletal muscle, liver, and more preferably
involving brain, pancreas, and skeletal muscle.
[0162] AL-2 is also useful for selection of cell populations
enriched for AL-2-receptor bearing cells. Such populations can be
useful in cellular therapy regimens where it is necessary to treat
patients that are depleted of certain cell types.
[0163] The human AL-2 is clearly also useful insofar as it can be
administered to a human having depressed levels of endogenous AL-2,
preferably in the situation where such depressed levels lead to a
pathological disorder.
[0164] The prominent expression of AL-2 DNA in the cerebellum,
cerebral cortex, medulla, spinal cord, occipital pole, frontal
lobe, temporal lobe, putamen, amygdala, caudate nucleus, corpus
callosum, hippocampus, substantia nigra, subthalamic nucleus, and
thalamus (see Examples) is consistent with the use of AL-2 to treat
neurodegenerative diseases or, other neuronal disorders or
conditions in which these structures, or neurons projecting to
these structures, are affected.
[0165] AL-2 finds use in treating neurodegenerative disorders or
nerve damage where nerve regeneration and (re)establishment of
neuronal pathways are desired outcomes, since it may play a role in
neuronal cell migration and axonogenesis. A critical stage in the
development of the nervous system, and during nerve regeneration as
might occur after injury, is the projection of axons to their
targets. Navigational decisions are made at the growth cones of the
migrating axons. As axons grow their growth cones extend and
retract filopodia and lamellipodia processes which are implicated
in the navigational decisions and pathfinding abilities of
migrating axons. Like peripheral nervous system axons, the growth
cones of neurons associated with the central nervous system follow
stereotyped pathways and apparently can selectively choose from a
number of possible routes (reviewed by Goodman et al., Cell,
72:77-98 (1993)). For example, subcellular localization of a Hek5
homolog, the murine Nuk receptor tyrosine kinase, indicates that
this receptor is concentrated at sites of cell-cell contact, often
involving migrating neuronal cells or their extensions (Henkemeyer
et al., Oncogene, 9:1001-14 (1994)). Most notably, high levels of
Nuk protein are found within initial axon outgrowths and associated
nerve fibers. The axonal localization of Nuk was transient and not
detected after migrations have ceased, which suggests a role for
this tyrosine kinase during the early pathfinding and/or
fasciculation stages of axonogenesis, which can be important
processes during recovery from neuronal damage.
[0166] Accordingly, AL-2 (and embodiments disclosed herein or
identified by the methods presented herein) is believed to be
useful in promoting the development, maintenance, regeneration,
migration, or process-outgrowth of neurons in vivo, including
central (brain and spinal chord), peripheral (sympathetic,
parasympathetic, sensory, and enteric neurons), and motoneurons.
The ligands, agonists and antagonists may accordingly be used to
stimulate or inhibit these activities associated with
neurodegenerative conditions and conditions involving trauma and
injury to the nervous system. Consequently, AL-2 may be utilized in
methods for the diagnosis and/or treatment of a variety of
neurologic diseases and disorders.
[0167] In some embodiments of the invention, purified AL-2 can be
administered to patients in whom the nervous system has been
damaged by trauma, surgery, stroke, ischemia, infection, metabolic
disease, nutritional deficiency, malignancy, or toxic agents, to
promote the survival or growth of neurons. For example, AL-2 can be
used to promote the survival or growth of motoneurons that are
damaged by trauma or surgery. Also, AL-2 can be used to treat
motoneuron disorders, such as amyotrophic lateral sclerosis (Lou
Gehrig's disease), Bell's palsy, and various conditions involving
spinal muscular atrophy, or paralysis. AL-2 can be used to treat
human neurodegenerative disorders, such as Alzheimer's disease,
Parkinson's disease, epilepsy, demyelinating diseases, such as
multiple sclerosis, Huntington's chorea, Down's Syndrome, nerve
deafness, Meniere's disease, and other disorders of the cerebellum
(Hefti, Neurobiol., 25(11):1418-35 (1994); Marsden, Lancet,
335:948-952 (1990); Agid, Lancet, 337:1321-1327 (1991); Wexler et
al., Ann. Rev. Neurosci., 14:503-529 1991)). AL-2 can be used as
cognitive enhancer, to enhance learning particularly in dementias
or trauma, since they can promote axonal outgrowth and synaptic
plasticity, particularly of hippocampal neurons that express
AL-2-binding Eph-family receptors and cortical neurons that express
AL-2. AL-2 can be used in bacterial and viral infections of the
nervous system, deficiency diseases, such as Wernicke's disease and
nutritional polyneuropathy, progressive supranuclear palsy, Shy
Drager's syndrome, multistem degeneration and olivo ponto
cerebellar atrophy, and peripheral nerve damage.
[0168] For example, in Alzheimer's disease there is a critical loss
of basal forebrain cholinergic neurons, cortical neurons, and
hippocampal neurons. Although maximally effective treatment of this
neurodegenerative condition may require protection of all
vulnerable neuronal populations, treatment with AL-2 alone is
expected to provide therapeutic benefit. Alzheimer's disease, which
has been identified by the National Institutes of Aging as
accounting for more than 50% of dementia in the elderly, is also
the fourth or fifth leading cause of death in Americans over 65
years of age. Four million Americans, 40% of Americans over age 85
(the fastest growing segment of the U.S. population), have
Alzheimer's disease. Twenty-five percent of all patients with
Parkinson's disease also suffer from Alzheimer's disease-like
dementia. And in about 15% of patients with dementia, Alzheimer's
disease and multi-infarct dementia coexist. The third most common
cause of dementia, after Alzheimer's disease and vascular dementia,
is cognitive impairment due to organic brain disease related
directly to alcoholism, which occurs in about 10% of alcoholics.
However, the most consistent abnormality for Alzheimer's disease,
as well as for vascular dementia and cognitive impairment due to
organic brain disease related to alcoholism, is the degeneration of
the cholinergic system arising from the basal forebrain (BF) to
both the codex and hippocampus (Big1 et al., in Brain Cholinergic
Systems, M. Steriade and D. Biesold, eds., Oxford University Press,
Oxford, pp. 364-386 (1990)). And there are a number of other
neurotransmitter systems affected by Alzheimer's disease (Davies,
Med. Res. Rev., 3:221 (1983)). However, cognitive impairment,
related for example to degeneration of the cholinergic
neurotransmitter system, is not limited to individuals suffering
from dementia. It has also been seen in otherwise healthy aged
adults and rats. Studies that compare the degree of learning
impairment with the degree of reduced cortical cerebral blood flow
in aged rats show a good correlation (Berman et al., Neurobiol.
Aging, 9:691 (1988)). In chronic alcoholism the resultant organic
brain disease, like Alzheimer's disease and normal aging, is also
characterized by diffuse reductions in cortical cerebral blood flow
in those brain regions where cholinergic neurons arise (basal
forebrain) and to which they project (cerebral cortex) (Lofti et
al., Cerebrovasc and Brain Metab. Rev. 1:2 (1989)).
[0169] The progressive nature of Parkinson's disease is due to a
loss of nigral dopaminergic neurons of the substantia nigra (Studer
et al., Eur. J. Neuroscience, 7:223-233 (1995)). ALS involves
progressive degeneration of motoneurons of the spinal cord, brain
stem and cerebral cortex.
[0170] Further, AL-2 can be used to treat neuropathy, and
especially peripheral neuropathy. "Peripheral neuropathy" refers to
a disorder affecting the peripheral nervous system, most often
manifested as one or a combination of motor, sensory, sensorimotor,
or autonomic neural dysfunction. The wide variety of morphologies
exhibited by peripheral neuropathies can each be attributed
uniquely to an equally wide number of causes. For example,
peripheral neuropathies can be genetically acquired, can result
from a systemic disease, or can be induced by a toxic agent.
Examples include but are not limited to distal sensorimotor
neuropathy, or autonomic neuropathies such as reduced motility of
the gastrointestinal tract or atony of the urinary bladder.
Examples of neuropathies associated with systemic disease include
post-polio syndrome; examples of hereditary neuropathies include
Charcot-Marie-Tooth disease, Refsum's disease,
Abetalipoproteinemia, Tangier disease, Krabbe's disease,
Metachromatic leukodystrophy, Fabry's disease, and Dejerine-Sottas
syndrome; and examples of neuropathies caused by a toxic agent
include those caused by treatment with a chemotherapeutic agent
such as vincristine, cisplatin, methotrexate, or
3'-azido-3'-deoxythymidine.
[0171] AL-2 may play a role in neurogenesis, for example in axon
bundling or process outgrowth. The following mechanism is not meant
to be limiting to the invention. Any role of AL-2 in axon fascicle
formation may be indirect, i.e. AL-2 and its receptor, may not
themselves function as adhesion molecules but rather are involved
in regulating the fasciculation process. Accordingly, activation of
AL-2-receptor expressed on the neurons, by AL-2 expressed on
astrocytes for example, might activate a signaling pathway that
promotes fasciculation, possibly by up-regulating or activating
adhesion molecules. Activation of AL-2-receptor may cause growth
cone repulsion and collapse, forcing axons together for
fasciculation. AL-2 expressed on astrocytes would serve as a
repulsive cue to axons driving axons together. This model is
compatible with the current view that astrocytes play an important
role during development of the CNS, where they are thought to
provide a substratum and trophic support for growing axons (Hatten
et al., Semin. Neurosci., 2:455-465 (1990)).
[0172] A tyrosine kinase is required for axon bundling. Neurons in
the developing or regenerating nervous system presumably require
two types of factors, those that promote growth and survival, and
those that provide spatial or directional guidance in the
establishment of neuronal pathways (Tessier-Lavigne, Curr. Opin.
Genet. Devel., 4:596-601 (1994)). Tyrosine kinases are known to
play a well-established role in the former and can participate in
the latter. Accordingly, AL-2 can play role in the formation of
neuronal pathways, a crucial feature of both development and
regeneration in the nervous system.
[0173] In still further embodiments of the invention, AL-2
antagonists, and especially anti-AL-2 antibodies, can be
administered to patients suffering from neurologic diseases and
disorders characterized by excessive production or activity of
AL-2. AL-2 antagonists can be used in the prevention of aberrant
regeneration of sensory neurons such as may occur post-operatively,
or in the selective ablation of sensory neurons, for example, in
the treatment of chronic pain syndromes.
[0174] In yet another embodiment AL-2 stimulates hematopoiesis and
thus find use in treating hematopoietic-related disorders. Htk, a
candidate receptor for AL-2, has a wide tissue distribution
including expression in several myeloid hematopoietic cell lines
(Bennett et al., J. Biol. Chem. 269:14211-8 (1994)). Hematopoietic
expression of Htk in the monocytic lineage (myeloid but not
lymphoid hematopoietic cells) indicates that AL-2, upon Htk binding
and activation, can activate differentiation and/or proliferation
of these cells, finding use in treating conditions such as anemia,
bone marrow transplant (autologous or otherwise) or as adjunct
therapy in chemo- or radiation-therapies. Furthermore, AL-2
antagonists can reduce or prevent differentiation and/or
proliferation of these cells, a function that finds particular use
in disease conditions involving malignant forms of these cells, for
example, in treating acute myeloid leukemia (AML), chronic myeloid
leukemia (CML), or myelodysplastic syndrome (MDS). AL-2 antagonists
can be administered in conjunction with other agents or therapies
for AML or CML.
[0175] The development of a vascular supply, angiogenesis, is
essential for the growth, maturation, and maintenance of normal
tissues, including neuronal tissues. It is also required for wound
healing and the rapid growth of solid tumors and is involved in a
variety of other pathological conditions. Current concepts of
angiogenesis, based in large part on studies on the vascularization
of tumors, suggest that cells secrete angiogenic factors which
induce endothelial cell migration, proliferation, and capillary
formation. Numerous factors have been identified which induce
vessel formation in vitro or in vivo in animal models. These
include FGF.alpha., FGF.beta., TGF-.alpha., TNF-.alpha., VPF or
VEGF, monobutyrin, angiotropin, angiogenin, hyaluronic acid
degradation products, and more recently, B61 for
TNF-.alpha.-induced angiogenesis (Pandey et al., Science,
268:567-569 (1995)). Inhibitors of angiogenesis include a
cartilage-derived inhibitor identified as TIMP, PF-4,
thrombospondin, laminin peptides, heparin/cortisone, minocycline,
fumagillin, difluoromethyl ornithine, sulfated chitin derivatives,
and B61 antibody. The major development of the vascular supply
occurs during embryonic development, at ovulation during formation
of the corpus luteum, and during wound and fracture healing. Many
patholoiogical disease states are characterized by augmented
angiogenesis including tumor growth, diabetic retinopathy,
neovascular glaucoma, psoriasis, and rheumatoid arthritis. During
these processes normally quiescent endothelial cells which line the
blood vessels sprout from sites along the vessel, degrade
extracellular matrix barriers, proliferate, and migrate to form new
vessels. Angiogenic factors, secreted from surrounding tissue,
direct the endothelial cells to degrade stromal collagens, undergo
directed migration (chemotaxis), proliferate, and reorganize into
capillaries.
[0176] AL-2 may stimulate either the growth or differentiation of
cells expressing an AL-2 receptor. AL-2 that induces
differentiation of AL-2-receptor bearing may be useful in the
treatment of certain types of cancers. AL-2 may be used alone or in
combination with standard chemotherapy or radiation therapy for
cancer treatment. Where an AL-2-receptor is shown to be involved in
the development of a cancerous state, either through stimulation of
cell growth or through promotion of metastasis by stimulating cell
mobility and adhesion, AL-2 antagonists as taught herein will find
use. Fragments or analogs of AL-2 that bind to but do not activate
the receptor are useful antagonists. Administration of an
antagonist having affinity for the receptor will block receptor
binding and activation by endogenous activators. Administration of
soluble AL-2 receptor may also be used to counteract the biological
effects of receptor activation. Soluble AL-2 receptor will compete
with endogenous cell surface receptors for binding to activators,
including AL-2, and thereby reduce the extent of AL-2 receptor
activation. In addition, monoclonal antibodies directed either to
AL-2 or to the receptor may be useful in blocking the interactions
of AL-2, or other activator, with AL-2 receptors on cell
surfaces.
[0177] Accordingly, AL-2 can find further use in promoting or
enhancing angiogenesis by receptor activation on endothelial or
stromal cells. The induction of vascularization is a critical
component of the wound healing process. Neovascularization, also
known as angiogenesis, is a complex process involving several
sequential steps including basement membrane degradation,
endothelial cell mobilization and proliferation, vessel
canalization, and new basement membrane formation (Mantovani, Int.
J. Cancer, 25:617 (1980)). Vascularization ensures that
proliferating and differentiating fibroblasts are supplied with
nutrients and oxygen, and that elements of humoral and cellular
immunity are delivered to sites of potential bacterial infection.
It is desirable to induce neovascularization as early as possible
in the course of wound healing, particularly in the case of
patients having conditions that tend to retard wound healing, e.g.,
bums, decubitus ulcers, diabetes, obesity and malignancies. Even
normal post-surgical patients will be benefited if they can be
released from hospital care at any earlier date because of
accelerated wound healing. This invention provides novel
compositions and methods for modulating angiogenesis. A patient
bearing a wound can be treated by applying an angiogenically active
dose of an AL-2 compound to the wound. This facilitates the
neovascularization of surgical incisions, burns, traumatized
tissue, skin grafts, ulcers and other wounds or injuries where
accelerated healing is desired. In individuals who have
substantially impaired wound healing capacity, thereby lack the
ability to provide to the wound site endogenous factors necessary
for the process of wound healing, the addition of exogenous AL-2
and compositions of the invention enable wound healing to proceed
in a normal manner. The proteins of the present invention are
expected to accelerate the healing process in a broad spectrum of
wound conditions. Novel topical compositions containing an Al-2
compound are provided for use in the inventive method, as are novel
articles such as sutures, grafts and dressings containing an AL-2
compound. The term "wound" is defined herein as any opening in the
skin, mucosa or epithelial linings, most such openings generally
being associated with exposed, raw or abraded tissue. There are no
limitations as to the type of wound or other traumata that can be
treated in accordance with this invention, such wounds including
(but are not limited to): first, second and third degree burns
(especially second and third degree); surgical incisions, including
those of cosmetic surgery; wounds, including lacerations,
incisions, and penetrations; and ulcers, e.g., chronic non-healing
dermal ulcers, including decubital ulcers (bed-sores) and ulcers or
wounds associated with diabetic, dental, hemophilic, malignant and
obese patients. Furthermore, normal wound-healing may be retarded
by a number of factors, including advanced age, diabetes, cancer,
and treatment with anti-inflammatory drugs or anticoagulants, and
the proteins described herein may be used to offset the delayed
wound-healing effects of such treatments.
[0178] Although the primary concern is the healing of major wounds
by neovascularization, it is contemplated that an AL-2 compound may
also be useful for minor wounds, and for cosmetic regeneration of
epithelial cells. Preferably, the wounds to be treated are burns
and surgical incisions, whether or not associated with viral
infections or tumors. In most cases wounds are not the result of a
tumor or a viral infection and ordinarily they do not include tumor
cells.
[0179] AL-2 is preferably delivered to wounds by topical
application, "topical" in this context meaning topical to the
wound, and does not necessarily refer to epidermal application.
When applied topically, the AL-2 compound is usually combined with
other ingredients, such as carriers and/or adjuvants. There are no
limitations on the nature of such other ingredients, except that
they must be pharmaceutically acceptable, efficacious for their
intended administration, and cannot degrade or inactivate AL-2.
AL-2 is applied to burns in the form of an irrigant or salve, and
if so then in an isotonic solution such as physiological saline
solution or D5W. AL-2 is particularly useful in accelerating the
growth and survival of skin grafts applied to bums. Ordinarily, an
AL-2-containing composition is impregnated into the grafts or
adherently coated onto the face of the graft, either on the side of
the graft to be applied to the burn or on the exterior side of the
graft. AL-2 also is included in burn debridement salves which
contain proteases so long as the debridement enzyme does not
proteolytically inactivate the AL-2.
[0180] AL-2 is impregnated into surgical articles in accordance
with this invention, such articles being defined as items to be
contacted with wounds which articles are typically water adsorbent
or hydratable and which have a therapeutic utility in treating
wounds. Examples of surgical articles are dressings, sutures,
pledgets, skin grafling films (including living skin grafts as well
as collagen-containing membranes or synthetic skin substitutes) and
the like as will be known to the clinician. Dressings for use
herein generally comprise water adsorbent laminates containing AL-2
to be adherently placed into contact with wounds. Improved
dressings for use with AL-2 as described herein preferably will
have a membrane such as a dialysis membrane interposed between the
wound surface and the adsorbent substance in the dressing, the
membrane containing pores sufficiently small for AL-2 to diffuse
into the wound but not sufficiently large for epithelial cells to
penetrate into the adsorbent. The degree of adsorbency will vary
considerably and in fact dressings are included herein which are
nonadsorbent, i.e., the AL-2 is deposited or stored in an aqueous
reservoir which is used to irrigate the wound on a continuous or
intermittent basis.
[0181] AL-2 also is formulated into ointments or suspensions,
preferably in combination with purified collagen, in order to
produce semisolid or suspension vehicles. Conventional oleaginous
formulations containing AL-2 are useful as salves. Such AL-2
carriers and formulations release AL-2 on a sustained basis at the
wound, thereby serving to create a chemotactic gradient that
directibnally orients neovascularization, e.g., into a skin graft.
Sustained release formulations for AL-2 include semipermeable
polymer matrices in the form of shaped articles, e.g., films, or
microcapsules. Implantable sustained release matrices include
copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman
et al., Biopolymers, 22(1):547-556 (1983)),
poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.
Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)),
ethylene vinyl acetate (Langer et al., Id.), or
poly-D-(-)-3-Hydroxybutyric acid (EP 133,988A). These formulations
may function as bioerodible matrices or as stable sources for the
passive diffusion of AL-2.
[0182] Sustained release AL-2 compositions for contact with wounds
also include liposomally entrapped AL-2. Liposomes containing AL-2
are prepared by methods known per se: DE 3,218,121A; Epstein et
al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); Hwang et al.,
Proc. Natl. Acad. Sci. USA, 77:4030-4034 (1980); EP 52322A; EP
36676A; EP 88046A; EP 143949A; EP 142641A; Japanese patent
application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and
EP 102,324A. Ordinarily the liposomes are of the small (about
200-800 Angstroms) unilamellar type in which the lipid content is
greater than about 30 mol. % cholesterol, the selected proportion
being adjusted for the optimal rate of AL-2 leakage.
[0183] AL-2 is formulated with other ingredients such as carriers
and/or adjuvants, e.g., albumin, nonionic surfactants and other
emulsifiers. There are no limitations on the nature of such other
ingredients, except that they must be pharmaceutically acceptable,
efficacious for their intended administration, and cannot degrade
the activity of the active ingredients of the compositions.
Suitable adjuvants include collagen or hyaluronic acid
preparations, fibronectin, factor XIII, or other proteins or
substances designed to stabilize or otherwise enhance the active
therapeutic ingredient(s).
[0184] AL-2 optionally is supplied with other known angiogenic
agents such as heparin, which has been shown to accelerate the
healing of thermal bums, TNF, TGF-.alpha., TGF-.beta., fibroblast
growth factor, epidermal growth factor, B61, angiogenin, platelet
factor 4, insulin, PDGF, and angiogenesis factor and the angiogenic
activity of the combinations observed for synergistic effects. AL-2
optionally also is combined with an with an IFN, e.g., IFN-.gamma.,
and other cytokines, or may be free of interferons such as
IFN-.gamma.. Where such cytokines or known angiogenic agents are
species-specific, the appropriate cytokine or agent will be
selected for the species to be treated.
[0185] Animals or humans are treated in accordance with this
invention. It is possible but not preferred to treat an animal of
one species with AL-2 of another species. A preferred AL-2 for use
herein is soluble AL-2-IgG.
[0186] The amount of AL-2 to be contacted with the wound depends
upon a great number of variables that will be taken into account by
the clinician, including the presence of other angiogenic agents in
the AL-2 formulations, the nature of the wound to be treated, the
condition of the patient, the AL-2 formulation selected, the
neovascularizing activity of the molecular species of AL-2 chosen
and the route of administration. Lesser amounts of AL-2 typically
are administered when the AL-2 is formulated into a sustained
release vehicle, e.g., dressing or ointment, and when the AL-2 is
administered by direct topical contact rather than impregnated into
a bandage or dressing. Concentrations in the range of 0.10
ng/ml-100 ug/ml may be used. The typical topical formulation will
be capable of delivering a concentration of AL-2-IgG, or
equivalent, at the neovascularization target site (for example, a
skin graft) in a range of about from 0.10 ng/ml to 10000 ng/ml,
more preferably 0.20 ng/ml to 1000 ng/ml, and even more preferably
0.25 ng/ml to 350 ng/ml, although this therapeutic dose range is
subject to considerable variation as noted above. Delivery of
concentrations outside of this range may offer certain of the
benefits of AL-2 neovascularization, but the clinician will be
expected to monitor dosages in order to optimize performance of
AL-2 in wound healing. It also should be noted that the weight
amount will vary for other AL-2 variants and forms if their
molecular weight and/or angiogenic potency differ from that of
AL-2-IgG. Potency differences are easily determined by comparing
the degree of neovascularization achieved with the candidate AL-2
and AL-2-IgG in any of the assays set forth in the Examples
herein.
[0187] Accordingly, a method for accelerating the
neovascularization of a wound is provided that includes the step of
applying to the wound an angiogenically effective dose of a
composition comprising tumor necrosis factor. The composition can
be applied topically by direct contact with the wound, particularly
when the wound is a fresh surgical incision. In a preferred
embodiment the composition further comprises collagen or a
synthetic skin substitute. The caregiver may further administer to
the wound a growth factor, an antibiotic, a debridement agent,
and/or angiogenin. A preferred composition for the debridement of
burns contains an AL-2 compound and a proteolytic enzyme which does
not inactivate the neovascularizing activity of AL-2. The
therapeutic compositions can be reapplied at
one-to-several-day-intervals until healing is complete.
[0188] Therapeutic options for patients with vascular disease,
particularly vascular obstructive disease, are sometimes limited.
Such patients are often refractory to conservative measures and
typically unresponsive t6 drug therapy (Takeshita et al., J. Clin.
Invest., 93:662-670 (1994)). When vascular obstruction is lengthy
and/or widespread, nonsurgical revascularization may not be
feasible. Surgical therapy, consisting of arterial bypass and/or
amputation, may be complicated by a variable morbidity and
mortality, and is often dependent for its efficacy upon short- and
long-term patency of the conduit used. Therapeutic angiogenesis
constitutes an alternative treatment strategy for such patients.
The present invention provides methods for enhancing angiogenesis
in a mammal comprising administering to the mammal an effective
amount of AL-2. The AL-2 alone may be administered to the mammal,
or alternatively, may be administered to the mammal in combination
with other therapies and/or pharmacologic agents. In particular
AL-2 finds use in patients suffering from vascular insufficiency or
limb ischemia secondary to arterial occlusive disease. The effects
of AL-2 proteins of the invention on angiogenesis can be tested,
for example, in a rabbit model of hindlimb ischemia. This rabbit
model was designed to simulate ischemia characteristics of patients
with severe lower extremity arterial occlusive disease and is
performed essentially as described in Takeshita et al., J. Clin.
Invest., 93:662-670 (1994). Measurements of calf blood pressure
(BP) index; angiographic score of collateral formation;
intravascular Doppler-wire analysis of blood flow; and
microsphere-based analysis of muscle perfusion at rest and during
stress are performed.
[0189] AL-2 antagonists can find use in inhibiting, preventing or
treating pathological angiogenesis, such as during tumor
vascularization. Tumor neovascularization is a vital stage in the
growth of solid tumors (Polverini et al., Lab. Invest., 51:635-642
(1985)). The progressive growth of solid tumors is strictly
dependent on their ability to attract new blood vessels that will
supply them with oxygen and essential nutrients (Bouck, Cancer
Cells, 2(6):179-185 (1990)). Angiogenesis has been shown to precede
or accompany malignancy. In the absence of neovascularization the
size of tumor grafts becomes limited. When angiogenesis is absent,
tumors tend to remain dormant. Therefore, angiogenic activity has
been directly correlated with tumor growth. AL-2 antagonist
compositions and methods of the invention can modulate (e.g.,
prevent or reduce) new capillary growth into tumors.
[0190] A variety of non-neoplastic diseases, previously thought to
be unrelated, can be considered "angiogenic diseases" because they
are dominated by the pathological growth of capillary blood
vessels. These diseases include diabetic retinopathy, arthritis,
hemangiomas, psoriasis, and ocular neovascularization. AL-2
antagonist compositions and methods of the invention can be used to
treat these conditions.
[0191] Vascularization also plays a critical role in chronic
inflammatory conditions such as rheumatoid arthritis (Koch et al.,
Arthr. Rheum., 29:471-479 (1986)). Rheumatoid arthritis ("RA") is a
chronic heterogeneous disorder in which a variety of etiological
agents may be responsible for initiating a series of events leading
to inflammation in multiple joints. The cause of the disease
remains unknown, although by analogy with other forms of arthritis
such as that accompanying Lyme disease, it has been postulated that
infection with as yet unidentified bacteria or viruses in a
genetically susceptible host is an initiating event. Persistence
could result from the presence of viral or bacterial antigens that
generate an immune response or cross-react with host tissues
together with amplification effects of cellular products of the
host. While many patients have systemic manifestations in RA, many
of the most serious consequences of RA stem from its effects on
articular connective tissues, which are characterized by
alterations of the synovial membrane with proliferation of lining
cells and infiltration by chronic inflammatory cells. Erosions of
bone occur in areas contiguous with the inflammatory cell mass as
well as in regions adjacent to bone marrow distant from the
inflammation. The bone erosions are probably produced through
induction of differentiation and activation of osteoclast
progenitors. The erosion of soft connective tissues, e.g.,
cartilage, joint capsules, tendons, and ligaments, results from
direct release of proteolytic enzymes from cells of the
inflammatory cell mass or from polymorphonuclear leukocytes that
are typically abundant in rheumatoid synovial fluids, although rare
in the synovial membrane. See, for example, Harris, W. N. Kelley et
al., eds., Textbook of Rheumatology, W. B. Saunders, Philadelphia,
pp. 886-915 (1985); Dayer et al., Clin. Rheum. Dis., 4:517-537
(1978); Krane, Arthritis and Allied Conditions. A textbook of
Rheumatology, ed. by D. J. McCarty, pp. 593-604, Lea and Febiger,
Philadelphia (1985); and Krane et al., Lymphokines, 7:75-136
(1982). Therapy for RA depends on the stage of the disease. Stage
1, where a postulated antigen is presented to T-cells with no
obvious arthritic symptoms, is not treated. Stage 2 involves T-cell
and B-cell proliferation and angiogenesis in synovial membrane,
resulting in malaise, mild joint stiffness, and swelling. During
Stage 3, neutrophils accumulate in synovial fluid and synovial
cells proliferate without polarization or invasion of cartilage,
resulting in joint pain and swelling, morning stiffness, malaise,
and weakness. Current therapy for Stages 2 and 3 includes bed rest,
application of heat, supplemental eicosapentaenoic and
docosahexanoic acid, and drugs. Nonsteroidal anti-inflammatory
drugs, including aspirin, continue to be the foundation of drug
therapy in treating Stages 2 and 3 of the disease. Those
anti-inflammatory drugs other than aspirin include indomethacin,
phenylbutazone, phenylacetic acid derivatives such as ibuprofen and
fenoprofen, naphthalene acetic acids (naproxen), pyrrolealkanoic
acid (tometin), indoleacetic acids (sulindac), halogenated
anthranilic acid (meclofenamate sodium), piroxicam, zomepirac, and
diflunisal. Second-line drugs for RA Stages 2 and 3 include
anti-malarial drugs such as hydroxychloroquine, sulfasalazine, gold
salts, and penicillamine, and low-dose methotrexate. These
alternatives frequently produce severe side effects, including
retinal lesions and kidney and bone marrow toxicity. The
irreversible destruction of cartilage occurs in Stage 4 of the
disease. Currently available drugs and treatments include total
lyrnphoid irradiation, high-dose intravenous methylprednisolone,
and cyclosporine. Cyclosporine is nephrotoxic and the other
treatments exert substantial toxicity as well. As a result, such
immunosuppressive agents heretofore have been used only in the
treatment of severe and unremitting RA. Other possible therapeutic
drugs for Stage 4 of RA include cyclic oligosaccharides
(cyclodextrins), which, when combined with a noninflammatory
steroid (cortexolone), inhibit angiogenesis in vivo. Folkman et
al., Science 243:1490-1493 (1989). Antibodies against crucial
components of the early phase of the immune response include
anti-Class II MHC antibodies (Gaston et al., Arthritis Rheum.,
31:21-30 (1988); Sany et al., Arthritis Rheum., 25:17-24 (1982)),
anti-interleukin-2 receptor antibodies (Kyle et al., Ann. Rheum.
Dis., 48:428-429 (1989)), anti-CD4 antibodies (Herzog et al., J.
Autoimmun., 2:627-642 (1989); Walker et al., J. Autoimmun.,
2:643-649 (1989)), and antithymocyte globulin (Shmerling et al.,
Arthritis Rheum., 32:1495-1496 (1989)). The last three of these
drugs have been used in patients with RA. The present invention
provides compositions and methods that down-regulate inflammatory
and proliferative pathways in RA by modulating the associated
angiogenesis. The compositions contain an angiogenesis-modulating
effective amount of an AL-2 antagonist. The compositions can
further comprise other angiogenesis-modulating agents, particularly
agents for treating RA as discussed above or agents for treating
tumors. The methods involve the step of administering an
angiogenesis-modulating effective amount of an AL-2 antagonist to a
mammal in need of such treatment. By "modulating" in the context of
conditions in which angiogenesis is undesirable is meant blocking,
inhibiting, preventing, reversing, or reducing angiogenesis, or
preventing further progression of angiogenesis.
[0192] There seems to be little or no biochemical difference
between angiogenic peptides expressed by tumors and those expressed
by normal tissues. Nor are there any morphological differences
between the new capillaries that respond to a malignancy and the
capillary growth that occurs during physiological
neovascularization (Folkman et al., Science, 235:442-447 (1987)).
As there is no qualitative difference between the angiogenic
capabilities of nonmalignant and malignant diseases, results from
normal and malignant vascularization assays can be easily compared,
and progress can be made in either area independently of the system
of investigation used (Paweletz et al., Critical Reviews in
Oncology/Hematology, 9(3):197-198 (1989)). Thus, the invention
relates to a method comprising inhibiting angiogenesis by
administering an effective amount of AL-2 antagonist. The invention
comprises a method for the treatment of angiogenesis-dependent
diseases by administering an effective amount of AL-2 antagonist to
a mammal. Angiogenesis dependent diseases include, but are not
limited to diabetic retinopathy, arthritis, tumor growth and
metastasis, neovascular glaucoma, retinopathy of prematurity,
senile macular degeneration, and hypergeneration of scars after
wound healing. The pharmaceutical composition of the present
invention exhibit therapeutic properties and preventative or
inhibitive properties against diseases associated with
angiogenesis, for example, inflammatory diseases (e.g., rheumatoid
arthritis), diabetic retinopathy, tumors such as malignant tumors
(e.g., cancer such as mastocarcinoma, hepatoma, colic carcinoma,
Kaposi's sarcoma, lung carcinomas and other epithelial
carcinomas).
[0193] Numerous methods, in vitro and in vivo, are available to
screen candidate AL-2 or AL-2 antagonists compounds for angiogenic
or angiogenesis-inhibiting properties. Several in vitro assays of
endothelial cell growth, migration, and capillary tube formation
are known and can be used with the compounds of the invention,
particularly as initial screening methods for angiogenic or
angiostatic substances. Further testing would typically use in vivo
animal testing. U.S. Pat. No. 5,382,514, which is incorporated
herein, describes numerous models for angiogenesis in vivo. For
example, the corneal pocket assay involves the surgical
implantation of polymer pellets containing angiogenic factors in
the cornea of larger animals such as rabbits. Since quantitation
can be difficult the assay is usually used for preferred candidate
compounds. The rabbit ear chamber assay requires the surgical
insertion of a glass or plastic viewing device and measurement of
capillary migration by microscopy. The rat dorsal air sac assay
involves implants of stainless steel chambers containing angiogenic
factors. An alginate assay which generates an angiogenic response
has been described which involves the injection of tumor cells
encased in alginate subcutaneously into mice. The accumulation of
hemoglobin in the injected gel is used to quantitate the angiogenic
response. A compound can be administered to the chorio-allantoic
membranes of aged, typically three-day-aged, fertilized chicken
eggs and the appearance of neovascularization after a lapse of
time, typically two days is observed (CAM assay; Ausprunk et al.,
Am. J. Pathol., 97:597 (1975)). The neovascularization inhibitory
rates are compared with an untreated control group. A more recent
assay method involves providing a liquid matrix material which
forms a matrix gel when injected into a host; adding an angiogenic
agent to the liquid matrix material; injecting the liquid matrix
material containing the angiogenic agent into a host to form a
matrix gel; recovering the matrix gel from the host; and
quantitating angiogenesis of the recovered matrix gel. A variation
of this can be used to test for inhibitors of vascularization in a
tissue by providing a liquid matrix material which forms a matrix
gel when injected into a host; adding an angiogenic inhibiting
agent to the liquid matrix material; and injecting the liquid
matrix material containing the angiogenic inhibiting agent into a
tissue situs of a host to form a matrix gel. This system can be
used with compounds of the invention when inducing vascularization
in a tissue is desired by providing a liquid matrix material which
forms a matrix gel when injected into a host; adding an angiogenic
inducing agent to the liquid matrix material; and injecting the
liquid matrix material containing the angiogenic inducing agent
into a tissue situs of a host to form a matrix gel. In a preferred
embodiment, a solution of basement membrane proteins supplemented
with fibroblast growth factor and heparin is injected
subcutaneously in a host, e.g., a mouse, where it forms a gel.
Sprouts from vessels in the adjacent tissue penetrate into the gel
within days connecting it with the external vasculature.
Angiogenesis is then quantitated by image analysis of vessels and
by measuring the hemoglobin present in the vessels within the gel.
This assay method facilitates the testing of both angiogenic and
angiostatic agents in vivo. In addition, the endothelial cells
responding to the angiogenic factors can be recovered in vitro for
further studies. Preferred compounds have 50-70% inhibition rates,
and more preferred compounds show 80-100% inhibition rates. As
described herein the angiogenically active proteins of the
invention provide use in in vitro and in vivo screens for compounds
that inhibit angiogenesis by measuring inhibition of AL-2
stimulated angiogenesis in the presence and absence of the
candidate inhibitor.
[0194] Therapeutic formulations of AL-2 and AL-2 antagonists for
treating neurologic diseases and disorders and for modulating
angiogenesis and other disorders are prepared by mixing AL-2 or
AL-2 antagonist, e.g., anti-AL-2 antibody or a soluble
AL-2-binding-Eph-receptor, having the desired degree of purity,
with optional physiologically acceptable carriers, excipients, or
stabilizers which are well known. Acceptable carriers, excipients
or stabilizers are nontoxic at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid; 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, arginine or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; salt-forming counterions such as sodium;
and/or nonionic surfactants such as Tween, Pluronics or
polyethylene glycol (PEG).
[0195] It may be desirable to adsorb AL-2 onto a membrane, such as
a silastic membrane, which can be implanted in proximity to damaged
neural tissue, or to incorporate AL-2 into liposomes (PCT Pat. Pub.
No. WO 91/04014, published Apr. 4, 1991).
[0196] AL-2 optionally is combined with or administered in concert
with other neurotrophic factors to achieve a desired therapeutic
effect. For example, AL-2 may be used together with NGF, NT-3,
BDNF, NT-4/5, an insulin-like growth factor (e.g., IGF-1, IGF-2, or
IGF-3) or another neurotrophic factor to achieve a synergistic
stimulatory effect on the growth of sensory neurons, wherein the
term "synergistic" means that the effect of the combination of AL-2
with a second substance is greater than that achieved with either
substance used individually.
[0197] AL-2 and AL-2 antagonists to be used for in vivo
administration must be sterile. This is readily accomplished by
filtration of a solution of AL-2 or anti-AL-2 antibody through
sterile filtration membranes. Thereafter, the filtered solution may
be 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 filtered solution
also may be lyophilized to produce sterile AL-2 or anti-AL-2
antibody in a powder form.
[0198] Methods for administering AL-2 and AL-2 antagonists in vivo
include injection or infusion by intravenous, intraperitoneal,
intracerebral, intrathecal, intramuscular, intraocular,
intraarterial, or intralesional routes, and by means of
sustained-release formulations.
[0199] Sustained-release formulations generally consist of AL-2 or
AL-2 antagonists and a matrix from which the AL-2 or AL-2
antagonists are released over some period of time. Suitable
matrices include semipermeable polymer matrices in the form of
shaped articles, for example, membranes, fibers, or microcapsules.
Sustained release matrices may comprise polyesters, hydrogels,
polylactides, U.S. Pat. No. 3,773,919, copolymers of L-glutamic
acid and gamma ethyl-L-glutamate, Sidman, et al., Biopolymers, 22:
547-556 (1983), poly(2-hydroxyethyl-methacrylate), or ethylene
vinyl acetate, Langer, et al., J. Biomed. Mater. Res., 15: 167-277
(1981); Langer, Chem. Tech., 12:98-105 (1982).
[0200] In one embodiment of the invention, the therapeutic
formulation comprises AL-2 or AL-2 antagonist entrapped within or
complexed with liposomes. For example, AL-2 covalently joined to a
glycophosphatidyl-inositol moiety may be used to form a liposome
comprising AL-2. In a further embodiment, the therapeutic
formulation comprises cells actively producing AL-2 or AL-2
antagonist. Such cells may be directly introduced into the tissue
of a patient, or may be encapsulated within porous membranes which
are then implanted in a patient, in either case providing for the
delivery of AL-2 or anti-AL-2 antagonist into areas within the body
of the patient in need of increased or decreased concentrations of
AL-2. Alternatively, an expression vector comprising AL-2 DNA may
be used for in vivo transformation of a patient's cells to
accomplish the same result.
[0201] An effective amount of AL-2 or AL-2 antagonist, e.g.,
anti-AL-2 antibody, to be employed therapeutically will depend, for
example, upon the therapeutic objectives, the route of
administration, and the condition of the patient. Accordingly, it
will be necessary for the therapist to titer the dosage and modify
the route of administration as required to obtain the optimal
therapeutic effect. A typical daily dosage might range from about 1
.mu.g/kg to up to 100 mg/kg or more, depending on the factors
mentioned above. Where possible, it is desirable to determine
appropriate dosage ranges first in vitro, for example, using assays
for neuronal cell survival or growth which are known in the art,
and then in suitable animal models, from which dosage ranges for
human patients may be extrapolated. In a specific embodiment of the
invention, a pharmaceutical composition effective in promoting the
survival or growth of neurons will provide a local growth promoting
activity concentration in vivo of between about 0.1 and 10 ng/ml.
Typically, the clinician will administer AL-2 until a dosage is
reached that achieves the desired effect. Therapeutic progress is
easily monitored by conventional assays.
[0202] In the treatment of tumors the compositions described herein
can be administered subcutaneously or intramuscularly, for example,
and the pharmacological activities of an AL-2 antagonist can be
maintained over a long period of time by the sustained-release
effect of a composition of the present invention. The number of
administrations can therefore be reduced. The composition can also
be by directly injecting the composition into a tumor-controlling
artery. In the case of treatment of an adult patient having a
tumor, the dose of the AL-2 antagonist can be appropriately
selected depending upon the kind of tumor, site, size, and kind of
AL-2 antagonist. For example, the dose of a protein AL-2
antagonists, particularly an antibody, can be about 0.1 mg to about
500 mg, typically about 1.0 mg to about 300 mg, more typically
about 25 mg to about 100 mg. The administration frequency can be
appropriately selected depending upon the kind of disease and
dosage form. In the case of injection into the tumor-controlled
artery or tumor itself, frequently repeated injections are not
required and a single injection once every one to 4 weeks may be
sufficient for the desired therapeutic effects.
[0203] The nucleic acid encoding the AL-2 may be used as a
diagnostic for tissue-specific typing. For example, such procedures
as in situ hybridization, Northern and Southern blotting, and PCR
analysis, can be used to determine whether DNA and/or RNA encoding
AL-2 is present in the cell type(s) being evaluated. AL-2 nucleic
acid or polypeptide may also be used as diagnostic markers for such
tissues. For example, the AL-2 may be labeled, using the techniques
described herein, and expression of AL-2-receptor, including the
preferred receptors disclosed herein, receptor can be quantified
via its binding to labelled AL-2.
[0204] AL-2 nucleic acid is also useful for the preparation of AL-2
polypeptide by recombinant techniques exemplified herein.
[0205] The invention also provides methods for studying the
function of the AL-2 protein. Cells, tissues, and non-human animals
lacking AL-2 expression, partially lacking in AL-2 expression, or
over-expressing AL-2 can be developed using recombinant molecules
of the invention having specific deletion or insertion mutations in
the AL-2 gene. For example, the extracellular domain or parts
thereof, the transmembrane region or parts thereof, and the
cytoplasmic domain can be deleted. A recombinant molecule may be
used to inactivate or alter the endogenous gene by homologous
recombination, and thereby create an AL-2 deficient (or
over-expressing) cell, tissue or animal.
[0206] Null alleles can be generated in cells, such as embryonic
stem cells by deletion mutation. A recombinant AL-2 gene may also
be engineered to contain an insertion mutation which inactivates
AL-2. Such a construct may then be introduced into a cell, such as
an embryonic stem cell, by a technique such as transfection,
electroporation, injection, etc. Cells lacking an intact AL-2 gene
can then be identified, for example by Southern blotting, Northern
blotting or by assaying for expression of AL-2 protein using the
methods described herein. Such cells may then be fused to embryonic
stem cells to generate transgenic non-human animals deficient in
AL-2. Germine transmission of the mutation may be achieved, for
example, by aggregating the embryonic stem cells with early stage
embryos, such as 8 cell embryos, in vitro; transferring the
resulting blastocysts into recipient females and; generating
germine transmission of the resulting aggregation chimeras. Such a
mutant animal may be used to define specific nerve cell
populations, developmental patterns of axonogenesis, neural tube
formation and nerve regeneration and in vivo processes, normally
dependent on AL-2 expression.
[0207] Methods for preparing cells, tissues, and non-human animals
lacking in AL-2 expression or partially lacking in AL-2 expression,
and deficient in the expression of other genes are provided. In one
embodiment, an animal may be generated which is deficient in AL-2
and another tyrosine kinase receptor ligand. Such animals could be
used to determine how the members of the family cooperate in
embryonic development, particularly development of the nervous
system. For example, an animal lacking or partially lacking AL-2
expression and Htk-L expression can be generated to determine how
the receptor tyrosine kinases cooperate in neurogenesis, e.g., the
segmental patterning of the hindbrain. Multiple deficient mice can
also be generated to study the interaction of AL-2 and other
proteins such as the ligands of the Src-family of cytoplasmic
tyrosine kinases. For example, an animal may be generated which
lacks or partially lacks AL-2 expression, and expression of one or
more Src family tyrosine kinases including Src or Fyn and their
ligands.
[0208] The binding characteristics of AL-2 (including variants) can
also be determined using purified receptor, e.g., conjugated,
soluble receptor (for example, .sup.125I-Htk-Fc or Hek5-IgG) in
competition assays as described herein. For example, either intact
cells expressing AL-2 or soluble AL-2 bound to a solid substrate
are used to measure the extent to which a sample containing a
putative AL-2-receptor competes for binding of a conjugated soluble
receptor to AL-2.
[0209] The AL-2 of the present invention can be used in a binding
assay to detect cells expressing an Eph-family receptor that binds
AL-2. For example, AL-2 or an extracellular domain or a fragment
thereof can be conjugated to a detectable moiety such as .sup.125I.
Radiolabeling with .sup.125I can be performed by any of several
standard methodologies that yield a functional .sup.125I-AL-2
molecule labeled to high specific activity. Alternatively, another
detectable moiety such as an enzyme that can catalyze a
colorimetric or fluorometric reaction, biotin or avidin may be
used. Cells or samples to be tested for AL-2-receptor expression
can be contacted with labeled AL-2. After incubation, unbound
labeled AL-2 is removed and binding is measured using the
detectable moiety.
[0210] The AL-2 proteins disclosed herein can be employed to
measure the biological activity of an AL-2 receptor in terms of
binding affinity for AL-2. For example, AL-2 can be employed in a
binding affinity study to measure the biological activity of a
receptor that has been stored at different temperatures, or
produced in different cell types. Thus, AL-2 proteins find use as
reagents in "quality assurance" studies, e.g., to monitor shelf
life and stability of receptor protein under different conditions.
Furthermore, AL-2 can be used in determining whether biological
activity is retained after modification of a receptor protein
(e.g., chemical modification, truncation, mutation, etc.). The
binding affinity of the modified receptor for an AL-2 is compared
to that of an unmodified receptor to detect any adverse impact of
the modifications on biological activity of the receptor.
[0211] Binding of AL-2 to an Eph-family receptor can be determined
using conventional techniques, including competitive binding
methods, such as RIAs, ELISAs, and other competitive binding
assays. Ligand/receptor complexes can be identified using such
separation methods as filtration, centrifugation, flow cytometry
(see, e.g., Lyman et al., Cell 75:1157-1167 (1993); Urdal et al.,
J. Biol. Chem. 263:2870-2877 (1988); and Gearing et a., EMBO J.
8:3667-3676 (1989)). Results from binding studies can be analyzed
using any conventional graphical representation of the binding
data, such as Scatchard analysis (Scatchard, Ann. NY Acad. Sci.
51:660-672 (1949); and Goodwin et a., Cell 73:447-456 (1993)), and
the like. Since the AL-2 induces receptor phosphorylation,
conventional tyrosine phosphorylation assayscan also be used.
[0212] Isolated AL-2 polypeptide may be used in quantitative
diagnostic assays as a standard or control against which samples
containing unknown quantities of AL-2 may be prepared.
[0213] AL-2 preparations are also useful in generating antibodies,
as standards in assays for AL-2 (e.g., by labeling AL-2 for use as
a standard in a radioimmunbassay, or enzyme-linked immunoassay),
for detecting the presence of an AL-2-receptor in a biological
sample (e.g., using a labelled AL-2), in affinity purification
techniques, and in competitive-type receptor binding assays when
labeled for example with radioiodine, enzymes, fluorophores, spin
labels, or branched DNA.
[0214] AL-2 polypeptide can be produced in prokaryotic cells using
the techniques taught herein, and the unglycosylated protein so
produced can be used as a molecular weight marker, for example.
Preferably unglycosylated, soluble AL-2 is used. AL-2 can be used
as a molecular weight marker in gel filtration chromatography or
SDS-PAGE, for example, either analytical or preparative modes,
where it is desirable to determine molecular weight(s) for
separated peptides. AL-2 is most preferably used in combination
with other known molecular weight markers as standards to provide a
range of molecular weights. Other known molecular weight markers
can be purchased commercially, e.g., from Amersham Corporation,
Arlington Heights, Ill., for example. The molecular weight markers
can be labelled to enable easy detection following separation.
Techniques for labelling antibodies and proteins are discussed
herein and are well known in the art. For example, the molecular
weight markers can be biotinylated and, following separation on
SDS-PAGE, for example, can be detected using
streptavidin-horseradish peroxidase.
[0215] AL-2 is used for competitive screening of potential agonists
or antagonists for binding to an AL-2 receptor. AL-2 variants are
useful as standards or controls in assays for AL-2, provided that
they are recognized by the analytical system employed, e.g., an
anti-AL-2 antibody.
[0216] One embodiment is a method for identifying compounds that
modulate the activity of an AL-2-binding-Eph-family receptor. The
method includes the steps of (a) exposing cells exhibiting the
receptor to ligand, i.e., AL-2 or modified or variant forms, for a
time sufficient to allow formation of receptor-ligand complexes and
induce signal transduction, (b) determining the extent of activity
within the cells, and (c) comparing the measured activity to the
activity in cells not exposed to the ligand. Receptor activity may
be detected by changes in target cell proliferation,
differentiation, metabolism, or other activity of interest (e.g.,
axon-targeting, neuronal outgrowth), preferably one predictive of
success of a therapeutic method. The receptor can be endogenous or
can be present as a result of expression of a recombinant
molecule.
[0217] AL-2 can be useful as a growth factor or differentiation for
cells having an AL-2 receptor. These cells, which can be grown ex
vivo, can simultaneously be exposed to other known growth factors
or cytokines. Exemplary cytokines include the interleukins (e.g.,
IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF),
macrophage colony-stimulating factor (M-CSF), granulocyte
colony-stimulating factor (GM-CSF), erythropoietin (Epo),
lymphotoxin, steel factor (SLF), tumor necrosis factor (TNF) and
gamma-interferon. This results in proliferation and/or
differentiation of the cells having a AL-2 receptor. For example,
human tumor cell lines for which it is desired to isolate certain
tumor associated factors (usually proteins) therefrom can be grown
ex vivo using AL-2. Also, antibodies against the tumor associated
factors can be generated which may be useful for diagnostic
purposes. Examples of such tumor cell lines which are candidates
for treatment with AL-2 include mammary cancer cells (e.g. MCF-7),
liver cell lines, Colo 205, NCI 69, HM-1 and HeLa, for example.
[0218] A different use of an AL-2 is as a reagent in protein
purification procedures. AL-2 or AL-2-IgG fusion proteins may be
attached to a solid support material by conventional techniques and
used to purify an AL-2-binding protein, e.g. receptor, by affinity
chromatography. AL-2 can be used for affinity purification of an
AL-2 receptor. Briefly, this technique involves covalently
attaching AL-2 to an inert and porous matrix (e.g., agarose reacted
with cyanogen bromide). A solution containing the AL-2 receptor can
then be passed through the chromatographic material and can he
subsequently released by changing the elution conditions (e.g. by
changing pH or ionic strength).
[0219] AL-2 polypeptides find use as carriers for delivering agents
to cells bearing an AL-2-binding cell surface receptor. Al-2 can be
used to deliver diagnostic or therapeutic agents to these cells (or
to other cell types found to express an AL-2 receptor on the cell
surface) in in vitro, ex vivo, or in vivo procedures. One example
of such use is to expose an AL-2 receptor expressing neoplastic
cell line to a therapeutic agent/AL-2 conjugate to assess whether
the conjugate exhibits a desired effect on the target cells. A
number of different therapeutic agents attached to AL-2 can be
included in an assay to detect and compare the effect of the agents
on the target cells. In a preferred embodiment the agent is a
cytotoxin; however, the agent can be a viral protease inhibitor or
the like. In another embodiment, a diagnostic, i.e. detectable
agent, is conjugated to AL-2 to detect the presence of
AL-2-receptor-expressing cells.
[0220] Diagnostic and therapeutic agents that may be attached to a
AL-2 polypeptide include, but are not limited to, drugs, toxins,
antiviral agents, radionuclides, chromophores, fluorescent
compounds, enzymes that catalyze a colorimetric or fluorometric
reaction, and the like, with the particular agent being chosen
according to the intended application. Examples of drugs include
those used in treating various forms of cancer, e.g., nitrogen
mustards such as L-phenylalanine nitrogen mustard or
cyclophosphamide, intercalating agents such as
cis-diaminodichloroplatinum, antimetabolites such as
5-fluorouracil, vinca alkaloids such as vincristine, and
antibiotics such as bleomycin, doxorubicin, daunorubicin, and
derivatives thereof. Among the toxins are ricin, abrin, diphtheria
toxin, Pseudomonas aeruginosa exotoxin A, ribosbmal inactivating
proteins, mycotoxins such as trichothecenes, and derivatives and
fragments (e.g., single chains) thereof. Radionuclides suitable for
diagnostic use include, but are not limited to, .sup.123I,
.sup.131I, .sup.99mTc, .sup.111In, and .sup.76Br. Radionuclides
suitable for therapeutic use include, but are not limited to,
.sup.131I, .sup.211At, .sup.77Br, .sup.186Re, .sup.188Re,
.sup.212Pb, .sup.212Bi, .sup.109Pd, .sup.64Cu, and .sup.67Cu.
[0221] Such agents may be attached to AL-2 by any suitable
conventional procedure. AL-2 contains functional groups on amino
acid side chains that can be reacted with functional groups on a
desired agent to form covalent bonds, for example. Alternatively,
the protein or agent may be derivatized to generate or attach a
desired reactive functional group, preferably a site-specific
reactive group. The derivatization may involve attachment of one of
the bifunctional coupling reagents available for attaching various
molecules to proteins (Pierce Chemical Company, Rockford, Ill.). A
number of techniques for radiolabeling proteins are known.
Radionuclide metals may be attached to AL-2 by using a suitable
bifunctional chelating agent, for example.
[0222] Conjugates comprising AL-2 and a suitable diagnostic or
therapeutic agent (preferably covalently linked) are administered
or otherwise employed in an amount appropriate for the particular
application.
[0223] In view of the sequence identity between AL-2 and Htk-L as
shown in FIGS. 4 and 5, which is determined for the first time
herein, and since Htk-L is a ligand of the transmembrane-sequence
type and binds an Eph-family receptor, namely Htk, the present
application provides embodiments of methods of treatment wherein an
effective amount of Htk-L is administered to a patient in need of
such treatments as discussed for the first time herein for AL-2.
Accordingly, WO 96/02645, published Feb. 1, 1996, is incorporated
by reference herein for its teachings regarding nucleic acid
sequences encoding Htk-L, Htk-L proteins and variants, and methods
for their production and formulation. Consequently, it is the
intent of the present inventors that new uses and methods of
administration of AL-2, as taught for the first time herein, are to
be applied to Htk-L. For example, in one embodiment Htk-L will find
use in methods of treatment of angiogenesis-related conditions as
taught herein for AL-2.
[0224] In summary, by providing nucleic acid molecules encoding
AL-2, the present invention enables for the first time the
production of AL-2 by recombinant DNA methods, thus providing a
reliable source of sufficient quantities of AL-2 for use in various
diagnostic and therapeutic applications. In view of its distinct
biological properties, purified recombinant AL-2 will be especially
useful in a variety of circumstances, such as in
angiogenesis-related conditions and where it is necessary or
desirable to assure neuronal function, growth, survival, or
cell-cell contact, but where other neurotrophic factors or
angiogenic agents either cannot be used or are less effective.
[0225] The following examples are offered by way of illustration
only and are not intended to limit the invention in any manner. All
patent and literature references cited herein are expressly
incorporated.
EXAMPLES
Example 1
Isolation of a Full-length cDNA Encoding AL-2
[0226] The Genbank EST database was screened with an AL-1 sequence
and with sequences from several other members of the Eph-receptor
ligand family, namely B61, Lerk2 and Htk-L. From this search, EST
sequence H10006 was identified (see FIG. 3A-3B) and selected to
provide a sequence from which a probe-based cloning approach for a
novel neurotrophic factor was devised.
[0227] Two 60-mer oligonucleotide probes were designed based on the
sequence of the EST H10006, namely sense-probe-H1006 (5'-GGA CAA
AGT CCC GAG GAG GGG CTG TCC CCC GAA AAC CTG TGT CTG AAA TGC CCA TGG
AAA-3') and antisense-probe-H1006 (5'-CAG GTT CTC CTT CCC CAG GCT
CCC AGG CTG TGG GCT GCC CCT CGG TCT CTT TCC ATG GGC-3'). The probes
are alternatively referred to as sense-probe-H10006 and
anti-sense-probe-H10006, respectively.
[0228] The two synthetic probes were labeled and used to screen a
human fetal brain cDNA library. Filters were hybridized in 50%
formamide and washed in 0.2% SSC/0.1% SDS at 55.degree. C. Six
double-positive clones, i.e., clones that hybridized with both
probes, were identified and selected. These clones were plaque
purified, and their cDNA inserts were transferred into a plasmid
vector and sequenced. Two distinct sequences encoding identical
proteins differing only at their C-termini were observed indicating
a novel neurotrophic factor designated AL-2. The shorter form,
which ends with the sequence "KV," was designated AL-2s
("AL-2-short"), and the longer form, which contains additional
amino acids at its C-terminal end, was designated AL-2l
("AL-2-long"). FIGS. 1A-1B depicts the AL-2l cDNA sequence and the
deduced AL-2l amino acid sequence. FIG. 2A-2B depicts the AL-2s
cDNA sequence and the deduced AL-2s amino acid sequence. FIG. 3A-3B
depicts alignment of the AL-2l nucleic acid sequence with the EST
H10006 sequence.
Example 2
Expression of AL-2 by Northern Blot Analysis
[0229] A Northern blot of poly(A)+ RNA isolated from pancreas,
kidney, skeletal muscle, liver, lung, placenta, brain and heart
tissue was screened with an AL-2 probe. The highest levels of AL-2
expression were in the brain, pancreas, and skeletal muscle. Lower
levels were detectable in kidney, liver, placenta and heart.
[0230] Within the brain AL-2 was expressed in every brain region
tested, including cerebellum, cerebral cortex, medulla, spinal
cord, occipital pole, frontal lobe, temporal lobe, putamen,
amygdala, caudate nucleus, corpus callosum, hippocampus, whole
brain, substantia nigra, subthalamic nucleus and thalamus.
Interestingly, a second, shorter RNA transcript was observed in the
brain samples. The shorter transcript may be the AL-2s mRNA. The
ratio between the observed long and short transcripts differed
amongst the brain regions screened.
Example 3
Construction and Production of an AL-2-IgG Fusion
[0231] A soluble AL-2-IgG fusion protein was constructed by
recombinant DNA techniques. DNA encoding a soluble AL-2-IgG chimera
was constructed by joining the DNAs encoding the extracellular
domain of AL-2 and the Fc domain of IgG.sub.1, similar to the
construction of Rek7-IgG and AL-1-IgG (see Winslow et al., Neuron,
14:973-981 (1995)), with the AL-2 sequence replacing the AL-1
sequence. The AL-2 coding region from its initiation methionine-1
to glycine-218 was fused at its 3' end to the 5'-end (at the
aspartic acid) of the 343 amino acid sequence of IgG2b.
[0232] AL-2-IgG fusion protein is generated by transfection of HEK
293 cells (Graham, et al., J. Gen. Virol. 36:59 (1977)) with the
plasmid nRKAL-2-IgG under conditions as described by Winslow et
al., Neuron, 14:973-981 (1995)). Conditioned media is collected
after 3 days and AL-2-IgG is purified by Protein A
chromatography.
Example 4
Biological Activity: Activation of Eph-Related Receptors by
AL-2
[0233] The ability of AL-2 or variant, e.g., AL-2-IgG, to activate
a Eph-family receptor can be determined by tyrosine
autophosphorylation of the receptor in a receptor-expressing cell
source as described herein. Cells expressing an Eph-family
receptor, preferably Hek2, Hek5, Hek6/elk/Cek6, or Htk, are
incubated with AL-2 and specific phosphorylation of the Eph-family
receptor is monitored. Specific phosphorylation indicates that AL-2
not only binds to the Eph-family receptor, but that it also
activates the Eph-family receptor.
[0234] Cells expressing an Eph-family receptor, e.g., cultured
primary cortical neurons, are detected and analyzed by in situ
hybridization and/or immunoprecipitation and immunoblotting with
anti-Eph-family-receptor antibodies, preferably anti-Hek2,
anti-Elk/Hek6/Cek6, anti-Hek5 or anti-Htk, and anti-phosphotyrosine
antibodies. Membrane-bound AL-2 is transiently expressed on the
surface of transfected 293 cells and its activation of the
endogenous Eph-family receptor in the receptor-expressing cell is
monitored. Activation of endogenous Eph-family receptor is
indicated by autophosphorylation of the receptor. Alternatively,
soluble AL-2 fusion, e.g., AL-2-IgG, dimers, multimers, as taught
herein, is provided to the receptor-expressing cells and tested for
activation of endogenous Eph-family receptor as described, for
example, by Winslow et al., Neuron, 14:973-981 (1995).
Membrane-attachment has been reported as required or preferred for
maximal receptor activation with other members of this ligand
family (Davis et al., Science, 266:816-819 (1994)).
[0235] HEK 293 cells are transfected with an AL-2 cDNA expression
plasmid using the calcium phosphate coprecipitation method
(Simonsen et al., Proc. Natl. Acad. Sci. USA, 80:2495-2499 (1983)).
Primary cortical neurons from E16 rats are plated at a density of
5.times.10.sup.6 cells/15 cm dish and cultured for 4 days. These
cells are then treated with purified soluble AL-2 or soluble
AL-2-IgG (0.1-1 .mu.g/ml) or a number of 293 cells expressing an
equivalent number of membrane-bound AL-2 for 10 min at 37.degree.
C. Immunoprecipitation of lysates with rabbit anti-Eph-family
receptors and immunoblotting with mouse anti-phosphotyrosine is
essentially as described (Kaplan et al., Science, 252:554-559
(1991); Kaplan et al., Nature, 350:158-160 (1991)). Immunoblotted
bands are visualized using a horseradish peroxidase-conjugated
sheep anti-mouse antibody and the ECL fluorescence detection system
(Amersham) as described by the manufacturer.
[0236] An ability of AL-2-IgG to activate the autophosphorylation
of receptor, preferably to a similar extent as membrane-bound AL-2,
indicates that the soluble AL-2-IgG fusion protein and the like can
be used as an agonist for Eph-family receptors in vitro, ex vivo,
and in vivo. An inability of soluble AL-2 (e.g., free ECD) to
activate receptor autophosphorylation despite its ability to bind
receptor indicates that certain soluble forms of AL-2 as taught
herein can act as antagonists of Eph-family receptors.
Sequence CWU 1
1
* * * * *