U.S. patent application number 11/327164 was filed with the patent office on 2006-10-12 for lymphocyte homing receptors.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Laurence A. Lasky, Steven D. Rosen, Mark S. Singer, Scott E. Stachel, Ted A. Yednock.
Application Number | 20060229435 11/327164 |
Document ID | / |
Family ID | 26979673 |
Filed Date | 2006-10-12 |
United States Patent
Application |
20060229435 |
Kind Code |
A1 |
Lasky; Laurence A. ; et
al. |
October 12, 2006 |
Lymphocyte homing receptors
Abstract
DNA isolates coding for the lymphocyte homing receptor and
methods of obtaining such DNA are provided, together with
expression systems for recombinant production of the lymphocyte
homing receptor useful in therapeutic or diagnostic
compositions.
Inventors: |
Lasky; Laurence A.;
(Sausalito, CA) ; Stachel; Scott E.; (Berkeley,
CA) ; Rosen; Steven D.; (San Francisco, CA) ;
Singer; Mark S.; (Berkeley, CA) ; Yednock; Ted
A.; (Fairfax, CA) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
26979673 |
Appl. No.: |
11/327164 |
Filed: |
January 5, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09119209 |
Jul 20, 1998 |
|
|
|
11327164 |
Jan 5, 2006 |
|
|
|
08513278 |
Aug 10, 1995 |
5840844 |
|
|
09119209 |
Jul 20, 1998 |
|
|
|
08059027 |
May 6, 1993 |
|
|
|
08513278 |
Aug 10, 1995 |
|
|
|
07786149 |
Oct 31, 1991 |
5216131 |
|
|
08059027 |
May 6, 1993 |
|
|
|
07315015 |
Feb 23, 1989 |
5098833 |
|
|
07786149 |
Oct 31, 1991 |
|
|
|
Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/69.1; 530/388.22 |
Current CPC
Class: |
C07K 2319/02 20130101;
C07K 2319/32 20130101; C07K 14/705 20130101; C07K 2319/705
20130101; C07K 2319/33 20130101; A61K 38/00 20130101 |
Class at
Publication: |
530/350 ;
530/388.22; 435/069.1; 435/320.1; 435/325 |
International
Class: |
C07K 14/72 20060101
C07K014/72; C07K 16/28 20060101 C07K016/28; C12P 21/06 20060101
C12P021/06 |
Claims
1-2. (canceled)
3. An isolated polypeptide encoded by a DNA able to hybridize under
stringent conditions to the complement of a DNA sequence encoding
the carbohydrate domain (Trp39 to Cys155), the epidermal growth
factor domain (Cys160 to Leu193); or a complement binding domain
(Cys197 to Glu328) of the leukocyte homing receptor (LHR) amino
acid sequence shown in FIG. 1 (SEQ ID NO:2), wherein the
polypeptide is devoid of a functional cytoplasmic domain, and
wherein the stringent hybridization conditions comprise 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardts solution, 10 %
dextran sulfate, and 20 micrograms per ml denatured, sheared salmon
sperm DNA, overnight at 42.degree. C., and wherein said polypeptide
has one or more of the following functional characteristics: a)
binds a known LHR ligand or anti-LHR antibody; b) induces anti-LHR
antibodies; c) mediates binding of lymphocytes to endothelium of
lymphoid tissue; or d) competes with normal binding of lymphocytes
to lymphoid tissue.
4. An isolated polypeptide consisting essentially of an amino acid
sequence that is at least 70% homologous to one or more of the
carbohydrate binding domain (Trp39 to Cys155), the epidermal growth
factor domain (Cys160 to Leu193), or a complement binding domain
(Cys197 to Glu328) of the leukocyte homing receptor having the
amino acid sequence of SEQ ID NO:2, and wherein said polypeptide
has one or more of the following functional characteristics: a)
binds a known LHR ligand or anti-LHR antibody; b) induces anti-LHR
antibodies; c) mediates binding of lymphocytes to endothelium of
lymphoid tissue; or d) competes with normal binding of lymphocytes
to lymphoid tissue.
5. The polypeptide of claim 4, having the amino acid sequence
spanning Trp39 to Cys155 of SEQ ID NO:2.
6. The polypeptide of claim 4, having the amino acid sequence
spanning Cys160 to Leu193 of SEQ ID NO:2.
7. The polypeptide of claim 4, having the amino acid sequence
spanning Cys197 to Glu328 of SEQ ID NO:2.
8. An isolated polypeptide comprising an amino acid sequence that
is at least 70% homologous to one or more of the carbohydrate
binding domain (Trp39 to Cys155), the epidermal growth factor
domain (Cys160 to Leu193), or a complement binding domain (Cys197
to Glu328) of the leukocyte homing receptor having the amino acid
sequence of SEQ ID NO:2, wherein the polypeptide lacks a functional
transmembrane domain, and wherein said polypeptide has one or more
of the following functional characteristics: a) binds a known LHR
ligand or anti-LHR antibody; b) induces anti-LHR antibodies; c)
mediates binding of lymphocytes to endothelium of lymphoid tissue;
or d) competes with normal binding of lymphocytes to lymphoid
tissue.
9. An isolated polypeptide comprising an amino acid sequence that
is at least 70% homologous to one or more of the carbohydrate
binding domain (Trp39 to Cys 155), the epidermal growth factor
domain (Cys160 to Leu193), or a complement binding domain (Cys197
to Glu328) of the leukocyte homing receptor having the amino acid
sequence of SEQ ID NO:2, wherein the polypeptide lacks a functional
cytoplasmic domain, and wherein said polypeptide has one or more of
the following functional characteristics: a) binds a known LHR
ligand or anti-LHR antibody; b) induces anti-LHR antibodies; c)
mediates binding of lymphocytes to endothelium of lymphoid tissue;
or d) competes with normal binding
10. An isolated polypeptide encoded by a DNA able to hybridize
under stringent conditions to the complement of a DNA sequence
encoding the carbohydrate binding domain (Trp39 to Cys155), the
epidermal growth factor domain (Cys160 to Leu193); or a complement
binding domain (Cys197 to Glu328) of the leukocyte homing receptor
(LHR) amino acid sequence shown in FIG. 1 (SEQ ID NO:2), wherein
the stringent conditions are overnight incubation at 42.degree. C.
in a solution comprising 20% formamide, 5.times.SSC (150 mM NaCl,
15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardts solution, 10 % dextran sulfate, and 20 micrograms per ml
denatured, sheared salmon sperm DNA, and wherein the polypeptide
lacks a functional transmembrane domain, a functional cytoplasmic
domain, or both, and wherein said polypeptide has one or more of
the following functional characteristics: a) binds a known LHR
ligand or anti-LHR antibody; b) induces anti-LHR antibodies; c)
mediates binding of lymphocytes to endothelium of lymphoid tissue;
or d) competes with normal binding of lymphocytes to lymphoid
tissue.
11. An isolated polypeptide encoded by a DNA able to hybridize
under stringent conditions to the complement of a DNA sequence
encoding the carbohydrate binding domain (Trp39 to Cys155), the
epidermal growth factor domain (Cys160 to Leu193); or a complement
binding domain (Cys197 to Glu328) of the leukocyte homing receptor
(LHR) amino acid sequence shown in FIG. 1 (SEQ ID NO:2), wherein
the polypeptide is devoid of a functional transmembrane domain, and
wherein the stringent conditions are overnight incubation at
42.degree. C. in a solution comprising 20% formamide, 5.times.SSC
(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH
7.6), 5.times. Denhardts solution, 10 % dextran sulfate, and 20
micrograms per ml denatured, sheared salmon sperm DNA, and wherein
said polypeptide has one or more of the following functional
characteristics: a) binds a known LHR ligand or anti-LHR antibody;
b) induces anti-LHR antibodies; c) mediates binding of lymphocytes
to endothelium of lymphoid tissue; or d) competes with normal
binding of lymphocytes to lymphoid tissue.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to novel lymphocyte homing receptors,
to methods for making these homing receptors, and to nucleic acids
encoding these receptors.
[0002] Lymphocytes are mediators of normal tissue inflammation as
well as pathologic tissue damage such as occurs in rheumatoid
arthritis and other autoimmune diseases. In order to fully exploit
the antigenic repertoire of the immune system, vertebrates have
evolved a mechanism for distributing lymphocytes with diverse
antigenic specificities to spatially distinct regions of the
organism (Butcher, E. C., Curr. Top. Micro. Immunol. 128, 85
(1986); Gallatin, W. M., et al., Cell 44, 673 (1986); Woodruff, J.
J., et al., Ann. Rev. Immunol. 5. 201 (1987); Duijvestijn, A., et
al., Immunol. Today 10, 23 (1989); Yednock, T. A., et al., Adv.
Immunol (in press) (1989)).
[0003] This mechanism involves the continuous recirculation of the
lymphocytes between the blood and the lymphoid organs. The
migration of lymphocytes between the blood, where the cells have
the greatest degree of mobility, and the lymphoid organs, where the
lymphocytes encounter sequestered and processed antigen, is
initiated by an adhesive interaction between receptors on the
surface of the lymphocytes and ligands on the endothelial cells of
specialized postcapillary venules, e.g., high endothelial venules
(HEV) and the HEV-like vessels induced in chronically inflamed
synovium.
[0004] The lymphocyte adhesion molecules have been termed homing
receptors, since they allow these cells to localize in or "home" to
particular secondary lymphoid organs.
[0005] Candidates for the lymphocyte homing receptor have been
identified in mouse, rat and human (Gallatin, W. M., et al., Nature
303, 30 (1983) Rasmussen, R. A., et al., J. Immunol. 135, 19
(1985); Chin, Y. H., et al., J. Immunol. 136, 2556 (1986);
Jalkanen, S., et al., Eur. J. Immunol. 10, 1195 (1986)). The
following literature describes work which has been done in this
area through the use of a monoclonal antibody, termed Mel 14,
directed against a purported murine form of a lymphocyte surface
protein (Gallatin, W. M., et al., supra; (Mountz, J. D., et al., J.
Immunol. 140, 2943 (1988); (Lewinsohn, D. M., et al., J. Immunol.
138, 4313 (1987); Siegelman, M., et al., Science 231, 823 (1986);
St. John, T., et al., Science 231, 845 (1986)).
[0006] Immunoprecipitation experiments have shown that this
antibody recognizes a diffuse, .sup.-90,000 dalton cell surface
protein on lymphocytes (Gallatin, W. M., et al., supra) and a
.sup.-100,000 dalton protein on neutrophils (Lewinsohn, D. M., et
al., supra).
[0007] A partial sequence--13 residues--for a purported lymphocyte
homing receptor identified by radioactively labeled amino acid
sequencing of a Mel-14 antibody-defined glycoprotein was disclosed
by Siegelman et al. (Siegelman, M., et al. Science 231, 823
(1986)).
[0008] Lectins are a carbohydrate-binding domain found in a variety
of animals, including humans as well as the acorn barnacle and the
flesh fly. The concept of lectins functioning in cell adhesion is
exemplified by the interaction of certain viruses and bacteria with
eucaryotic host cells (Paulson, J. C., The Receptors Vol. 2 P. M.
Conn, Eds. (Academic Press. NY, 1985), pp. 131; Sharon, N., FEBS
Lett. 217, 145 (1987)). In eucaryotic cell-cell interactions,
adhesive functions have been inferred for endogenous lectins in a
variety of systems (Grabel, L., et al., Cell 17, 477 (1979);
Fenderson, B., et al., J. Exp. Med. 160, 1591 (1984); Kunemund, V.,
J. Cell Biol. 106, 213 (1988); Bischoff, R., J. Cell Biol. 102,
2273 (1986); Crocker, P. R., et al., J. Exp. Med. 164, 1862 (1986);
including invertebrate (Glabe, C. G., et al., J. Cell. Biol. 94,
123 (1982); DeAngelis, P., et al., J. Biol. Chem. 262, 13946
(1987)) and vertebrate fertilization (Bleil, J. D., et al., Proc.
Natl. Acad. Sci., U.S.A. 85, 6778 (1988); Lopez, L. C., et al., J.
Cell Biol. 101, 1501 (1985)). The use of protein-sugar interactions
as a means of achieving specific cell recognition appears to be
well known.
[0009] The literature suggests that a lectin may be involved in the
adhesive interaction between the lymphocytes and their ligands
(Rosen, S. D., et al., Science 228, 1005 (1985); Rosen, S. D., et
al., J. Immunol. (in press) (1989); Stoolman, L. M., et al., J.
Cell Biol 96, 722 (1983); Stoolman, L. M., et al., J. Cell Biol.
99, 1535 (1984); Yednock, T. A., et al., J. Cell Bio. 104, 725
(1987); Stoolman, L. M., et al., Blood 70, 1842 (1987); A related
approach by Brandley, B. K., et al., J. Cell Biol. 105, 991 (1987);
Yednock, T. A., et al., in preparation; and Yednock, T. A., et al.,
J. Cell Biol. 104, 725 (1987)).
[0010] The character of a surface glycoprotein that may be involved
in human lymphocyte homing was investigated with a series of
monoclonal and polyclonal antibodies generically termed Hermes.
These antibodies recognized a .sup.-90,000 dalton surface
glycoprotein that was found on a large number of both immune and
non-immune cell types and which, by antibody pre-clearing
experiments, appeared to be related to the Mel 14 antigen.
(Jalkanen, S., et al., A.N.N. Rev. Med., 38, 467-476 (1987);
Jalkanen, S., et al., Blood, 66 (3), 577-582 (1985); Jalkanen, S.,
et al., J. Cell Biol., 105, 983-990 (1987); Jalkanen, S., et al.,
Eur. J. Immunol., 18, 1195-1202 (1986).
[0011] Epidermal growth factor-like domains have been found on a
wide range- of proteins, including growth factors, cell surface
receptors, developmental gene products, extracellular matrix
proteins, blood clotting factors, plasminogen activators, and
complement (Doolittle, R. F., et al., CSH Symp. 51, 447
(1986)).
[0012] The inventors have characterized the lymphocyte cell surface
glycoprotein (referred to hereafter as the "LHR") which mediates
the binding of lymphocytes to the endothelium of lymphoid
tissue.
[0013] Accordingly, it is an object of this invention to provide
nucleic acid sequences encoding the LHR.
[0014] It is another object to provide a method for expression of
the LHR in recombinant cell culture.
[0015] A further object is to enable the preparation of the LHR
having variant amino acid sequences or glycosylation not otherwise
found in nature, as well as other derivatives of the LHR having
improved properties including enhanced specific activity and
enhanced plasma half-life.
SUMMARY OF THE INVENTION
[0016] The LHR of this invention is full-length, mature LHR, having
the amino acid sequence described herein at FIGS. 1 and 2, and
naturally occurring alleles, or predetermined amino acid sequence
or derivitization or glycosylation variants thereof.
[0017] The objects of this invention have been accomplished by a
method comprising providing nucleic acid encoding the LHR;
transforming a host cell with the nucleic acid; culturing the host
cell to allow the LHR to accumulate and recovering the LHR.
[0018] Full length cDNA clones and DNA encoding the human and the
murine LHR (HuLHR and MLHR, respectively) have been identified and
isolated, and moreover this DNA is readily expressed by recombinant
host cells.
[0019] Analysis of the cDNA sequence reveals that the LHR is a
glycoprotein which contains the following protein domains: a signal
sequence, a carbohydrate binding domain, an epidermal growth
factor-like (egf) domain, at least one complement binding domain
repeat, a transmembrane binding domain (TMD), and a charged
intracellular domain. The LHR of this invention contains at least
one but not necessarily all of these domains.
[0020] Also provided are LHR having variant amino acid sequences or
glycosylation not otherwise found in nature, as well as other
derivatives of the LHR having improved properties including
enhanced specific activity and modified-plasma half-life, as well
as enabling methods for the preparation of such variants.
[0021] Polynucleotide probes are provided which are capable of
hybridizing under stringent conditions to the LHR gene.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 depicts the amino acid and DNA sequence of the Human
LHR (HuLHR).
[0023] FIG. 2 depicts the amino acid and DNA sequence of the Murine
LHR (MLHR).
[0024] FIG. 3 shows a comparison between the amino acid sequences
for the mature HuLHR and MLHR.
[0025] FIGS. 4A-4C show the isolation and N-terminal sequencing of
the MLHR. FIG. 4A shows an SDS-polyacrylamide gel of material
purified from a detergent extract of murine spleens by Mel 14
monoclonal antibody affinity chromatography. FIG. 4B shows the
results of the subjection of the 90,000 dalton band of FIG. 4A to
gas phase Edman degradation. The residues underlined between amino
acids 7 and 15 were chosen to produce the oligonucleotide probe
shown in FIG. 4C. FIG. 4C shows as 32-fold redundant 26-mer
oligonucleotide probe.
[0026] FIG. 5 shows the transient expression of the MLHR cDNA
clone. Lanes A-F signify the following: --A. Lysates of 293 cells
transfected with a MLHR expression plasmid immunoprecipitated with
Mel 14 monoclonal antibody. --B. Supernatants of 293 cells
transfected with a MLHR expression plasmid immunoprecipitated with
Mel 14 monoclonal antibody. --C. Lysates of 293 cells transfected
with a plasmid expressing the HIV gp120 envelope glycoprotein
immunoprecipitated with the Mel 14-monoclonal antibody. --D.
Supernatants of 293 cells transfected with the HIV envelope
expression plasmid immunoprecipitated with the Mel 14 monoclonal
antibody. --E. Supernatants of 38C13 cells immunoprecipitated with
the Mel 14 monoclonal antibody. --F. Lysates of 38C13 cells surface
labeled with I.sup.125 and immunoprecipitated with the Mel 14
monoclonal antibody.
[0027] FIGS. 6A-6C show protein sequences which are heterologous
but functionally comparable to the MLHR. Those lines labelled
"MLHR" correspond to the MLHR of FIG. 2. FIG. 6A compares
carbohydrate-binding domains; FIG. 6B compares epidermal growth
factor domains; and FIG. 6C compares complement binding factor
domains.
[0028] FIG. 7 is a schematic of protein domains found in the LHR,
including the signal sequence, carbohydrate binding domain,
epidermal growth factor (egf) domain, two complement binding domain
repeats (arrows), transmembrane binding domain (TMD), and charged
intracellular domain.
DETAILED DESCRIPTION
[0029] The LHR is defined as a polypeptide having a qualitative
biological activity in common with-the LHR of FIG. 1 or FIG. 2 and
which contains a domain greater than about 70% homologous,
preferably greater than about 75% homologous, and most preferably
greater than about 80% homologous with the carbohydrate binding
domain, the epidermal growth factor domain, or the carbohydrate
binding domain of the LHR of FIG. 1 or FIG. 2.
[0030] Homologous is defined herein as the percentage of residues
in the candidate sequence that are identical with the residues in
the carbohydrate binding domain, the epidermal growth factor
domain, or the complement binding domains in FIG. 1 or FIG. 2 after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent homology.
[0031] Included within the scope of the LHR as that term is used
herein are LHRs having the amino acid sequences of the HuLHR or
MLHR as set forth in FIG. 1 or 2, deglycosylated or unglycosylated
derivatives of the LHR, homologous amino acid sequence variants of
the sequence of FIG. 1 or 2, and homologous in-vitro-gent ed
variants and derivatives of the LHR, which are capable of
exhibiting a biological activity in common with the LHR of FIG. 1
or FIG. 2.
[0032] LHR biological activity is defined as either 1)
immunological cross-reactivity with at least one epitope of the
LHR, or 2) the possession of at least one adhesive, regulatory or
effector function qualitatively in common with the LHR.
[0033] One example of-the qualitative biological activities of the
LHR is its binding to ligands on the specialized high endothelial
cells of the lymphoid tissues. Also, it frequently requires a
divalent cation such as calcium for ligand binding.
[0034] Immunologically cross-reactive as used herein means that the
candidate polypeptide is capable of competitively inhibiting the
qualitative biological activity of the LHR having this activity
with polyclonal antisera raised against the known active analogue.
Such antisera are prepared in conventional fashion by injecting
goats or rabbits, for example, subcutaneously with the known active
analogue in complete Freund's adjuvant, followed by booster
intraperitoneal or subcutaneous injection in incomplete
Freunds.
[0035] Structurally, as shown in FIG. 3, the LHR includes several
domains which are identified as follows (within .+-.10 residues):
a. signal sequence (residues 20-32), which is followed by a
carbohydrate binding domain (identified in FIG. 3 as a "lectin"
domain) (residues 39-155), an epidermal growth factor (egf) domain
(residues 160-193), a complement factor binding domain (residues
197-317), a transmembrane binding domain (TMD) (residues 333-355),
and a cytoplasmic domain (residues 356-372). The boundary for the
LHR extracellular domain generally is at, or within about 30
residues of, the N-terminus of the transmembrane domain, and is
readily identified from an inspection of the LHR sequence.
[0036] FIGS. 6A-6C show a variety of proteins having some homology
to three of these domains. FIG. 6A shows carbohydrate binding
domains, FIG. 6B shows epidermal growth factor domains, and FIG. 6C
shows somewhat homologous complement binding domains.
[0037] A first embodiment of this invention is the HuLHR, whose
nucleotide and amino acid sequence is shown in FIG. 1.
[0038] Another embodiment of the LHR of this invention is the MLHR
whose nucleotide and amino acid sequence is shown in FIG. 2.
[0039] A comparison of the amino sequences of HuLHR and MLHR is
presented in FIG. 3, and shows a high degree of overall sequence
homology (.sup.-83%). The degrees of homology between the various
domains found in the HuLHR versus the MLHR, however, are variable.
For example, the degree of sequence conservation between the MLHR
and the HuLHR in both the carbohydrate-binding and egf domains is
approximately 83%, while the degree of conservation in the first
complement binding repeat falls to 79% and only 63% in the second
repeat, for an overall complement binding domain homology of
.sup.-71%. Furthermore, while the two MLHR complement binding
domain repeats are identical, those in the MLHR have differences,
and differ as well to the murine repeats. Interestingly, the degree
of conservation between the two receptors in the transmembrane
sequence and surrounding regions is virtually identical, with only
one conservative hydrophobic substitution, probably within the
transmembrane anchor region.
[0040] Finally, comparison of the amino acid sequence found for the
HuLHR with that- recently reported (Zhov, D., B. Secd., submitted
for publication) for the human Hermes/CD44 antigen showed a
complete lack of homology between these proteins (data not
shown).
[0041] This invention is particularly concerned with amino acid
sequence variants of the LHR. Amino acid sequence variants of the
LHR are prepared with various objectives in mind, including
increasing the affinity of the LHR for its binding partner,
facilitating the stability, purification and preparation of the
LHR, modifying its plasma half life, improving therapeutic efficacy
and lessening the severity or occurrence of side effects during
therapeutic use of the LHR.
[0042] Amino acid sequence variants of the LHR fall into one or
more of three classes: Insertional, substitutional, or deletional
variants. These variants ordinarily are prepared by site specific
mutagenesis of nucleotides in the DNA encoding the LHR, by which
DNA encoding the variant is obtained, and thereafter expressing the
DNA in recombinant cell culture. However, variant LHR fragments
having up to about 100-150 amino acid residues are prepared
conveniently by In vitro synthesis.
[0043] The amino acid sequence variants of the LHR are
predetermined variants not found in nature or naturally occurring
alleles. The LHR variants typically exhibit the same qualitative
biological--for example, ligand binding--activity as the naturally
occurring HuLHR or MLHR analogue. However, the LHR variants and
derivatives that are not capable of binding to their ligands are
useful nonetheless (a) as a reagent in diagnostic assays for the
LHR or antibodies to the LHR, (b) when insolubilized in accord with
known methods, as agents for purifying anti-LHR antibodies from
antisera or hybridoma culture supernatants, and (c)as immunogens
for raising antibodies to the LHR or as immunoassay kit components
(labelled, as a competitive reagent for the native LHR or
unlabelled as a standard for the LHR assay) so long as at least one
LHR epitope remains active.
[0044] While the site for introducing an amino acid sequence
variation is predetermined, the mutation per se need not be
predetermined. For example, in order to optimize the performance of
a mutation at a given site, random or saturation mutagenesis (where
all 20 possible residues are inserted) is conducted at the target
codon and the expressed LHR variant is screened for the optimal
combination of desired activities. Such screening is within the
ordinary skill in the art.
[0045] Amino acid insertions usually will be on the order of about
from 1 to 10 amino acid residues; substitutions are typically
introduced for single residues; and deletions will range about from
1 to 30 residues. Deletions or insertions preferably are made in
adjacent pairs, i.e. a deletion of 2 residues or insertion of 2
residues. It will be amply apparent from the following discussion
that substitutions, deletions, insertions or any combination
thereof are introduced or combined to arrive at a final
construct.
[0046] Insertional amino acid sequence variants of the LHR are
those in which one or more amino acid residues extraneous to the
LHR are introduced into a predetermined site in the target LHR and
which displace the preexisting residues.
[0047] Commonly, insertional variants are fusions of heterologous
proteins or polypeptides to the amino or carboxyl terminus of the
LHR. Such variants are referred to as fusions of the LHR and a
polypeptide containing a sequence which is other than that which is
normally found in the LHR at the inserted position. Several groups
of fusions are contemplated herein.
[0048] Immunologically active LNR derivatives and fusions comprise
the LHR and a polypeptide containing a non-LHR epitope, and are
within the scope of this invention. The non-LHR epitope is any
immunologically competent polypeptide, i.e., any polypeptide which
is capable of eliciting an immune response in the animal to which
the fusion is to be administered or which is capable of being bound
by an antibody-raised against the non-LHR polypeptide.
[0049] Typical non-LHR epitopes will be those which are borne by
allergens, autoimmune epitopes, or other potent immunogens or
antigens recognized by pre-existing antibodies in the fusion
recipient, including bacterial polypeptides such as trpLE,
beta-galactosidase, viral polypeptides such as herpes gD protein,
and the like.
[0050] Immunogenic fusions are produced by cross-linking in vitro
or by recombinant cell culture transformed with DNA encoding an
immunogenic polypeptide. It is preferable that the immunogenic
fusion be one in which the immunogenic sequence is joined to or
inserted into the LHR or fragment thereof by a peptide bond(s).
These products therefore consist of a linear polypeptide chain
containing the LHR epitope and at least one epitope foreign to the
LHR. It will be understood that it is within the scope of this
invention to introduce the epitopes anywhere within the LHR
molecule or fragment thereof.
[0051] Such fusions are conveniently made in recombinant host cells
or by the use of bifunctional cross-linking agents. The use of a
cross-linking agent to fuse the LHR to the immunogenic polypeptide
is not as desirable as a linear fusion because the cross-linked
products are not as easily synthesized in structurally homogeneous
form.
[0052] These immunogenic insertions are particularly useful when
formulated into a pharmacologically acceptable carrier and
administered to a subject in order to raise antibodies against the
LHR, which antibodies in turn are useful in diagnostics or in
purification of the LHR by immunoaffinity techniques known per se.
Alternatively, in the purification of the LHR, binding partners for
the fused non-LHR polypeptide, e.g. antibodies, receptors or
ligands, are used to adsorb the fusion from impure admixtures,
after which the fusion is eluted and, if desired, the LHR is
recovered from the fusion, e.g. by enzymatic cleavage.
[0053] Other fusions, which may or may not also be immunologically
active, include fusions of the mature LHR sequence with a signal
sequence heterologous to the LHR, and fusions of the LHR to
polypeptides having enhanced plasma half life (ordinarily >about
20 hours) such as immunoglobulin chains or fragments thereof.
[0054] Signal sequence fusions are employed in order to more
expeditiously direct the secretion of the LHR. The heterologous
signal replaces the native LHR signal, and when the resulting
fusion is recognized, i.e. processed and cleaved by the host cell,
the LHR is secreted. Signals are selected based on the intended
host cell, and may include bacterial yeast, mammalian and viral
sequences. The native LHR signal or the herpes gD glycoprotein
signal is suitable for use in mammalian expression systems.
[0055] Plasma proteins which have enhanced plasma half-life longer
than that of the transmembrane modified LHR include serum albumin,
immunoglobulins, apolipoproteins, and transferrin, and desirably
are fused with the LHR. Preferably, the LHR-plasma protein fusion
is not significantly immunogenic in the animal in which it is used
(i.e., it is homologous to the therapeutic target) and the plasma
protein does not cause undesirable side effects in patients by
virtue of its normal biological activity.
[0056] The LHR extracellular domain generally is fused at its
C-terminus to the immunoglobulin constant region. The precise site
at which the fusion is made is not critical; other sites
neighboring or within the extracellular region may be selected in
order to optimize the secretion or binding characteristics of the
soluble LHR. The optimal site will be determined by routine
experimentation. The fusion may typically take the place of either
or both the transmembrane and cytoplasmic domains.
[0057] Substitutional variants are those in which at least one
residue in the FIG. 1 or 2 sequence has been removed and a
different residue inserted in its place. Such substitutions
generally ate made in accordance with the following Table 1 when it
is desired to finely modulate the characteristics of the LHR.
TABLE-US-00001 TABLE 1 Original Residue Exemplary Substitutions Ala
ser Arg lys Asn gln; his Asp glu Cys ser; ala Gln asn Glu asp Gly
pro His asn; gln Ile leu; val Leu ile; val Lys arg; gln; glu Met
leu; ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val
ile; leu
[0058] Novel amino acid sequences, as well as isosteric analogs
(amino acid or otherwise), as included within the scope of this
invention.
[0059] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those in Table 1, i.e., selecting residues that differ more
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site or (c) the bulk
of the side chain. The substitutions which in general are expected
to produce the greatest changes in LHR properties will be those in
which (a) a hydrophilic residue, e.g. seryl or threonyl, is
substituted for (or by) a hydrophobic residue, e.g. leucyl,
isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline
is substituted for (or by) any other residue; (c) a residue having
an electropositive side chain, e.g., lysyl, arginyl, or histidyl,
is substituted for (or by) an electronegative residue, e.g.,
glutamyl or aspartyl; or (d) a residue having a bulky side chain,
e.g., phenylalanine, is substituted for (or by) one not having a
side chain, e.g., glycine.
[0060] Some deletions, insertions, and substitutions will not
produce radical changes in the characteristics of the LHR molecule.
However, when it is difficult to predict the exact effect of the
substitution, deletion, or insertion in advance of doing so, for
example when modifying the LHR carbohydrate binding domain or an
immune epitope, one skilled in the art will appreciate that the
effect will be evaluated by routine screening assays. For example,
a variant typically is made by site specific mutagenesis of the
LHR-encoding nucleic acid, expression of the variant nucleic acid
in recombinant cell culture and, optionally, purification from the
cell culture for example by immunoaffinity adsorption on a
polyclonal anti-LHR column (in order to adsorb the variant by at
least one remaining immune epitope). The activity of the cell
lysate or purified LHR variant is then screened in a suitable
screening assay for the desired characteristic. For example, a
change in the immunological character of the LHR, such as affinity
for a given antibody such as Mel-14, is measured by a
competitive-type immunoassay. As more becomes known about the
functions in vivo of the LHR other assays will become useful in
such screening. Modifications of such protein properties 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 to the artisan.
[0061] Substitutional variants of the LHR also include variants
where functionally homologous (having at least .sup.-70% homology)
to domains of other proteins are substituted by routine methods for
one or more of the above-identified LHR domains. FIGS. 6A-6C may be
used by those skilled in the art for sources for such substitutable
domains. For example, the flesh fly lectin whose sequence is shown
in FIG. 6A may be modified to rise to the level of at least
.sup.-70% homology with the carbohydrate binding domain of the LHR,
and then substituted for that domain. Similarly, coagulation Factor
X, whose sequence is shown in FIG. 6B may be modified to rise to
the level of at least .sup.-70% homology with the egf-domain of the
LHR, and then substituted for that domain. Similar substitutions
may desirably be made for the signal sequence, the complement
binding domain, the transmembrane domain, and for the cytoplasmic
domain. Only substitutions of such functionally homologous domains
of other proteins which are free from all flanking regions of
proteins other than the LHR are within the scope of this
invention.
[0062] Another class of LHR variants are deletional variants.
Deletions are characterized by the removal of one or more amino
acid residues from the LHR sequence. Typically, the transmembrane
and cytoplasmic domains, or only the cytoplasmic domains of the LHR
are deleted. However, deletion from the LHR. C-terminal to any
other suitable site N-terminal to the transmembrane region which
preserves the biological activity or immune cross-reactivity of the
LHR is suitable. Excluded from the scope of deletional variants are
the protein digestion fragments heretofore obtained in the course
of elucidating amino acid sequences of the LHR, and protein
fragments having less than .sup.-70% sequence homology to any of
the above-identified LHR domains.
[0063] Embodiments of this invention include DNA sequences encoding
fragments of the LHR, such as the complement binding domain, the
carbohydrate domain, and the epidermal growth factor domain. The
complement binding domain finds usefulness in the diagnosis and
treatment of complement-mediated diseases, as well as in the
oligomerization of the LHR with itself or with other components on
the lymphocyte surface.
[0064] Deletions of cysteine or other labile residues also may be
desirable, for example in increasing the oxidative stability of the
LHR. Deletion or substitutions of potential proteolysis sites, e.g.
Arg Arg, is accomplished by deleting one of the basic residues or
substituting one by glutaminyl or histidyl residues.
[0065] In one embodiment, the LHR is comprised of the carbohydrate
binding domain in the absence of a complement binding domain and/or
the egf domain. This embodiment may or may not contain either or
both the transmembrane and cytoplasmic regions.
[0066] A preferred class of substitutional or deletional variants
are those involving a transmembrane region of the LHR.
Transmembrane regions of LHR subunits are highly hydrophobic or
lipophilic domains that are the proper size to span the lipid
bilayer of the cellular membrane. They are believed to anchor the
LHR in the cell membrane, and allow for homo- or heteropolymeric
complex formation with the LHR.
[0067] Inactivation of the transmembrane domain, typically by
deletion or substitution of transmembrane domain hydroxylation
residues, will facilitate recovery and formulation by reducing its
cellular or membrane lipid affinity and improving its aqueous
solubility. If the transmembrane and cytoplasmic domains are
deleted one avoids the introduction of potentially immunogenic
epitopes, either by exposure of otherwise intracellular
polypeptides that might be recognized by the body as foreign or by
insertion of heterologous polypeptides that are potentially
immunogenic. Inactivation of the membrane binding function is
accomplished by deletion of sufficient residues to produce a
substantially hydrophilic hydropathy profile at this site or by
substituting with heterologous residues which accomplish the same
result.
[0068] A principal advantage of the transmembrane inactivated LHR
is that it may be secreted into the culture medium of recombinant
hosts. This variant is soluble in body fluids such as blood and
does not have an appreciable affinity for cell membrane lipids,
thus considerably simplifying its recovery from recombinant cell
culture.
[0069] As a general proposition, all variants will not have a
functional transmembrane domain and preferably will not have a
functional cytoplasmic sequence.
[0070] For example, the transmembrane domain may be substituted by
any amino acid sequence, e.g. a random or predetermined sequence of
about 5 to 50 serine, threonine, lysine, arginine, glutamine,
aspartic acid and like hydrophilic residues, which altogether
exhibit a hydrophilic hydropathy profile. Like the deletional
(truncated) LHR, these variants are secreted into the culture
medium of recombinant hosts.
[0071] Examples of HuLHR amino acid sequence variants are described
in the table below. The residue following the residue number
indicates the replacement or inserted amino acids. TABLE-US-00002
TABLE 2 Substitutions Arg58-Asp59: Lys-Glu Ala71: Ser Lys78: Gln
Asp116: Glu Leu150: Val His168: Gln Ile174: Leu Asn181: Gln Thr211:
Ser Phe214: Leu Ser226: Thr Phe244: Met Thr282: Ser Ile288: Val
Lys298-Lys299: Arg-Arg Ile302: Leu Deletions Gly96-Ile97 Asn136
Ser166 Ser220 Asn271 Ile296 Insertions 67-Glu-Ser-Ala
83-Gly-Thr-Thr 209-Asn 241-Val-Glu-Asn 292-Tyr-Tyr-Tyr
[0072] Preferably, the variants represent conservative
substitutions. It will be understood that some variants may exhibit
reduced or absent biological activity. These variants nonetheless
are useful as standards in immunoassays for the LHR so long as they
retain at least one immune epitope of the LHR.
[0073] Glycosylation variants are included within the scope of the
HuLHR. They include variants completely lacking in glycosylation
(unglycosylated) and variants having at least one less glycosylated
site than the native form (deglycosylated) as well as variants in
which the glycosylation has been changed. Included are
deglycosylated and unglycosylated amino acid sequence variants,
deglycosylated and unglycosylated LHR having the native, unmodified
amino acid sequence of the LHR, and other glycosylation variants.
For example, substitutional or deletional mutagenesis is employed
to eliminate the N- or O-linked glycosylation sites of the LHR,
e.g., the asparagine residue is deleted or substituted for by
another basic residue such as lysine or histidine. Alternatively,
flanking residues making up the glycosylation site are substituted
or deleted, even though the asparagine residues remain unchanged,
in order to prevent glycosylation by eliminating the glycosylation
recognition site.
[0074] Additionally, unglycosylated LHR which has the amino acid
sequence of the native LHR is produced in recombinant prokaryotic
cell culture because prokaryotes are incapable of introducing
glycosylation into polypeptides.
[0075] Glycosylation variants are produced by selecting appropriate
host cells or by In vitro methods. Yeast, for example, introduce
glycosylation which varies significantly from that of mammalian
systems. Similarly, mammalian cells having a different species
(e.g. hamster, murine, insect, porcine, bovine or ovine) or tissue
origin (e.g. lung, liver, lymphoid, mesenchymal or epidermal) than
the source of the LHR are routinely screened for the ability to
introduce variant glycosylation as characterized for example by
elevated levels of mannose or variant ratios of mannose, fucose,
sialic acid, and other sugars typically found in mammalian
glycoproteins. In vitro processing of the LHR typically is
accomplished by enzymatic hydrolysis, e.g. neuraminidase
digestion.
[0076] Covalent modifications of the LHR molecule are included
within the scope hereof. Such modifications are introduced by
reacting targeted amino acid residues of the recovered protein with
an organic derivatizing agent that is capable of reacting with
selected side chains or terminal residues, or by harnessing
mechanisms of post-translational modification that function in
selected recombinant host cells. The resulting covalent derivatives
are useful in programs directed at identifying residues important
for biological activity, for immunoassays of the LHR or for the
preparation of anti-LHR antibodies for immunoaffinity purification
of the recombinant LHR. For example, complete inactivation of the
biological activity of the protein after reaction with ninhydrin
would suggest that at least one arginyl or lysyl residue is
critical for its activity, whereafter the individual residues which
were modified under the conditions selected are identified by
isolation of a peptide fragment containing the modified amino acid
residue. Such modifications are within the ordinary skill in the
art and are performed without undue experimentation.
[0077] Derivatization with bifunctional agents is useful for
preparing intermolecular aggregates of the protein with immunogenic
polypeptides as well as for cross-linking the protein to a water
insoluble support matrix or surface for use in the assay or
affinity purification of antibody. In addition, a study of
intrachain cross-links will provide direct information on
conformational structure. Commonly used cross-linking agents
include 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
(succinimidyl-propionate), and bifunctional maleimides such as
bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-[(p-azido-phenyl)dithio] propioimidate yield
photoactivatable intermediates which are capable of forming
cross-links in the presence of light. Alternatively, reactive water
insoluble matrices such as cyanogen bromide activated carbohydrates
and the systems reactive substrates described in U.S. Pat. Nos.
3,959,080; 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;
4,055,635; and 4,330,440 are employed for protein immobilization
and cross-linking.
[0078] Certain post-translational derivatizations are the result of
the action of recombinant host cells on the expressed polypeptide.
Glutaminyl and asparaginyl residues are frequently
post-translationally deamidated to the corresponding glutamyl and
aspartyl residues. Alternatively, these residues are deamid ed
under mildly acidic conditions. Either form of these residues falls
within the scope of this invention.
[0079] Other post-translational 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 (T. E. Creighton,
Proteins: Structure and Molecular Properties, W.H. Freeman &
Co., San Francisco pp 79-86 [1983]), acetylation of the N-terminal
amine and, in some instances, amidation of the C-terminal
carboxyl.
[0080] Other derivatives comprise the polypeptide of this invention
covalently bonded to a nonproteinaceous polymer. The
nonproteinaceous polymer ordinarily is a hydrophilic synthetic
polymer, i.e., a polymer not otherwise found in nature. However,
polymers which exist in nature and are produced by recombinant or
in vitro methods are useful, as are polymers which are isolated
from nature. Hydrophilic polyvinyl polymers fall within the scope
of this invention, e.g. polyvinylalcohol and polyvinylpyrrolidone.
Particularly useful are polyalkylene ethers such as polyethylene
glycol, polypropylene glycol, polyoxyethylene esters or methoxy
polyethylene glycol; polyoxyalkylenes such as polyoxyethylene,
polyoxypropylene, and block, copolymers of polyoxyethylene and
polyoxypropylene (Pluronics); polymethacrylates; carbomers;
branched or unbranched polysaccharides which comprise the
saccharide monomers D-mannose, D- and L-galactose, fucose,
fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid,
D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid,
or alginic acid), D-glucosamine. D-galactosamine, D-glucose and
neuraminic acid including homopolysaccharides and
heteropolysaccharides such as lactose, amylopectin, starch,
hydroxyethyl starch, amylose, dextran sulfate, dextran, dextrins,
glycogen, or the polysaccharide subunit of acid
mucopoly-saccharides, e.g. hyaluronic acid; polymers of sugar
alcohols such as polysorbitol and polymannitol; and heparin or
heparon.
[0081] Where the polysaccharide is the native glycosylation or the
glycosylation attendant on recombinant expression, the site of
substitution may be located at other than a native N or O-linked
glycosylation site wherein an additional or substitute N or
O-linked site has been introduced Into the molecule. Mixtures of
such polymers may be employed, or the polymer may be homogeneous.
The polymer prior to crosslinking need not be, but preferably is,
water soluble, but the final conjugate must be water soluble. In
addition, the polymer should not be highly immunogenic in the
conjugate form, nor should it possess viscosity that is
incompatible with intravenous infusion or injection if it is
intended to be administered by such routes.
[0082] Preferably the polymer contains only a single group which is
reactive. This helps to avoid cross-linking of protein molecules.
However, it is within the scope herein to optimize reaction
conditions to reduce cross-linking, or to purify the reaction
products through gel filtration or chromatographic sieves to
recover substantially homogeneous derivatives.
[0083] The molecular weight of the polymer may desirably range from
about 100 to 500,000, and preferably is from about 1,000 to 20,000.
The molecular weight chosen will depend upon the nature of the
polymer and the degree of substitution. In general, the greater the
hydrophilicity of the polymer and the greater the degree of
substitution, the lower the molecular weight that can be employed.
Optimal molecular weights will be determined by routine
experimentation.
[0084] The polymer generally is covalently linked to the
polypeptide herein through a multifunctional crosslinking agent
which reacts with the polymer and one or more amino acid or s
residues of the protein. However, it is within the scope of this
invention to directly crosslink the polymer by reacting a
derivatized polymer with the protein, or vice versa.
[0085] The covalent crosslinking site on the polypeptide includes
the N-terminal amino group and epsilon amino groups found on lysine
residues, as well as other amino, imino, carboxyl, sulfhydryl,
hydroxyl or other hydrophilic groups. The polymer may be covalently
bonded directly to the protein without the use of a multifunctional
(ordinarily bifunctional) crosslinking agent. Covalent bonding to
amino groups is accomplished by known chemistries based upon
cyanuric chloride, carbonyl diimidazole, aldehyde reactive groups
(PEG alkoxide plus diethyl acetal of bromoacetaldehyde; PEG plus
DMSO and acetic anhydride, or PEG chloride plus the phenoxide of
4-hydroxybenzaldehyde, succinimidyl active esters, activated
dithiocarbonate PEG, 2,4,5-trichlorophenylchloroformate or
p-nitrophenylchloroformate activated PEG. Carboxyl groups are
derivatized by coupling PEG-amine using carbodiimide.
[0086] Polymers are conjugated to oligosaccharide groups by
oxidation using chemicals, e.g. metaperiodate, or enzymes, e.g.
glucose or galactose oxidase, (either of which produces the
aldehyde derivative of the carbohydrate), followed by reaction with
hydrazide or amino-derivatized polymers, in the same fashion as is
described by Heitzmann et al., P.N.A.S., 71:3537-3541 (1974) or
Bayer et al., Methods in Enzymology, 62:310 (1979), for the
labeling of oligosaccharides with biotin or avidin. Further, other
chemical or enzymatic methods which have been used heretofore to
link oligosaccharides and polymers are suitable. Substituted
oligosaccharides are particularly advantageous because, in general,
there are fewer substitutions than amino acid sites for
derivatization, and the oligosaccharide products thus will be more
homogeneous. The oligosaccharide substituents also are optionally
modified by enzyme digestion to remove sugars, e.g. by
neuraminidase digestion, prior to polymer derivatization.
[0087] The polymer will bear a group which is directly reactive
with an amino acid side chain, or the N- or C-terminus of the
polypeptide herein, or which is reactive with the multifunctional
cross-linking agent. In general, polymers bearing such reactive
groups are known for the preparation of immobilized proteins. In
order to use such chemistries here, one should employ a water
soluble polymer otherwise derivatized in the same fashion as
insoluble polymers heretofore employed for protein immobilization.
Cyanogen bromide activation is a particularly useful procedure to
employ in crosslinking polysaccharides. "Water soluble" in
reference to the starting polymer means that the polymer or its
reactive intermediate used for conjugation is sufficiently water
soluble to participate in a derivatization reaction.
[0088] "Water soluble" in reference to the polymer conjugate means
that the conjugate is soluble in physiological fluids such as
blood.
[0089] The degree of substitution with such a polymer will vary
depending upon the number of reactive sites on the protein, whether
all or a fragment of the protein is used, whether the protein is a
fusion with a heterologous protein, the molecular weight,
hydrophilicity and other characteristics of the polymer, and the
particular protein derivatization sites chosen. In general, the
conjugate contains about from 1 to 10 polymer molecules, while any
heterologous sequence may be substituted with an essentially
unlimited number of polymer molecules so long as the desired
activity is not significantly adversely affected. The optimal
degree of crosslinking is easily determined by an experimental
matrix in which the time, temperature and other reaction conditions
are varied to change the degree of substitution, after which the
ability of the conjugates to function in the desired fashion is
determined.
[0090] The polymer, e.g. PEG, is crosslinked by a wide variety of
methods known per se for the covalent modification of proteins with
nonproteinaceous polymers such as PEG. Certain of these methods,
however, are not preferred for the purposes herein. Cyanuric
chloride chemistry leads to many side reactions, including protein
cross-linking. In addition, it may be particularly likely to lead
to inactivation of proteins containing sulfhydryl groups. Carbonyl
diimidazole chemistry (Beauchamp et al., "Anal. Biochem." 131:25-33
[1983]) requires high pH (>8.5), which can inactivate proteins.
Moreover, since the "activated PEG" intermediate can react with
water, a very large molar excess of "activated PEG" over protein is
required. The high concentrations of PEG required for the carbonyl
diimidazole chemistry also led to problems with purification, as
both gel filtration chromatography and hydrophobic interaction
chromatography are adversely effected. In addition, the high
concentrations of "activated PEG" may precipitate protein, a
problem that per se has been noted previously (Davis, U.S. Pat. No.
4,179,337). On the other hand, aldehyde chemistry (Royer, U.S. Pat.
No. 4,002,531) is more efficient since it requires only a 40 fold
molar excess of PEG and a 1-2 hr incubation. However, the manganese
dioxide suggested by Royer for preparation of the PEG aldehyde is
problematic "because of the pronounced tendency of PEG to form
complexes with metal-based oxidizing agents" (Harris et al., "J.
Polym. Sci., Polym. Chem. Ed." 22:341-352 [1984]). The use of a
moffatt oxidation, utilizing DMSO and acetic anhydride, obviates
this problem. In addition, the sodium borohydride suggested by
Royer must be used at a high pH and has a significant tendency to
reduce disulfide bonds. In contrast, sodium cyanoborohydride, which
is effective at neutral pH and has very little tendency to reduce
disulfide bonds is preferred.
[0091] The conjugates of this invention are separated from
unreacted starting materials by gel filtration. Heterologous
species of the conjugates are purified from one another in the same
fashion.
[0092] The polymer also may be water insoluble, as a hydrophilic
gel or a shaped article such as surgical tubing in the form of
catheters or drainage conduits.
[0093] DNA encoding the LHR is synthesized by In vitro methods or
is obtained readily from lymphocyte cDNA libraries. The means for
synthetic creation of the DNA encoding the LHR, either by hand or
with an automated apparatus, are generally known to one of ordinary
skill in the art, particularly in light of the teachings contained
herein. As examples of the current state of the art relating to
polynucleotide synthesis, one is directed to Maniatis et al.,
Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratory (1984), and Horvath et al., An Automated DNA Synthesizer
Employing Deoxynucleoside 3'-Phosphoramidites, Methods in
Enzymology 154: 313-326, 1987, hereby specifically incorporated by
reference.
[0094] Alternatively, to obtain DNA encoding the LHR from sources
other than murine or human, since the entire DNA sequence for the
preferred embodiment of the HuLHR (FIG. 1) and of the MLHR (FIG. 2)
are given, one needs only to conduct hybridization screening with
labelled DNA encoding either HuLHR or HuLHR or fragments thereof
(usually, greater than about 20, and ordinarily about 50 bp) in
order to detect clones which contain homologous sequences in the
cDNA libraries derived from the lymphocytes of the particular
animal, followed by analyzing the clones by restriction enzyme
analysis and nucleic acid sequencing to identify full-length
clones. If full length clones are not present in the library, then
appropriate fragments are-recovered from the various clones and
ligated at restriction sites common to the fragments to assemble a
full-length clone. DNA encoding the LHR from other animal species
is obtained by probing libraries from such species with the human
or murine sequences, or by synthesizing the genes in vitro.
[0095] Included within the scope hereof are nucleic acid sequences
that hybridize under stringent conditions to a fragment of the DNA
sequence in FIG. 1 or FIG. 2, which fragment is greater than about
10 bp, preferably 20-50 bp, and even greater than 100 bp. Also
included within the scope hereof are nucleic acid sequences that
hybridize under stringent conditions to a fragment of the LHR other
than the signal, or transmembrane, or cytoplasmic domains.
[0096] Included also within the scope hereof are nucleic acid
probes which are capable of hybridizing under stringent conditions
to the cDNA of the LHR or to the genomic gene for the LHR
(including introns and 5' or 3' flanking regions extending to the
adjacent genes or about 5,000 bp, whichever is greater).
[0097] Identification of the genomic DNA for the LHR is a
straight-forward matter of probing a particular genomic library
with the cDNA or its fragments which have been labelled with a
detectable group, e.g. radiophosphorus, and recovering clone(s)
containing the gene. The complete gene is pieced together by
"walking" if necessary. Typically, such probes do not encode
sequences with less than 70% homology to HuLHR or MLHR, and they
range from about from 10 to 100 bp in length.
[0098] In general, prokaryotes are used for cloning of DNA
sequences in constructing the vectors useful in the invention. For
example, E. coli K12 strain 294 (ATCC No. 31446) is particularly
useful. Other microbial strains which may be used include E. coli B
and E. coli X1776 (ATCC No. 31537). These examples are illustrative
rather than limiting. Alternatively, in vitro methods of cloning.
e.g. polymerase chain reaction, are suitable.
[0099] The LHR of this invention are expressed directly in
recombinant cell culture as an N-terminal methionyl analogue, or as
a fusion with a polypeptide heterologous to the LHR, preferably a
signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the LHR. For example, in constructing a
prokaryotic secretory expression vector for the LHR, the native LHR
signal is employed with hosts that recognize that signal. When the
secretory leader is "recognized" by the host, the host signal
peptidase is capable of cleaving a fusion of the leader polypeptide
fused at its C-terminus to the desired mature LHR. For host
prokaryotes that do not process the LHR signal, the signal is
substituted by a prokaryotic signal selected for example from the
group of the alkaline phosphatase, penicillinase, lpp or heat
stable enterotoxin II leaders. For yeast secretion the human LHR
signal may be substituted by the yeast invertase, alpha factor or
acid phosphatase leaders. In mammalian cell expression the native
signal is satisfactory for mammalian LHR, although other mammalian
secretory protein signals are suitable, as are viral secretory
leaders, for example the herpes simplex gD signal.
[0100] The LHR may be expressed in any host cell, but preferably
are synthesized in mammalian hosts. However, host cells from
prokaryotes, fungi, yeast, insects and the like are also are used
for expression. Exemplary prokaryotes are the strains suitable for
cloning as well as E. coli W3110 (F.sup.-, .lamda..sup.-,
ptototrophic, ATTC No. 27325), other enterobacteriaceae such as
Serratia marcescans, bacilli and various pseudomonads. Preferably
the host cell should secrete minimal amounts of proteolytic
enzymes.
[0101] Expression hosts typically are transformed with DNA encoding
the LHR which has been ligated into an expression vector. Such
vectors ordinarily carry a replication site (although this is not
necessary where chromosomal integration will occur). Expression
vectors also include marker sequences which are capable of
providing phenotypic selection in transformed cells, as will be
discussed further below. For example, E. coil is typically
transformed using pBR322, a plasmid derived from an E. coil species
(Bolivar, et al., Gene 2: 95 [1977]). pBR322 contains genes for
ampicillin and tetracycline resistance and thus provides easy means
for identifying transformed cells, whether for purposes of cloning
or expression. Expression vectors also optimally will contain
sequences which are useful for the control of transcription and
translation, e.g., promoters and Shine-Dalgarno sequences (for
prokaryotes) or promoters and enhancers (for mammalian cells). The
promoters may be, but need not be, inducible; surprisingly, even
powerful constitutive promoters such as the CMV promoter for
mammalian hosts have been found to produce the LHR without host
cell toxicity. While it is conceivable that expression vectors need
not contain any expression control, replicative sequences or
selection genes, their absence may hamper the identification of LHR
transformants and the achievement of high level LHR expression.
[0102] Promoters suitable for use with prokaryotic hosts
illustratively include the .mu.-lactamase and lactose promoter
systems (Chang et al., "Nature", 275: 615 [1978]; and Goeddel et
al., "Nature" 281: 544 [1979]), alkaline phosphatase, the
tryptophan (trp) promoter system (Goeddel "Nucleic Acids Res." 8:
4057 [1980] and EPO Appln. Publ. No. 36,776) and hybrid promoters
such as the tac promoter (H. de Boer et al., "Proc. Natl. Acad.
Sci. USA" 80: 21-25 [1983]). However, other functional bacterial
promoters are suitable. Their nucleotide sequences are generally
known, thereby enabling a skilled worker operably to ligate them to
DNA encoding the LHR (Siebenlist et al., "Cell" 20: 269 [1980])
using linkers or adaptors to supply any required restriction sites.
Promoters for use in bacterial systems also will contain a
Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding
the LHR.
[0103] In addition to prokaryotes, eukaryotic microbes such as
yeast or filamentous fungi are satisfactory. Saccharomyces
cerevisiae is the most commonly used eukaryotic microorganism,
although a number of other strains are commonly available. The
plasmid YRp7 is a satisfactory expression vector in yeast
(Stinchcomb, et al. Nature 282: 39 [1979]; Kingsman et al, Gene 7:
141 [1979]; Tschemper et al., Gene 10: 157 [1980]). This plasmid
already contains the trpl gene which 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 (Jones, Genetics
85: 12 [1977]). The presence of the trpl lesion as a characteristic
of the yeast host cell genome then provides an effective
environment for detecting transformation by growth in the absence
of tryptophan.
[0104] Suitable promoting sequences for use with yeast hosts
include the promoters for 3-phosphoglycerate kinase (Hitzeman et
al., "J. Biol. Chem." 255: 2073 [1980]) or other glycolytic enzymes
(Hess et al., "J. Adv. Enzyme Reg." 7: 149 [1968]; and Holland,
"Biochemistry" 17: 4900 [1978]), 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.
[0105] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase. degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in R.
Hitzeman et al., European Patent Publication No. 73,657A.
[0106] Expression control sequences are known for eukaryotes.
Virtually all eukaryotic genes have an AT-rich region located
approximately 25 to 30 bases upstream from the site where
transcription is initiated. Another sequence found 70 to 80 bases
upstream from the start of transcription of many genes is a CXCAAT
region where X may be any nucleotide. At the 3' end of most
eukaryotic genes is an AATAAA sequence which may be the signal for
addition of the poly A tail to the 3' end of the coding sequence.
All of these sequences are inserted into mammalian expression
vectors.
[0107] Suitable promoters for controlling transcription from
vectors in mammalian host cells are readily obtained from various
sources, for example, the genomes of viruses such as polyoma virus,
SV40, adenovirus, MMV (steroid inducible), retroviruses (e.g. the
LTR of HIV), hepatitis-B virus and most preferably cytomegalovirus,
or from heterologous mammalian promoters, e.g. the beta actin
promoter. The early and late promoters of SV40 are conveniently
obtained as an SV40 restriction fragment which also contains the
SV40 viral origin of replication. Fiers et al., Nature, 273: 113
(1978). The immediate early promoter of the human cytomegalovirus
is conveniently obtained as a HindIII E restriction fragment.
Greenaway, P. J. et al., Gene 18: 355-360 (1982).
[0108] Transcription of a DNA encoding the LHR by higher eukaryotes
is increased by inserting an enhancer sequence into the vector.
Enhancers are cis-acting elements of DNA, usually about from 10-300
bp, that act on a promoter to increase its transcription. Enhancers
are relatively orientation and position independent having been
found 5' (Laimins, L. et al., PNAS 78: 993 [1981]) and 3' (Lusky,
M. L., et al., Mol. Cell Bio. 3: 1108 [1983]) to the transcription
unit, within an intron (Banerji, J. L. et al., Cell 33: 729
[19831]) as well as within the coding sequence itself (Osborne, T.
F., et al., Mol. Cell Bio. 4: 1293 [1984]). Many enhancer sequences
are now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers.
[0109] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription which may affect mRNA expression.
These regions are transcribed as polyadenylated segments in the
untranslated portion of the =RNA encoding the LWR. The 3'
untranslated regions also include transcription termination
sites.
[0110] Expression vectors may contain a selection gene, also termed
a selectable marker. Examples of suitable selectable markers for
mammalian cells are dihydrofolate reductase (DHFR), thymidine
kinase (TK) or neomycin. When such selectable markers are
successfully transferred into a mammalian host cell, the
transformed mammalian host cell is able to survive if placed under
selective pressure. There are two widely used distinct categories
of selective regimes. The first category is based on a cell's
metabolism and the use of a mutant cell line which lacks the
ability to grow independent of a supplemented media. Two examples
are CHO DHFR.sup.- cells and mouse LTK.sup.- cells. These cells
lack the ability to grow without the addition of such nutrients as
thymidine or hypoxanthine. Because these cells lack certain genes
necessary for a complete nucleotide synthesis pathway, they cannot
survive unless the missing nucleotides are provided in a
supplemented media. An alternative to supplementing the media is to
introduce an intact DHFR or TK gene into cells lacking the
respective genes, thus altering their growth requirements.
Individual cells which were not transformed with the DHFR or TK
gene will not be capable of survival in non supplemented media.
[0111] The second category of selective regimes is dominant
selection which refers to a selection scheme used in any cell type
and does not require the use of a mutant cell line. These schemes
typically use a drug to arrest growth of a host cell. Those cells
which are successfully transformed with a heterologous gene express
a protein conferring drug resistance and thus survive the selection
regimen. Examples of such dominant selection use the drugs neomycin
(Southern et al., J. Kolec. Appl. Genet. 1: 327 (1982)),
mycophenolic acid (Mulligan et al., Science 209: 1422 (1980)) or
hygromycin (Sugden et al., Mol. Cell. Biol. 5: 410-413 (1985)). The
three examples given above employ bacterial genes under eukaryotic
control to convey resistance to the appropriate drug G418 or
neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin,
respectively.
[0112] Suitable eukaryotic host cells for expressing the LHR
include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL
1651); human embryonic kidney line (293 or 293 cells subcloned for
growth in suspension culture, Graham, F. L. et al., J. Gen Virol.
36: 59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10);
chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin, PNAS
(USA) 77: 4216, [1980]); mouse sertoli cells (TM4, Mather, J. P.,
Biol. Reprod. 23: 243-251 [1980]); monkey kidney cells (CV1 ATCC
CCL 70); african green monkey kidney cells (VERO-76, ATCC
CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);
canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells
(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75);
human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT
060562, ATCC CCL51); and, TRI cells (Mather, J. P. et al., Annals
N. Y. Acad. Sci. 38: 44-68 [1982]).
[0113] Construction of suitable vectors containing the desired
coding and control sequences employ standard ligation techniques.
Isolated plasmids or DNA fragments are cleaved, tailored, and
religated in the form desired to form the plasmids required.
[0114] For analysis to confirm correct sequences in plasmids
constructed, the ligation mixtures are used to transform E. coli
K12 strain 294 (ATCC 31446) and successful transformants selected
by ampicillin or tetracycline resistance where appropriate.
Plasmids from the transformants are prepared, analyzed by
restriction and/or sequenced by the method of Messing et al.,
Nucleic Acids Res. 9: 309 (1981) or by the method of Maxam et al.,
Methods in Enzymology 65: 499 (1980).
[0115] Host cells are transformed with the expression vectors of
this invention and cultured in conventional nutrient media modified
as appropriate for inducing promoters, selecting transformants or
amplifying the LHR gene. The culture conditions, such as
temperature, pH and the, like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0116] The host cells referred to in this disclosure encompass
cells in In vitro culture as well as cells which are within a host
animal.
[0117] "Transformation" means introducing DNA into an organism so
that the DNA is replicable, either as an extrachromosomal element
or by chromosomal integration. Unless indicated otherwise, the
method used herein for transformation of the host cells is the
method of Graham, F. and van der Eb, A., Virology 52: 456-457
(1973). However, other methods for introducing DNA into cells such
as by nuclear injection or by protoplast fusion may also be used.
If prokaryotic cells or cells which contain substantial cell wall
constructions are used, the preferred method of transfection is
calcium treatment using calcium chloride as described by Cohen, F.
N. et al., Proc. Natl. Acad. Sci. (USA). 69: 2110 (1972).
[0118] "Transfection" refers to the introduction of DNA into a host
eell whether or not any coding sequences are ultimately expressed.
Numerous methods of transfection are known to the ordinarily
skilled artisan, for example, CaPO.sub.4 and electroporation.
Transformation of the host cell is the indicia of successful
transfection.
[0119] The LHR is recovered and purified from recombinant cell
cultures by known methods, including ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, immunoaffinity
chromatography, hydroxyapatite chromatography and lectin
chromatography. Other known purification methods within the scope
of this invention utilize immobilized carbohydrates, epidermal
growth factor, or complement domains. Moreover, reverse-phase HPLC
and chromatography using anti-LHR antibodies are useful for the
purification of the LHR. Desirably, low concentrations
(approximately 1-5 mM) of calcium ion may be present during
purification. The LHR may preferably be purified in the presence of
a protease inhibitor such as PMSF.
[0120] The LHR is employed therapeutically to compete with the
normal binding of lymphocytes to lymphoid tissue. The LHR is
therefore particularly useful for organ or graft rejection, and for
the treatment of patients with inflammations, such as are for
example due to rheumatoid arthritis or other autoimmune diseases.
The LHR also finds application in the control of lymphoma
metastasis. Finally, the LHR is useful in treating conditions in
which there is an accumulation of lymphocytes.
[0121] The LHR, and the LHR variants and derivatives are also
useful as reagents in diagnostic assays for the LHR, antibodies to
the LHR, or competitive. inhibitors of LHR biological activity.
When insolubilized in accord with known methods, they are useful as
agents for purifying anti-U[R antibodies from antisera or hybridoma
culture supernatants. The LHR which may or may not have binding
activity find use as immunogens for raising antibodies to the LHR
or as immunoassay kit components (labelled, as a competitive
reagent for the native LHR, or unlabelled as a standard for a LHR
assay).
[0122] The LHR is placed into sterile isotonic formulations
together with required cofactors, and optionally are administered
by standard means well known in the field. The formulation of the
LHR is preferably liquid, and is ordinarily a physiologic salt
solution containing 0.5-10 mM calcium, non-phosphate buffer at pH
6.8-7.6, or may be lyophilized powder.
[0123] It is envisioned that intravenous delivery, or delivery
through catheter or other surgical tubing will be the primary route
for therapeutic administration. Alternative routes include tablets
and the like, commercially available nebulizers for liquid
formulations, and inhalation of lyophilized or aerosolized
receptors. Liquid formulations may be utilized after reconstitution
from powder formulations.
[0124] The LHR may also be administered via microspheres,
liposomes, other microparticulate delivery systems or sustained
release formulations placed in certain tissues including blood.
Suitable examples of sustained release carriers include
semipermeable polymer matrices in the form of shaped articles, e.g.
suppositories, or microcapsules. Implantable or microcapsular
sustained release matrices include polylactides (U.S. Pat. No.
3,773,919, EP 58,481) copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (U. Sidman et al., 1985, Biopolymers 22(1):
547-556), poly (2-hydroxyethyl-methacrylate) or ethylene vinyl
acetate (R. Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277
and R. Langer, 1982, Chem. Tech. 12: 98-105). Liposomes containing
the LHR are prepared by well-known methods: DE 3,218,121A; Epstein
et al. 1985, Proc. Natl. Acad. Sci. USA, 82:3688-3692; Hwang er
1980, Proc. Natl. Acad. Sci. USA, 77:4030-4034; EP 52322A; EP
36676A; EP 88046A; EP 143949A; EP 142541A; Japanese patent
application 83-11808; U.S. Pat. Nos. 4,485,045 and 4,544,545; and
UP 102,342A. Ordinarily the liposomes are of the small (about
200-800 Angstroms) unilamelar type in which the lipid content is
greater than about 30 mol. % cholesterol, the selected proportion
being adjusted for the optimal rate of the LHR leakage.
[0125] Sustained release LHR preparations are implanted or injected
into proximity to the site of inflammation or therapy, for example
adjacent to arthritic joints or peripheral lymph nodes.
[0126] The dose of the LHR administered will be dependent upon the
properties of the LHR employed, e.g. its activity and biological
half-life, the concentration of the LHR in the formulation, the
administration route for the LHR, the site and rate of dosage, the
clinical tolerance of the patient involved, the pathological
condition afflicting the patient and the like, as is well within
the skill of the physician.
[0127] LHR may also be administered along with other pharmacologic
agents used to treat the conditions listed above, such as
antibiotics, anti-inflammatory agents, and anti-tumor agents. It
may also be useful to administer the LHR along with other
therapeutic proteins such as gamma-interferon and other
immunomodulators.
[0128] In order to facilitate understanding of the following
examples certain frequently occurring methods and/or terms will be
described.
[0129] "Plasmids" are designated by a lower case p preceded and/or
followed by capital letters and/or numbers. The starting plasmids
herein are either commercially available, publicly available on an
unrestricted basis, or can be constructed from available plasmids
in accord with published procedures. In addition, equivalent
plasmids to those described are known in the art and will be
apparent to the ordinarily skilled artisan.
[0130] In particular, it is preferred that these plasmids have some
or all of the following characteristics: (1) possess a minimal
number of host-organism sequences; (2) be stable in the desired
host; (3) be capable of being present in a high copy number in the
desired host; (4) possess a regulatable promoter; and (5) have at
least one DNA sequence coding for a selectable trait present on a
portion of the plasmid separate from that where the novel DNA
sequence will be inserted. Alteration of plasmids to meet the above
criteria are easily performed by those of ordinary skill in the art
in light of the available literature and the teachings herein. It
is to be understood that additional cloning vectors may now exist
or will be discovered which-have the above-identified properties
and are therefore suitable for use in the present invention and
these vectors are also contemplated as being within the scope of
this invention.
[0131] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 .mu.g of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 .mu.l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 .mu.g of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation
times of about 1 hour at 37.degree. C. are ordinarily used, but may
vary in accordance with the supplier's instructions. After
digestion the reaction is electrophoresed directly on a
polyacrylamide gel to isolate the desired fragment.
[0132] Size separation of the cleaved fragments is performed using
8 percent polyacrylamide gel described by Goeddel, D. et al.,
Nucleic Acids Res., 8: 4057 (1980).
[0133] "Dephosphorylation" refers to the removal of the terminal 5'
phosphates by treatment with bacterial alkaline phosphatase (BAP).
This procedure prevents the two restriction cleaved ends of a DNA
fragment from "circularizing" or forming a closed loop that would
impede insertion of another DNA fragment at the restriction site.
Procedures and reagents for dephosphorylation are conventional.
Maniatis, T. et al., Molecular Cloning pp. 133-134 (1982).
Reactions using BAP are carried out in 50 mM Tris at 68.degree. C.
to suppress the activity of any exonucleases which are present in
the enzyme preparations. Reactions are run for 1 hour. Following
the reaction the DNA fragment is gel purified.
[0134] "Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide without adding a phosphate with an ATP in
the presence of a kinase. A synthetic oligonucleotide will ligate
to a fragment that has not been dephosphorylated.
[0135] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Maniatis,
T. et al., Id., p. 146). Unless otherwise provided, ligation is
accomplished using known buffers and conditions with 10 units of T4
DNA ligase ("ligase") per 0.5 .mu.g of approximately equimolar
amounts of the DNA fragments to be ligated.
[0136] "Filling" or "blunting" refers to the procedures by which
the single stranded end in the cohesive terminus of a restriction
enzyme-cleaved nucleic acid is converted to a double strand. This
eliminates the cohesive terminus and forms a blunt end. This
process is a versatile tool for converting a restriction cut end
that may be cohesive with the ends created by only one or a few
other restriction enzymes into a terminus compatible with any
blunt-cutting restriction endonuclease or other filled cohesive
terminus. Typically, blunting Is accomplished by incubating 2-15
.mu.g of the target DNA in 10 mM MgCl.sub.2, 1 mM dithiothreitol,
50 mM NaCl, 10 mM Tris (pH 7.5) buffer at about 37.degree. C. in
the presence of 8 units of the Klenow fragment of DNA polymerase I
and 250 .mu.M of each of the four deoxynucleoside triphosphates.
The incubation generally is terminated after 30 min. phenol and
chloroform extraction and ethanol precipitation.
[0137] It is presently believed that the three-dimensional
structure of the compositions of the present invention is important
to their functioning as described herein. Therefore, all related
structural analogs which mimic the active structure of those formed
by the compositions claimed herein are specifically included within
the scope of the present invention.
[0138] It is understood that the application of the teachings of
the present invention to a specific problem or situation will be
within the capabilities of one having ordinary skill in the art in
light of the teachings contained herein. Examples of the products
of the present invention and representative processes for their
isolation, use, and manufacture appear below, but should not be
construed to limit the invention. All literature citations herein
are expressly incorporated by reference.
EXAMPLES
[0139] Throughout these examples, all references to the "Mel 14"
monoclonal antibody or to "Mel 14" refer to a monoclonal antibody
directed against a purported murine form of a lymphocyte surface
protein, as described by Gallatin, et al., supra, Nature 303, 30
(1983), specifically incorporated by reference. The use of Mel 14
is no longer needed to practice this invention, however, due to the
provision herein of full sequences for the DNA and amino acids of
the LHR.
Example 1
Purification and Cloning of MLHR
Isolation of a cDNA Clone Encoding the MLHR.
[0140] MLHR was isolated from detergent-treated mouse spleens by
immunoaffinity chromatography using the Mel 14 monoclonal
antibody.
[0141] In a typical preparation, 300 spleens from ICR female mice
(16 weeks old) were minced and then homogenized with a
Potter-Elvehjem tissue grinder in 180 ml of 2% Triton X-100 in
Dulbecco's PBS containing 1 mM PMSF and 1% aprotinin. Lysis was
continued for 30 minutes on a shaker at 4.degree. C. The lysate was
centrifuged successively at 2,000.times.G for 5 minutes and at
40,000.times.G for 30 minutes.
[0142] The supernatant was filtered through Nitex screen and then
precleared by adsorption with rat serum coupled to cyanogen
bromide-activated Sepharose 4B (10 ml of packed gel). The rat serum
was diluted 1:10 for coupling with conjugation carried out
according to the manufacturer's instructions. The flow through was
applied to a 3 ml column of MEL-14 antibody coupled at 0.5 mg per
ml to Sepharose 4B. All column buffers contained sodium azide at
0.02%.
[0143] The column was washed with 25 ml of 2% Triton X-100 in PBS
followed by 25 ml of 10 mM CHAPS in the same buffer. Antigen was
released by addition of 10 ml of 10 mM CHAPS in 100 mM glycine, 200
mM NaCl, pH 3 and neutralized by collection into 1 ml of 1M TRIS
HCl, pH 7.6. After the column was washed with 20 mM triethylamine,
200 mM NaCl, pH 11 and re-equilibrated in 10 mM CHAPS in PBS, the
neutralized antigen, diluted into 100 ml of the column buffer, was
re-applied and the wash and release steps were repeated.
[0144] The purified protein was concentrated in a Centricon 30
(Amicon, Inc.) and analyzed by SDS-PAGE (7.5% acrylamide) with the
use of silver staining for visualization. A typical purification
yielded 30-40 .mu.g of antigen per 300 mice based upon comparisons
with orosomucoid standards.
[0145] As can be seen in FIG. 4A, a polyacrylamide gel of the
purified material showed a diffuse band migrating at approximately
90,000 daltons, and a higher molecular weight protein at around
180,000 daltons. The ratio of the 90,000 dalton to the 180,000
dalton component was 10:1 or greater in all of a large series of
preparations. The material was visualized by silver staining of a
10% polyacrylamide gel.
[0146] Gas phase Edman degradation of the 90,000 dalton band
resulted in the identification of a single N-terminal sequence
(FIG. 4B), including the very N-terminal amino acid. 38 N-terminal
amino acids were identified, with four gaps (X) at positions
1,19,33, and 34. The asparagine (N) at position 22 was inferred
from the absence of an amino acid signal at this position combined
with the following tyrosine (Y) and threonine (T) residues,
resulting in an N-linked glycosylation site consensus sequence
(NXT/S).
[0147] The 13-sequence residue shown in FIG. 4B above the 38
residue long N-terminus is that previously deduced by Siegelman et
al., supra, using radioactively-labelled amino acid sequencing,
which shows a high degree of homology (11 of 13 residues) with the
sequence of the LHR determined here.
[0148] No ubiquitin sequence was obtained in any of the three
sequencing runs that were done with two separate MLHR preparations.
Conceivably, this modification was absent in the mouse splenocytes
or the N-terminus of the ubiquitin is blocked to Edman degradation
in the LHR from this source.
[0149] The amino acid sequences of FIG. 2 were compared with known
sequences in the Dayhoff protein data base, through use of the
algorithm of Lipman, D. et al., Science 227, 1435-1441 (1981).
[0150] The residues in FIG. 4B which are underlined between amino
acids 7 and 15 were chosen to produce the oligonucleotide probe
shown in FIG. 4C. A 32-fold redundant 26-mer oligonucleotide probe
was designed from these residues and synthesized on an Applied
Biosystems oligonucleotide synthesizer. All of the possible codon
redundancies were included in this probe, with the exception of the
proline at position 9, where the codon CCC was chosen based upon
mammalian codon usage rules.
[0151] Screening of a murine spleen cDNA library obtained from
dissected mouse spleens with this probe resulted in the isolation
of a single hybridizing cDNA clone. Procedurally, 600,000 plaques
from an oligo dT-primed lambda gt 10 murine spleen cDNA library
produced from mM isolated from murine splenocytes with 5 .mu.g/ml
Concanavalin A for 6 hours were plated at 50,000 phage per plate
(12 plates) and hybridized with the p.sup.32 labeled 32-fold
redundant 26-mer oligonucleotide probe shown in FIG. 4C, in 20%
formamide, 5.times.SSC (150 mM NaCl. 15 mM trisodium citrate), 50
mM sodium phosphate (pH7.6), 5.times. Denhardts solution, 10%
dextran sulfate, and 20 micrograms/ml denatured, sheared salmon
sperm DNA overnight at 42.degree. C. These parameters are referred
to herein as "stringent conditions". The filters were washed in
1.times.SSC, 0.1% SDS at 42.degree. C. for 2.times.30 minutes and
autoradiographed at -70.degree. C. overnight. A single duplicate
positive clone was rescreened. the EcoRl insert was isolated and
inserted into M13 or PUC 118/119 vectors and the nucleotide
sequence determined from single stranded templates using
sequence-specific primers.
[0152] FIG. 2 shows the complete DNA sequence of the 2.2 kilobase
EcoRl insert contained in this bacteriophage. The longest open
reading frame begins with a methionine codon at position 106-108. A
Kozak box homology is found surrounding this methionine codon,
suggesting that this codon probably functions in initiating protein
translation. A protein sequence containing 373 amino acids of
approximately 42,200 daltons molecular weight is encoded within
this open reading frame. The translated protein shows a sequence
from residues 40 to 76 that corresponds exactly with the N-terminal
amino acid sequence determined from the isolated MLHR.
[0153] This result suggests that the mature N-terminus of the MLHR
begins with the tryptophan residue at position 39. However, it is
believed that some proteolytic processing of the actual N-terminus
of the LHR may have occurred during the isolation-of the
protein.
[0154] A hydrophobicity profile of the protein reveals an
N-terminally located hydrophobic domain that could function as a
signal sequence for insertion into the lumen of the endoplasmic
reticulum. The deduced sequence for positions 39 to 333 is
predominantly hydrophilic followed by a 22 residue hydrophobic
domain, which is characteristic of a stop transfer or membrane
anchoring domain.
[0155] The putative intracellular region at the very C-terminus of
the protein is quite short, only 17 residues in length. On the
immediate C-terminal side of the predicted membrane-spanning domain
are several basic amino acids, a feature typical of junctions
between membrane anchors and cytoplasmic domains of cell surface
receptors, Yarden et al., Nature. A single serine residue,
potentially a site for phosphorylation, is present within the
putative cytoplasmic domain.
[0156] The protein contains ten potential N-linked glycosylation
sites, all of which are within the projected extracellular domain.
The absence of asparagine at position 60 (residue 22 of the mature
protein) in the peptide sequencing analysis confirms glycosylation
at this site and establishes the extracellular orientation of this
region. The coding region contains a total of 25 cysteine residues,
although 4 of these cysteine residues are located within the
putative leader sequence.
Protein Motifs within the MLHR
[0157] As shown in FIG. 6, comparison of the deduced MLHR amino
acid sequence to other proteins in the Dayhoff protein sequence
databank by using the fastp program (Lipman, D., and Pearson, W.,
Science 227, 1435-1441, 1985) revealed a number of interesting
sequence homologies.
[0158] Proteins with the highest sequence homology scores are shown
with boxes surrounding the regions of greatest sequence homology.
The numbers at the beginning of the sequences show the position
within the proteins where these homologous sequences are
located.
[0159] FIG. 6A shows that the N-terminal motif of the LHR (residues
39 to 155) has certain carbohydrate binding protein homologies, as
listed (the percentage of homology of these sequences to the MuLHR
are given in parentheses, and the references indicated are provided
after the Examples): Drickamer; the amino acid residues found by
Drickamer et al. (1), MLHR; the MLHR sequence, Hu.HepLec (27.8%);
human hepatic lectin (2), Barn.Lec (25%); acorn barnacle lectin
(3), Ra. HepLec (23.5%); rat hepatic lectin (4), Ch.HepLec (27.5%);
chicken hepatic lectin (5), Hu.IgERec (28.6%); human IgE receptor
(6), RaHepLec2 (22.6%); rat hepatic lectin 2 (7), Ra.ASGRec
(22.6%); rat asialoglycoprotein receptor (8), Ra.IRP (25.6%); rat
islet regenerating protein (9), Ra.MBP (26.1%); rat mannose binding
protein (10), Ra.MBDA (26.1%); rat mannose binding protein
precursor A (11). Ra.KCBP (27%); rat Kuppfer cell binding protein
(12), FlyLec (23.1%); flesh fly (Sarcophaga) lectin (13), and
Rab.Surf (20.9%); rabbit lung surfactant (14).
[0160] As can be seen, FIG. 6A shows that the most N-terminally
localized motif of the LHR shows a high degree of homology with a
number of calcium-dependent animal lectins, i.e., C-type lectins
(1). These include but are not limited to, various hepatic sugar
binding proteins from chicken, rat, and human, soluble
mannose-binding lectins, a lectin from Kupffer cells, the
asialoglycoprotein receptor, a cartilage proteoglycan core protein,
pulmonary surfactant apoproteins, and two invertebrate lectins from
the flesh fly and acorn barnacle. Although the complement of
"invariant" amino acids initially recognized by Drickamer and
colleagues, supra, as common to C-type animal lectins are not
completely conserved in the carbohydrate binding domain of the
MLHR, the degree of homology at these residues and at other
positions is apparent. The known lectins belonging to the C-type
family exhibit a range of sugar-binding specificities including
oligosaccharides with terminal galactose, N-acetylglucosamine, and
mannose (1).
[0161] Interestingly, the lectin domains of all of these proteins
except the acorn barnacle lectin and the flesh fly lectin are
located in their respective carboxy-termini, suggesting that this
MLHR domain may be contained-in an exon that can be shuffled to
different proteins for different functions.
[0162] The fact that there are many residues that are found to be
invariant in all of these carbohydrate binding proteins, strongly
suggests that this region functions as a carbohydrate binding
domain in the MLHR and apparently explains the observed ability of
lymphocytes to bind to the specialized endothelium of lymphoid
tissue in a sugar- and calcium-dependent manner. It is believed
that the carbohydrate binding domain of the LHR alone, without any
flanking LHR regions, is desirably used in the practice of this
invention.
[0163] The next motif (residues 160-193) that is found almost
immediately after the completion of the carbohydrate binding domain
shows a high degree of homology to the epidermal growth factor
(egf) family. FIG. 6B shows epidermal growth factor (egf)
homologies: MLHR; the MLHR sequence, Notch (38.5%); the Drosophila
melanogaster notch locus (15), S.purp (31.7%); Strongylocentrotur
purpuratus egf-like protein (16), Pro.Z (34.1%); bovine protein Z
(17), Fact.X (34.2%); coagulation factor X (18), Fact.VII (27.3%);
coagulation factor VII (19), Fact.IX (33.3%); coagulation factor IX
(20), Lin-12 (32.1%); Caenorhabditis elegans Lin-12 locus (21),
Fact. XII (26%); coagulation factor XII (22), and Mu.egf (30%);
murine egf (23).
[0164] As can be seen in FIG. 6B, the greatest degree of homology
in this region of the MLHR is found with the Drosophila neurogenic
locus, notch, although there is also significant homology to a
number of other members of this large family. The variable location
of this domain among the members of this family suggests that this
region may be contained within a genomic segment that can be
shuffled between different proteins for different functions.
[0165] In addition to 6 cysteine residues, virtually all members of
this family share three glycine residues. The conservation of
cysteine and glycine residues is consistent with the possibility of
a structural role for this region in the LHR. It is believed that
this domain may place the N-terminally localized carbohydrate
binding region in an appropriate orientation for ligand
interaction. It is further believed that this domain may serve to
strengthen the interaction between the lymphocyte and endothelium
by binding to an egf-receptor homologue on the endothelium
surface.
[0166] The final protein motif in the extracellular region of the
MLHR is encoded from amino acids 197 to 328. This region of the
glycoprotein contains two direct repeats of a 62 residue sequence
that contains an amino acid motif that bears a high degree of
homology to a number of complement factor binding proteins (FIG.
6C).
[0167] FIG. 6C shows complement binding protein homologies: MLHR;
MLHR sequence, HuComH (31.9%); human complement protein H precursor
(24), MuComl (28.9t); murine complement protein H precursor (25),
HuBeta (25.6%); human beta-2-glycoprotein I (26), HuCR1 (29.9%);
human CR1 (27), EBV/3d (25%)6; human Epstein-Barr virus/C3d
receptor (28), HuC2 (27.1%); human complement C2 precursor (29),
HuB (23.1%); human complement factor B (30), MuC4b (22%); murine
C4b-binding precursor (31), HuCls (29.2%); human Cls zymogen (32),
HuC4b (26.1%); human C4b binding protein (33), HuDAF (27.1%); human
decay accelerating factor (34), VacSecP (26.2%); vaccinia virus
secretory peptide (35).
[0168] These proteins, which encode a wide range of multiples of
this-repeated domain, include, among others, the human and murine
complement H precursors, the human beta 2 glycoprotein, the Epstein
Barr virus/C3d receptor, the human C4b binding protein, the decay
accelerating factor, and the vaccinia virus secretory
polypeptide.
[0169] FIG. 7C shows the homologies between the two direct repeats
in the MLHR and the direct repeats found in proteins contained
within the complement binding family. Many of the amino acids that
are conserved in this class of complement binding proteins,
including a number of conserved cysteine residues, are also found
in the 2 repeats in this region of the MLHR.
[0170] Interestingly, the two repeats contained within the MLHR are
not only exact duplications of each other at the amino acid level,
they also show exact homology at the nucleotide sequence level
(nucleotide residues 685-865 and 866-1056). While it is possible
that this result is due to a cloning artifact, a duplicated region
has been found in a number of other clones isolated from a separate
cDNA library produced from the MLHR expressing cell line, 38C13
(available from Stanford University, Palo Alto, Calif., U.S.A.), as
well as in a human homologue of the MLHR (discussed, infra.).
Furthermore, a number of other genes, most notably the Lp(a) gene,
show an even higher degree of intragenic repeat sequence
conservation of this domain. These results suggest that the MLHR,
like other members of the complement binding family, contains
multiple repeats of this binding domain.
[0171] In conclusion, it appears that the extracellular region of
the MLHR contains three separate protein motifs that have been
joined together to serve a new function or functions. A summary of
the protein motifs contained within this glycoprotein is shown in
FIG. 7.
Example 2
Cloning of HuLHR
[0172] Generally as described in the previous example, the 2.2 kb
EcoRl insert of the murine Mal 14 antigen cDNA clone described
above was isolated, labeled to high specific activity by randomly
primed DNA polymerase synthesis with P.sup.32 triphosphates, and
used to screen 600,000 clones from an oligo dT primed lambda gt10
cDNA library derived from human peripheral blood lymphocyte mRNA
obtained from primary cells. The filters were hybridized overnight
at 42.degree. C. in 40% formamide, 5.times.SSC (1.times.SSC is 30
mM NaCl, 3 mM trisodium citrate), 50 mM sodium phosphate (pH6.8),
10% dextran sulfate, 5.times. Denhardt's solution and 20
micrograms/ml sheared, boiled salmon sperm DNA. They were washed
2.times.40 minutes in 0.2.times.SSC, 0.1% sodium dodecyl sulfate at
55.degree. C. 12 clones (approximately 1 positive per plate of
50,000 phage) were picked, and the largest EcoR1 insert (.sup.-2.2
kilobases) was isolated and the DNA sequence was determined by
didoxynucleotide sequencing in the bacteriophage m13 using
sequence-specific primers.
[0173] This .sup.-2.2 kb clone encoded an open reading frame of 372
amino acids with a molecular weight of approximately 42,200 daltons
that began with a methionine which was preceded by a Kozak box
homology. The encoded protein contained 26 cysteine residues and 8
potential N-linked glycosylation sites. A highly hydrophobic region
at the N-terminus of the protein (residues 20-33) was a potential
signal sequence, while another highly hydrophobic C-terminally
located region of 22 amino acids in length (residues 335-357) was a
potential stop transfer or membrane anchoring domain. This
C-terminal hydrophobic region was followed by a charged, presumably
cytoplasmic, region.
[0174] Comparison of the nucleotide sequence of this human clone
with that previously found for the MLHR showed a high degree of
overall DNA sequence homology (.sup.-83%). The relative degrees of
amino acid sequence conservation between the MLHR and the HuLHR in
each of the LHR domains are: carbohydrate binding domain--83%;
egf-like domain--82%; complement binding repeat 1--79%; complement
binding repeat 2--63%; overall complement binding domain--71%; and
transmembrane domain--96%.
[0175] Comparison of the published Hermes sequence, Jalkanen,
supra, with the HuLHR sequence of FIG. 1 reveals a lack of sequence
homology.
Example 3
Expression of the MLHR
[0176] In order to conclusively prove that the murine cDNA clone
isolated here encoded the MLHR, the clone was inserted into an
expression vector and analyzed in a transient cell transfection
assay. Expression of the HuLHR was performed in a similar
fashion.
[0177] The Eco R1 fragment containing the open reading frame
described above (the .sup.-2.2 kilobase EcoR1 fragment whose
sequence is shown in FIG. 2) was isolated and ligated into the pRK5
vector which contains a cytomegalovirus promoter (Eaton, D., et
al., Biochemistry 25, 8343-8347, 1986; U.S. Ser. No. 07/097,472). A
plasmid containing the inserted cDNA in the correct orientation
relative to the promoter was selected and transfected onto 293
human embryonic kidney cells using CaPo.sub.4 precipitation.
[0178] After 2 days the cells were incubated with 500 microcuries
each of S.sup.35 cysteine and methionine. Lysates and supernatants
were prepared as previously described (Lasky, L., et al., Cell 50,
975-985, 1987) and immunoprecipitated with Mel 14 monoclonal
antibody (purified by immunoaffinity chromatography) by utilizing
an anti-rat IgG polyclonal antibody in a sandwich between the Mel
14 monoclonal antibody and protein A sepharose.
[0179] At the same time, the B-cell lymphoma, 38C13, a cell known
to express the MLHR, were either labeled metabolically with either
methionine or cysteine, for analysis of the supernatant MLHR, or
the cell-surface glycoproteins were labeled with I.sup.125 and
lactoperoxidase for analysis of cell-associated LHR and analyzed by
Mel 14 antibody immunoprecipitation.
[0180] The resultant iummoprecipitates were analyzed on 7.5%
polyacrylamide SDS gels and autoradiographed overnight at
-70.degree. C.
[0181] The results of these assays are shown in FIG. 5. In that
figure, the lanes A-F signify the following: [0182] --A. Lysates of
293 cells transfected with a MLHR expression plasmid
immunoprecipitated with Mel 14 monoclonal antibody. [0183] --B.
Supernatants of 293 cells transfected with a MLHR expression
plasmid immunoprecipitated with Mel 14 monoclonal antibody. [0184]
--C. Lysates of 293 cells transfected with a plasmid expressing the
HIV gp120envelope glycoprotein immunoprecipitated with the Mel 14
monoclonal antibody. [0185] --D. Supernatants of 293 cells
transfected with the HIV envelope expression plasmid
immunoprecipitated with the Mel 14 monoclonal antibody. [0186] --E.
Supernatants of 38C13 cells immunoprecipitated with the Mel 14
monoclonal antibody. [0187] --F. Lysates of 38C13 cells surface
labeled with I125 and immunoprecipitated with the Mel 14 monoclonal
antibody.
[0188] As can be seen in FIG. 5, cell transfected with this
construct produce two cell-associated proteins that reacted
specifically with the Mel 14 antibody. The cell associated proteins
migrated at approximately .sup.-70,000 daltons and .sup.-85,000
daltons, suggesting that the .sup.-42,200 dalton core protein
becomes glycosylated in the transfected cells. The larger band was
shifted in molecular weight following sialidase treatment (data not
shown), suggesting that it is a relatively nature form of the
glycoprotein, whereas the lower molecular weight band was resistant
to the enzyme, indicating that it may be a precursor form.
[0189] FACs analysis of transiently transfected cell lines with the
Mel 14 antibody showed that a portion of the LHR expressed in these
cells was detectable on the cell surface (data not shown).
[0190] The higher molecular weight glycoprotein produced in the
transfected cell line was found to be slightly smaller than that
produced by the Peripheral Lymph Node-homing B-cell lymphoma, 38C13
(FIG. 5, lane F), a result that has been found in other transfected
cell lines and may be due to cell-specific differences in
glycosylation.
[0191] Interestingly, both the 38C13 cells and the transfected
human cells appeared to shed a smaller molecular weight form of the
MLHR into the medium (FIG. 5, lanes B and E). The nature of this
shed molecule is unclear, although its reduced molecular weight
suggests that it may be a cleavage product of the cell surface form
resulting from proteolysis near the membrane anchor.
[0192] In conclusion, these results convincingly demonstrate that
the cDNA clone that we have isolated encodes the MLHR.
REFERENCES TO THE EXAMPLE
[0193] 1. Drickamer, K., J. Biol. Chem, 263, 9557 (1988);
Drickamer, K., Kidney Int. 32, S167 (1987). [0194] 2. Spiess. M.,
et al., Proc. Natl. Acad. Scl., U.S.A. 82, 6465 (1985). [0195] 3.
Muramoto, K., et al., Biochem Biophys. Acta 874, 285 (1986). [0196]
4. Leung, J., et al., J. Biol. Chem. 260, 12523 (1985); Holland,
E., et al., Proc. Natl. Acad. Sci., U.S.A. 81, 7338 (1984). [0197]
5. Drickamer, K., J. Biol. Chem. 256, 5827 (1981). [0198] 6.
Kikutani, H., et al., Cell 47, 657 (1986). [0199] 7. McPhaul, H.,
et al., Molec. Cell. Biol. 7, 1841 (1987). [0200] 8. Halberg, D.,
et al., J. Biol. Chem. 262, 9828 (1987). [0201] 9. Terzaono, K., et
al., J. Biol. Chem. 263, 2111 (1988). [0202] 10. Drickamer, K., et
al., J. Biol. Chem. 262, 2582 (1987). [0203] 11. Drickamer, K., et
al., J. Biol. Chem. 261, 6878 (1986). [0204] 12. Hoyle, G., et al.,
J. Bloi. Chem. 263, 7487 (1988). [0205] 13. Takahashi, H., et al.,
J. Biol. Chem. 260, 12228 (1985). [0206] 14. Boggaram. V., et al.,
J. Biol. Chem. 263, 2939 (1988). [0207] 15. Kidd, S., et al., Mol.
Cell. Biol. 6, 3094 (1986). [0208] 16. Hursh, D., et al., Science
237, 1487 (1987). [0209] 17. Hojrup, P., et al., FEBS Lett. 184,
333 (1985). [0210] 18. Fung, M., et al., Nucl. Acids Res. 12, 4481
(1984). [0211] 19. Takeya, H. et al., Proc. Natl. Acad. Sci.,
U.S.A. 76, 4990 (1979). [0212] 20. McMullen, B., et al., Biochem
Biophys. Res. Commun. 115, 8 (1983). [0213] 21. Greenwald, I., Cell
43, 583 (1985). [0214] 22. Cool, D., et al., Biol. Chem. 260, 13666
(1985). [0215] 23. Gray, A., et al., Nature 303, 722 (1983). [0216]
24. Scbulz, T., et al., Eur. J. Immunol. 16, 1351 (1986). [0217]
25. Kristensen, T., et al., Proc. Natl. Acad. Sci. U.S.A. 83, 3963
(1986). [0218] 26. Lozier, J., et al., Proc. Natl. Acad. Sci.,
U.S.A. 81, 3640 (1984). [0219] 27. Bentley, D. Biochem. J. 239, 339
(1986). [0220] 28. Moore, M., et al., Proc. Natl. Acad. Sci., U.S.A
84, 9194 (1987). [0221] 29. Bentley, D., et al. Proc. Natl. Acad.
Sci., U.S.A., 81, 1212 (1984). [0222] 30. Mole, J., et al., J.
Biol. Chem. 259, 3407 (1984). [0223] 31. DiScipio, R., et al., J.
Biol. Chem. 263, 549 (1988). [0224] 32. Kusomoto, H., et al., Proc.
Natl. Acad. Sci., U.S.A. 85, 7307 (1988). [0225] 33. Lintin, S., et
al., FEBS Lett. 204, 77 (1986). [0226] 34. Caras, I., et al.,
Nature 325, 545 (1987). [0227] 35. Kotwal, G., et al., Nature 335,
176 (1988).
Sequence CWU 1
1
* * * * *