Note: Descriptions are shown in the official language in which they were submitted.
-~ 2~2~7~
COMPOSITIONS AND METHODS FOR DETECTION
AND TREATMENT OF EPSTEIN-BARR VIRUS
INFECTION AND IMMUNE DISORDERS
Descri~tion
Technical Field
The present invention relates to synthetic `~
polypeptides, and polypeptide aggregates, that
correspond to a B lymphocyte membrane receptor
protein (CR2) for Epstein-Barr virus.
Backaround of the Invention
A 145 kilodalton (kDa) B lymphocyte ~ ;
membrane glycoprotein, designated CR2, serves as a
receptor for Epstein-Barr virus (EBV) and the C3d and
C3dg fragments of the third component of complement.
Epstein-Barr virus (EBV), a human ~ `
herpesvirus, is the causative agent of infectious
mononucleosis (Henle, G., et al, Proc. Natl. Acad.
~ , 59:94-101, 1968), a benign lymphoproliferative
disease. Under certain conditions, EBV infection may
facilitate the development of malignant diseases
including nasopharyngeal carcinoma (Henle, W., -~
Science 157:1064-1065, 1967 and Raab-Traub, N., et
al, Int. J. Cancer 39:25-29, 1987), Burkitt's ;
lymphoma (De-The, G., et al, Advances in Com~arative
Leukemia Research, Eds., D.S. Yohn and J.R.
,:
Blakeslee, Elsevier, NY, 1981) and the X-linked
lymphoproliferative syndrome (Harada, S., et al, J.
Immunol. 129:2532-2535, 1982). EBV is also an
opportunistic pathogen often associated with
immunocompromised patients receiving allografts or
with the acquired immune deficiency syndrome (AIDS)
(Pelicci, P.G., et al, J. Exp. Med. 164:2049-2060,
1986; Yarchoan, R., et al, J. Clin. Invest. 78:439-
447, 1986; and Montagnier, L., et al, Science 225:63-
66, 1984).
2~2~7~
EBV is unique among the human herpes
viruses in its selective tropism for B lymphocytes
and epithelial cells. This is largely due to
expression on the membrane of these cells of a
5- specific EBV receptor which has shown to be
antigenically, structurally and functionally
identical to the CR2 glycoprotein which binds the ~
C3d/C3dg complement fragments (Fingeroth, J.D., et - ;
al, Proc. Natl Acad. Sci.. USA 81:4510-4516, 1984;
Nemerow, G.R., et al, J. Virol. 55:347-351, 1985;
Frade, R., et al, Proc. Natl. Acad. Sci., USA
82:1490-1493, ls85; and Mold, C., et al, J. Immunol.
136:4140-4145, 1986). Molecular cloning of CR2 has
revealed that the entire extracellular domain of this
receptor is comprised of tandemly arranged repeating
elements which share significant sequence similarity
with a number of other complement- and noncomplement~
binding plasma and membrane proteins (Moore, M.P., et
al, Proc. Natl. Acad. Sci. USA 84:9194-9198, 1987;
and Weis, J.J., et al, J. Exp. Med. 167:1047-1066.
1988).
In addition to its dual ligand binding
function, CR2 is involved in a pathway of B cell ~ -
activation (Nemerow, G.R., et al, supra, 1985;
Chagelian & Fearnon, J. Exp. Med 163:101-115, 1986;
~ . . .
Wilson, et al, Blood 66:824-829, 1985; Frade, R., et
al, Proc. Natl. Acad. Sci.. USA 82:1490-1493, 1985;
~ . .
~- and Melchers, et al, Nature 317:264-267, 1985). This
property may play a crucial role in preparing the
30 cell for transformation by EBV.
The major envelope protein of EBV,
designated gp350/220, is a primary target of
;~ neutralizing antibody in man and sub-human primates
;~ (Thorley-Lawson & Geilinger, Proc. Natl. Acad. Sci.
35 U.S.A. 77:5307-5311, 1980). Gp350/220 is also the
ligand which mediates attachment of the virus to CR2
, ~
~ 2~23~7~
on B cells (Nemerow, G., et al, J. Virol. 61:1
1420, 1987 and Tanner, J., et al, Cell 50:203-213,
1987) and as such represents one of the few human
herpesvirus glycoproteins for which a receptor ~-~
binding function has been identified. The binding of
EBV to CR2 is specifically mediated by a 9 amino acid
residue epitope located near the N-terminus of ;
gp350/220 (Nemerow, G.R., et al., Cell 56:369-377,
1989). Binding of EBV, via gp350/220, to CR2 is
followed by endocytosis of the virus, de-envelopment ~`;
and transformation (Nemerow, G.R., et al, Virology ~;
132:186-198, 1984; Tedder, T.F., et al, J. Immunol.
137:1387-1391, 1986; and Tanner, et al, supra, 1987).
Purified CR2 (Nemerow, G.R. et al., J.
Virol. 61:1416-1420, 1987 and Nemerow, G.R. et al., ~i
J. Virol. 58:709-712, 1986) and cells transfected
with cDNA encoding CR2 (Ahern, J.M. et al., Proc.
Natl. Acad. Sci.. U.S.A. 85:9307-9311, 1988) bind
~ EBV, and gp350/220 selectively absorbs CR2 from B
`~ 20 lymphocyte membrane extracts (Fingeroth, J.D. et al.,
supra, 1984 and Tanner, J. et al., Cell 50:203-213,
1987). Molecular cloning and sequencing of cDNA
encoding CR2 has revealed that the extracellular
domain of the receptor contains 16 tandemly arranged
short repeating regions of about 60 to 75 amino
acids, followed by a hydrophobic transmembrane domain
and a short, 34 amino acid residue cytoplasmic tail
(Moore, M.D. et al., supra 1987, Weis, J.J. et al.,
J. Exp. Med. 167:1047-jlO66, 1988, and Fujisaku, A. et
al., J. Biol. Chem. 264:2il8-2125, 1989).
It has now been discovered that regions of
the extracellular portion of CR2 located at the amino
terminus of the CR2 protein participate in the
binding interaction of CR2 with EBV, gp35/220 or
C3d/C3dg complement fragments
Summary of the Invention
:' ',', '~ .;':
.
- 2~2~77~
The present invention is directed to
diagnostic and treatment methods that utilize
synthetic polypeptides that have amino acid residue
sequences which correspond to EBV-binding regions of
the extracellular domain of CR2, polypeptide
aggregates that contain amino acid residue sequences
corresponding to the EBV-binding regions of CR2 and
pharmacological compositions.
Methods for the inhibition and treatment of
EBV infections are provided by the present invention.
In these methods the aqueous medium, such as blood,
in contact with cells at risk for infection is
admixed with a CR2 polypeptide of the present
invention. The CR2 polypeptide-containing admixture
so formed is maintained for a time period sufficient
to allow any Epstein-Barr virus present in the
.. ..
suspension to bind to the polypeptide. In one ; ; ;~
embodiment a pharmacological composition containing
CR2 polypeptides is preferably injected into the ~ -
blood of a patient at risk for Epstein-Barr virus
infection. .-
A method of treatment for immune disorders
is also provided by the present invention. This `
method is directed to the administration to a patient -~
of a therapeutically effective amount of a
pharmacological composition of the present invention
to bind to either a C3dg fragment of complement or
antibodies directed against CR2.
In the present invention, a CR2 polypeptide
is defined by the presence of an amino acid residue
sequence corresponding to CR2 amino acid residues l-
256 as shown in Figure 9. In one embodiment, a
polypeptide aggregate is contemplated by the precent -~
invention that contains at least two CR2 polypeptides
of the present invention operatively linked together.
~` 2~2~7~
;~;
A pharmacological composition is pro~ided
by the present invention that contains a
physiologically tolerable carrier together with a ;
substantially purified CR2 polypeptide and/or CR2
5- polypeptide aggregate as described herein as an
active ingredient.
Methods for the detection of Epstein-Barr -
virus and/or anti-CR2 antibodies present in a fluid
sample and diagnostic kits are provided by the
present invention.
A method for the detection of Epstein-Barr ~;
virus in a fluid sample is provided in which a fluid
sample is contacted with a solid matrix that has a
CR2 polypeptide attached thereto for a time period
sufficient to allow any Epstein-Barr virus present in
the sample to specifically bind to the polypeptide.
The solid matrix is then separated from the liquid
sample, and washed (rinsed) to remove any unbound
material. The washed matrix is then contacted with a
label that is capable of indicating the presence of
any Epstein-Barr virus that is specifically bound to ;
the matrix.
~i A method of detecting the presence of
antibodies directed against CR2 in a fluid sample is
- 25 contemplated. In this method a fluid sample is
contacted with a solid matrix having a CR2
polypeptide of the present invention linked thereto
for a time period sufficient to allow the formation
of an immunoreaction product between the CR2
" ~ , .
polypeptide and any anti-CR2 antibodies present in
the sample. The solid matrix is separated from the
fluid sample and washed (rinsed) to remove any
unbound material. The washed matrix is then
contacted with a label capable of indicating the
presence of any antibodies bound to the matrix.
: ,.'; :'. :: '
~ ,, . ." ,',.
:~ .. .: :~.
2~237~
A diagnostic system in kit form is provided
that contains a package containing a polypeptide of
the present invention, preferably attached to a solid
matrix and a label for indicating the presence of
either an Epstein-Barr virus or an anti-CR2 antibody
that binds to the polypeptide.
Brief Description of the Drawinas
Figure 1 illustrates the circular dichroism ~;
spectral analysis of rCR2 performed as described in
lo Example 3.
Figure 2 illustrates the binding of rCR2
polypeptide to gp350/220 (triangles), C3dg (circles3
and ovalbumin (squares) coated onto 96 well plates
and determined as described in Example 4.
Figure 3 illustrates an ultracentrifugation
analysis of rCR2 binding to gp350/220 (Panel A) and
C3dg (Panel B) as described in Example 5. In Panel
A, [12sI]-gp350/220 was applied to linear sucrose ~-
gradients containing no rCR2 (open circles), 3 nM -~
rCR2 (closed circles), 30 nM rCR2 (closed squares),
or 100 nM rCR2 (open squares). In Panel B, [~25I]-
rCR2 was applied to linear sucrose gradients
containing either 1 uM C3dg (open circles) or no C3dg
(closed circles).
Figure 4 illustrates gp350/220 binding to
rCR2 as determined in Example 5. In Panel A, The
relative amount (r) of rCR2 bound per gp350/220 was
plotted versus the concentration of rCR2. The data
were derived from two experiments. Panel B shows a
Scatchard plot analysis of the same rCR2-gp350/220
binding data. Panel C shows a Hill plot analysis of
the same rCR2-gp350/220 data.
Figure 5 illustrates C3dg binding to rCR2
at low ionic strength as determined in Example 5.
Panel A shows the results of ultracentrifugation of
C3dg and rCR2 admixtures in 5-15% sucrose density
~: :.: ~
,: ~ ,..;:
2~2377~
gradients with 150 mM NaCl (open circles), 25 mM NaCl
(closed circles), and 10 mM NaCl (closed squares),
and the insert shows the relative amount (r) of C3dg
bound per CR2 plotted against NaCl concentration.
Panel B shows a determination of the dissociation
constant (KD) f rCR2 for C3dg by extrapolation from
lower salt concentrations. In Panel B the open
circles represent the data from Panel A and the
closed circles represent the data from a similar
study performed using 1 uM C3dg.
Figure 6 illustrates rCR2 inhibition of
binding of gp350/220 to B-lymphoblastoid cells as
determined in Example 6. Fluorescent microspheres
coated with puri~ied gp350/220 were reacted with CR2-
negative SF9 cells (Panel A) or with CR2-positive
Raji cells (Panels B, C and D). Gp350/220 coated
microspheres were reacted with 2.0 ug (Panel C) or 10
ug (Panel D) of purified rCR2 prior to incubation
with Raji cells.
Figure 7 illustrates the inhibition of EBV
infectivity of human peripheral blood mononuclear
cells (B cells) by rCR2 as determined in Example 7.
The outgrowth of transformed B cell colonies (closed
circles) and stimulation of 3H-thymidine (closed
triangles) incorporation were each measured to
indicate the extent of EBV infection. In control
experiments EBV was incubated with cells prior to the
addition of rCR2 (open circles and triangles).
Figure 8 illustrates the inhibition of EBV
,
infectivity by rCR2 for increasing amounts of EBV
present as determined in Example 7. Varying amounts ; ;
of EBV supernate were incubated with media alone
(solid lines) or with 10 ug of purified rCR2 (broken
lines). Samples were then reacted with peripheral
blood lymphocytes as described in FIGURE 7 and
2~2~
assessed for B cell transformation by colony
outgrowth and stimulation of DNA synthesis.
Figure 9 illustrates the nucleotide
sequence of a cDNA that codes for CR2, shown from
left to right and in the direction of 5' terminus to
3' terminus using the single letter nucleotide base
code. The mature, leaderless, structural gene for
CR2 begins at base 123 and ends at base 3320, with
the numbers for base residue positions indicated in
the right margin.
The amino acid residue sequence for CR2 is
indicated by the single letter code above the
nucleotide base sequence, with the numbers for each
residue position indicated in the left margin. The
reading frame is indicated by placement of the
deduced amino acid residue sequence above the
nucleotide sequence such that the single letter that
represents each amino acid is located above the
middle base in the corresponding codon.
Figure 10 illustrates in schematic form the
construction and resulting structure of the CR2
polypeptide expression vectors for producing rCR2-4,
rCR2 and rCR2-10, where the vectors are labeled as
pAC373 CR2(1-4), pAC373 CR2(1-16) and pAC373 CR2(1-
10~, respectively. The expression vectors
manipulated and produced by the construction process
are indicated in the figure by the circles. The
construction proceeds by a series of steps as
indicated by the arrows connecting the circles and
the intermediate nucIeic acid fragments in the
figure, and the steps are described in detail in
Example 1. Landmark and utilized restriction enzyme
recognition sites are indicated on the circles and
fragments by words or by labeled lines intersecting
;~ 35 the circles. Individual genes and their direction of ;~
~ 2~7~3
g ., ;
transcription are indicated by the arrows in the
circles.
Detailed Description of the Invention ;~ ~-
A. CR2 Poly~eptides
5- Substantially purified polypeptides that
correspond in amino acid residue sequence to regions
of the extracellular domain of CR2 have been
synthesized in the present invention, and are
designated herein as CR2 polypeptides.
A CR2 polypeptide of the present invention
includes an amino acid residue sequence that
corresponds to the amino acid residue sequence shown
in Figure 9 from residue 1 to residue 70.
In preferred embodiments a CR2 polypeptide
includes an amino acid residue sequence that
corresponds to the amino acid residue sequence shown
in Figure 9 from residue 1 to residue 133, from
residue 1 to residue 256, from residue 1 to residue
632, or from residue 1 to residue 1005.
Exemplary of two of the preferred
embodiments are the CR2 polypeptides rCR2 and rCR2-4
that are described in Example 1.
In one embodiment, the CR2 polypeptide can ~-
be produced as a fusion protein wherein one portion
of the polypeptide has a sequence that corresponds to
CR2 and another portion corresponds to the amino acid
sequence of a protein other than CR2. Representative ';
of this embodiment is the CR2 polypeptide rCR2-10
which includes as its carboxy terminus a portion of ~1
the polyhedrin gene as described in Example 1.
Compositions and diagnostic kits containing
the CR2 polypeptides of the present invention are
;~ provided, together with methods for the detection and
treatment of EBV infections and immune disorders.
All amino acid residues identified herein
are in the natural L-conformation. In keeping with
~2~7~
standard polypeptide nomenclature, J. Biol. Chem.,
243:3557-59 (1969), abbreYiations for amino acid - -
residues are as shown in the following Table of -~
Correspondence:
TABLE OF CORRESPONDENCE
SYMBOL AMINO ACID
1_Letter 3-Letter
Y Tyr L-tyrosine
G Gly glycine
F Phe L-phenylalanine
M Met L-methionine
A Ala L-alanine
S Ser L-serine
I Ile L-isoleucine
L Leu L-leucine
T Thr L-threonine
V Val L-valine
P Pro L-proline
K Lys L-lysine
H His L-histidine
Q Gln L-glutamine
E Glu L-glutamic acid
W TrP L-tryptophan
~. Arg L-arginine
D Asp L-Aspartic Acid
N Asn L-asparagine
C Cys L-cysteine
CR2 polypeptides are preferably produced by `
recombinant expression in the present invention. For
example, a baculovirus expression system is used to
generate milligram amounts of CR2 polypeptides. In
the baculovirus expression system a plasmid
containing a cDNA that encodes a portion of the
~; extracellular domain of CR2 was prepared and utilized ~ ~
.
~` 2 ~ 7 ~
........ : ~'';
11 ., ` . .,~ .
to produce recombinant CR2 polypeptides as described
in Example 1.
In the baculovirus expression system, a -
recombinant DNA plasmid, containing a cDNA that
5- encodes CR2, is truncated to encode a CR2 polypeptide
that comprises a region of the extracellular domain
of CR2. The CR2 polypeptide construct was then
inserted downstream of an appropriate promoter in a
transfer vector and integrated into a baculovirus.
The recombinant baculovirus is used to infect host
insect cells which then secrete recombinantly
produced CR2 polypeptides. Examples of the
baculovirus-expression system can be found in Example
1. :: :
As used herein, the term "CR2 polypeptide ~
aggregate" refers to a polymer containing at least ;
two CR2 polypeptides corresponding to regions of the
extracellular domain of CR2 operatively linked either
in tandem or to a polypeptide carrier.
As used herein, in reference to polypeptide ;~
aggregates, the term "operatively linked" refers to ~ : -
the covalent attachment of a CR2 polypeptide to
another CR2 polypeptide or to a peptide, polypeptide
or protein carrier. ;
The CR2 polypeptide aggregates of the present
invention can contain a plurality of the same or
different CR2 polypeptides, wherein the CR2
polypeptides are bound to each other or to a peptide,
polypeptide or protein carrier. ~ `
B. Pharmacological Compositions ;
Pharmacological compositions of the present
invention contain a physiologically tolerable carrier
together with a substantially purified CR2
polypeptide, or CR2 polypeptide aggregate, as
described above, dissolved or dispersed therein as an
active ingredient. In a preferred embodiment, the
`-
; ~'~ ''`~'
77~ ~
pharmacological composition is not immunogenic when
administered to a mammal or human patient.
As used herein, the terms "pharmaceutically
acceptable", "physiologically tolerable" and
grammatical variations thereof, as they refer to
compositions, carriers, diluents and reagents, are
used interchangeably and represent that the materials
are capable of administration to or upon a mammal
without the production of undesirable physiological
effects such as nausea, dizziness, gastric upset and
the like.
The preparation of a pharmacological
composition that contains active ingredients
dissolved or dispersed therein is well understood in
the art. Typically such compositions are prepared as
injectables either as liquid solutions or
suspensions, however, solid forms suitable for
solution, or suspensions, in liquid prior to use can
also be prepared.
The active ingredient can be mixed with -~
excipients which are pharmaceutically acceptable and
compatible with the active ingredient. Suitable
excipients are, for example, water, saline, dextrose,
glycerol, ethanol or the like and combinations
thereof. In addition, if desired, the composition
can contain minor amounts of auxiliary substances
such as wetting or emulsifying agents, pH buffering
agents and the like which enhance the effectiveness
of the active ingredient.
The pharmacological composition of the
present invention can include pharmaceutically
acceptable salts of the components therein.
Pharmaceutically acceptable salts include the acid
addition salts (formed with the free amino groups of
the polypeptide) that are formed with inorganic acids
such as, for example, hydrochloric or phosphoric
~`` 2~2~7~
13
acids, or such organic acids as acetic, tartaric,
mandelic and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic
bases such as, for example, sodium, potassium,
5- ammonium, calcium or ferric hydroxides, and such
organic bases as isopropylamine, trimethylamine, 2-
ethylamino ethanol, histidine, procaine and the like.
Physiologically tolerable carriers are well
known in the art. Exemplary of liquid carriers are
sterile aqueous solutions that contain no materials
in addition to the active ingredients and water, or
contain a buffer such as sodium phosphate at ;
physiological pH value, physiological saline or both,
such as phosphate-buffered saline. Still further,
aqueous carriers can contain more than one buffer
salt, as well as salts such as sodium and potassium ~ - -
chlorides, dextrose and other solutes.
Liquid compositions can also contain liquid
phases in addition to and to the exclusion of water.
Exemplary of such additional liquid phases are ;
glycerin, vegetable oils such as cottonseed oil, and
water-oil emulsions. ;
In a method of treatment, the pharmacological -
compositions are conventionally administered
intravenously, as by injection of a unit dose, for
example.
The term "unit dose" and its grammatical
equivalents, as used herein, refers to physically
discrete units suitable as unitary dosages for human
patients and other warm-blooded animals, each unit ;
containing a predetermined effective and potentiating
amount of active material calculated to produce the
desired therapeutic effect in association with the
required physiologically tolerable carrier.
The compositions are administered in a manner
compatible with the dosage formulation, and in a
2~2~
14
therapeutically effective amount. The quantity to be
administered depends on the subject to be treated,
and the capacity of the subject to utilize the active
ingredient. Precise amounts of active ingredients
required to be administered depend on the judgment of
the practitioner and are peculiar to each individual,
and usually are in a range of about 100 ug to about
100 mg of CR2 polypeptide per ml of patient blood.
Alternatively, continuous intravenous infusion
sufficient to maintain therapeutically effective
concentrations in the blood are contemplated.
C. Methods of Inhibition and Treatment of
Epstein-Barr Virus Infection
Epstein-sarr virus infection of mammalian
cells in an aqueous medium, such as blood, is
inhibited by methods of the present invention. ~ -
An aqueous medium containing mammalian cells,
such as B lymphocytes, is admixed with a
therapeutically effective amount of a pharmacological
composition of the present invention and maintained
for a time period sufficient to allow the CR2 ;~
polypeptides of the composition to specifically bind ~ -
any Epstein-Barr virus present in the aqueous medium.
When the admixture contains a concentration of
Epstein-Barr virus that is sufficient to infect the
mammalian cells under normal physiological conditions
when no pharmacological intervention or treatment is
undertaken, the therapeutically effective amount of
the CR2 polypeptide-containing composition utilized
in this method is that which produces a concentration
of CR2 polypeptide in the aqueous medium sufficient
to bind essentially all of the EBV present, and
usually is at a concentration of about 100 ug to
about 1 mg per ml.
When the method of treatment of the present
invention is utilized to inhibit EBV infection in
~i, ~ ~1,'!' , . ,
~ `77 ~
vivo the therapeutically effective amount of the
pharmacological composition administered is that
which produces a blood concentration of CR2
polypeptides sufficient to specifically bind the
circulating EBV present. Such a blood concentration
is usually about 100 ug to about 1 mg of CR2
polypeptide per ml. It is contemplated that multiple
administrations of the pharmacological composition of -
this invention over an appropriate time period and at
a dosage level determined by a medical practitioner ;
for the patient will be undertaken for the inhibition
of EBV infection in a human patient.
As used herein, the terms "specifically
bind", and "specifically attach", and grammatical ~;
forms thereof are used interchangeably and refer to ;~
non-random ligand binding, such as that which occurs
between gp350/220 and CR2. -~
In the method of treatment of the present
invention, a pharmacological composition, as
described above, is administered to a patient in any
manner that will efficaciously inhibit the infection ~
of mammalian cells, such as B lymphocytes, by EBV. ~ ~ -
Preferably, the composition is administered by either
intravenous injection of a unit dosage or continuous
intravenous infusion of a predetermined concentration
of CR2 polypeptides or CR2 polypeptide aggregates to
a patient.
D. Methods of Treatment
for Immune Disorders
! ' ~ ~ ' . i ' , .
Methods for the treatment of immunological
conditions in a patient are contemplated by the
present invention.
In one embodiment, a method for treating an
immune disorder characterized by the presence in a
patient of circulating antibodies directed against
CR2 present on B lymphocytes is provided. In this
~ : :,
77~
16
method CR2 polypeptide or CR2 polypeptide aggregate
is administered to the patient in a therapeutically
effective amount to effectively bind to the
antibodies and thus inhibit or prevent the
immunoreaction of the antibodies with B lymphocytes.
It is preferred that a pharmacological
composition administered in the present invention is
not immunogenic.
In another embodiment, a method for treating
an immunological disorder or condition in a patient, ;~
in which an immunoreaction product containing a C3d
or C3dg fragment of complement is present, is
provided. In this method, a patient is administered i -~
a therapeutically effective amount of a CR2 ~ -
polypeptide or polypeptide aggregate of the present
invention to bind to the c3d or C3dg fragment of ~;
complement present in the immunoreaction product, and
thus facilitate the alleviation of the symptoms of
the disorder. ;~
E. Diagnostic Systems and Methods
A diagnostic system in kit form of the
present invention includes, in an amount sufficient ;
for at least one assay, a CR2 polypeptide and/or CR2
polypeptide aggregate and/or composition of the
present invention, as a separately packaged reagent.
Instructions for use of the packaged reagent are also
typically included.
"Instructions for use" typically include a
tangible expression describing the reagent
concentration or at least one assay method parameter
such as the relative amounts of reagent and sample to
be admixed, maintenance time periods for
r~agent/sample admixtures, temperature, buffer
conditions and the like.
In one embodiment, a diagnostic system for
assaying for the presence of or to quantitate anti-
~ .
77~
17
CR2 antibodies in a sample, such as blood, plasma or
serum, comprises a package containing at least one
CR2 polypeptide of this invention. In another
embodiment, a diagnostic system of the present ~ ;;
5- invention for ~ssaying for the presence or amount of
EBV comprises a package containing a CR2 polypeptide,
CR2 polypeptide aggregate or pharmacological
composition of this invention.
In preferred embodiments, a diagnostic system
of the present invention further includes a label or
indicating means capable of signaling the formation
of a specifically bound complex containing a CR2
polypeptide or polypeptide aggregate of the present ; -~
invention.
As used herein, the terms "label" and
"indicating means" in their various grammatical forms
refer to single atoms and molecules that are either ~ -
directly or indirectly involved in the production of
a detectable signal to indicate the presence of a
complex. Illustrative labels include radionuclides,
such as 125I, and fluorescent radicals such as
fluorescein isothiocyanate and the like. --~
The linking of labels, i.e., labeling of,
polypeptides and proteins is well known in the art.
The techniques of protein conjugation or coupling
through activated functional groups are particularly
applicable. See, for example, Aurameas, et al.
Scand. J. Immunol., Vol. 8 Suppl. 7:7-23 (1978),
~odwell, et al, Biotech., 3:889-894 (1984) and U.S.
Pat. No. 4,493,795 to Nestor et al., which are
incorporated herein by reference.
The diagnostic systems can also include,
preferably as a separate package, a specific binding
agent. A "specific binding agent" is a molecular
entity capable of selectively binding a reagent
species of the present invention but is not itself a
~ 2~23~7~
protein expression product, polypeptide, or
polypeptide aggregate of the present invention.
Exemplary specific binding agents are antibody
molecules, complement proteins or fragments thereof, ~ ~
5- protein A and the like. For detecting EBV, the ~ ~ -
specific binding agent can bind the CR2 polypeptide
of this invention when it is present as part of a
complex. When detecting patient anti-CR2 antibodies, -~
anti-human Fc antibodies are conveniently used. In ~ ;
preferred embodiments the specific binding agent is ;
labeled. However, when the diagnostic system ~ ~
includes a specific binding agent that is not -
labeled, the agent is typically used as an amplifying
means or reagent. In these embodiments, the labeled
specific binding agent is capable of specifically
binding the amplifying means when the amplifying
means is bound to a reagent species-containing
complex. - `
The diagnostic kits of the present invention
can be used in an "ELISA" format to detect the
presence or quantity of EBV or anti-CR2 antibodies in
a body fluid sample such as blood serum, plasma or
urine. "ELISA" refers to an enzyme-linked
immunosorbent assay that employs an antibody or
antigen bound to a solid phase and an enzyme-antigen
or enzyme-antibody conjugate to detect and quantify
the amount of an antigen or antibody present in a
sample. A description of the ELISA technique is
found in Chapter 22 of the 4th Edition of Basic and
Clinical Immunology by D.P. Sites, et al, published
by Lange Medical Publications of Los Altos, CA in -
1982, and in U.S. Patents No. 3,654,090; No.
3,850,752; and No. 4,016,043, which are all
incorporated herein by reference. Thus, in preferred
embodiments, the CR2 polypeptide, or polypeptide
aggregate, of the present invention can be affixed to
-~ 2~2~77~
19 ,"~ ' ~
a solid matrix to form a solid support that is ~ -
separately packaged in the subject diagnostic
systems. ;
The reagent is typically affixed to the solid
5- matrix by adsorption from an aqueous medium although
other modes of affixation, well known to those
skilled in the art can be used. -
Useful solid matrices are well known in the -~
art. Such materials include the cross-linked dextran
available under the trademark SEPHADEX from Pharmacia
Fine Chemicals (Piscataway, NJ); agarose; beads of
polystyrene beads about 1 micron to about 5
millimeters in diameter available from Abbott ;~
Laboratories of North Chicago, IL; polyvinyl
chloride, polystyrene, cross-linked polyacrylamide,
nitrocellulose- or nylon-based webs such as sheets,
strips or paddles; or tubes, plates or the wells of a
microliter plate such as those made from polystyrene
or polyvinylchloride.
~; 20 The reagent species, labeled specific binding
agent or amplifying reagent of any diagnostic system
described herein can be provided in solution, as a
liquid diRpersion or as a substantially dry power,
e.g., in lyophilized form. Where the indicating
means is an enzyme, the enzyme's substrate can also
be provided in a separate package of a system. A
solid support such as the before-described microtiter
plate and one or more buffers can also be included as
separately packaged elements in this diagnostic assay
system.
The packages discussed herein in relation to
diagnostic systems are those customarily utilized in
diagnostic systems. Such packages include glass and
plastic ~e.g., polyethylene, polypropylene and
polycarbonate) bottles, vials, plastic and plastic-
foil laminated envelopes and the like.
::
~ 2~377~
, :~
-~
The present invention contemplates any method
that results in detecting EBV or anti-C~2 antibodies
in a body fluid sample using CR2 polypeptides or
polypeptide aggregates of this invention. Thus, ;
while exemplary methods are described herein, the
invention is not so limited.
The presence of anti-CR2 antibodies in an
antibody-containing bodily fluid is indicative of an
autoimmune disorder in the host animal.
To detect either EBV infection or the
presence of anti-CR2 antibodies in a patient, a
bodily fluid such as blood, plasma or serum from the
patient is contacted under biological assay
conditions with a CR2 polypeptide or aggregate
thereof for a period of time sufficient to form a
polypeptide containing complex. The presence of the
complex is indicative of infection by EBV or of a CR2
autoimmune disorder, respectively. The complex can
be detected as described herein.
Biological assay conditions are those that
maintain the biological activity of the CR2
polypeptide molecules of this invention and the EBV
or anti-CR2 antibodies sought to be assayed. Those
conditions include a temperature range of about 4 C
to about 45 C, preferably about 37 C, a pH value
range of about 5 to about 9, preferably about 7, and
an ionic strength varying from that of distilled
water to that of about one molar sodium chloride,
preferably about that of physiological saline.
Methods for optimizing such conditions are well known
in the art.
F. Expression Constructs
In living organisms, the amino acid residue
sequence of a protein or polypeptide is directly
related via the genetic code to the deoxyribonucleic
acid (DNA~ sequence of the structural gene that codes
~ ' '~, '~''',',''
::
~ 2~23~7~
for the protein and the mRNA from which it is
translated. Thus, a nucleotide sequence can be
defined in terms of the amino acid residue sequence,
i.e., protein or polypeptide, for which it codes.
An important and well known feature of the
genetic code is its redundancy. That is, for most of
the amino acids used to make proteins, more than one
coding nucleotide triplet (codon) can code for or
designate a particular amino acid residue.
Therefore, a number of different nucleotide sequences
can code for a particular amino acid residue
sequence. Such nucleotide sequences are considered
functionally equivalent since they can result in the
production of the same amino acid residue sequence in
all organisms. Occasionally, a methylated variant of -
a purine or pyrimidine may be incorporated into a ; ~;
given nucleotide sequence. However, such -
methylations do not affect the coding relationship in
any way.
A nucleotide sequence of the present
invention encodes a CR2 polypeptide sequence of this
` invention. Representative nucleotide sequences that
encode a CR2 polypeptide of the present invention can
include nucleotide sequences that correspond to the
nucleotide base sequence shown in Figure 9. Thus, a
preferred CR2 polypeptide-encoding nucleotide
se~uence includes a sequence that corresponds to the
sequence shown in Figure 9 from nucleotide base 123
to base 332, from base 123 to base 521, from base 123
to base 2018, or from base 123 to base 3137.
A DNA segment of the present invention that
encodes an extracellular CR2 amino acid residue
sequence and preferably an amino terminal sequence,
can easily be synthesized by chemical techniques, for
example, the phosphotriester method of Matteucci, et
al, J. Am. Chem. Soc., 103:3185 (1981). Of course,
,-:
~37~ :
22
by chemically synthesizing the coding sequence, any
desired modifications can be made simply by
substituting the appropriate bases for those encoding
the native amino acid residue sequence.
5- DNA segments consisting essentially of
structural genes encoding the CR2 extracellular
domain can be obtained by digesting B lymphocytes.
A nucleic acid construction of the present
invention can be produced by operatively linking an
expression vector to the nucleic acid sequence of the
present invention, preferably in a baculovirus such
as the Autoarapha californica nuclear polyhedrosis
virus.
As used herein, the term "operatively
linked", in reference to cDNA integration, describes
that the nucleotide sequence is joined to the vector
so that the sequence is under the transcriptional and
translation control of the expression vector and can ;
he expressed in a suitable host cell.
As used herein, the term "vector" refers to a
DNA molecule capable of autonomous replication in a ;
cell and to which another nucleotide sequence can be
operatively linked so as to bring about replication
of the attached segment. Vectors capable of
directing the expression of genes encoding CR2 amino
acid residue sequences are referred to herein as
"expression vectors". Thus, a recombinant DNA
molecule (rDNA) is a hybrid DNA molecule comprising
at least two nucleotide sequences not normally found
together in nature.
The choice of vector to which a DNA segment
of the present invention is operatively linked
depends directly, as is well known in the art, on the
functional properties desired, e.g., protein
expression, and the host cell to be transformed, ~
- ~,
2 &~ 2 ~ r~
23
these being limitations inherent in the art of
constructing recombinant DNA molecules.
Expression vectors which are relatively
harmless in a host animal, including primates, are
well known and can be used to induce an immune
response to an expressed foreign polypeptide included
in the vector. Typically, such vectors are provided
containing convenient restriction sites for insertion
of the desired DNA segment. Typical of such vectors
are vaccinia virus vectors and baculovirus vectors.
A variety of methods have been developed to ` ~ ~
operatively link DNA to vectors via complementary -~ '
cohesive termini. For instance, complementary ~i-
homopolymer tracts can be added to the DNA segment to
be inserted and to the vector DNA. The vector and
DNA segment are then joined by hydrogen bonding
between the complementary homopolymeric tails to form
recombinant DNA molecules.
Synthetic linkers containing one or more
restristion sites provide an alternative method of
joining the DNA segment to vectors. The DNA segment,
generated by endonuclease restriction digestion is
treated with bacteriophage T4 DNA polymerase or E.
coli DNA polymerase I, enzymes that remove
pro~ruding, 3', single-stranded termini with their
3'-5' exonucleolytic activities and fill in recessed
3' ends with their polymerizing activities. The
combination of these activities therefore generates
blunt-ended DNA segments. The blunt-ended segments
are then incubated with a large molar excess of
linker molecules in the presence of an enzyme that is
able to catalyze the ligation of blunt-ended DNA
molecules, such as bacteriophage T4 DNA ligase.
Thus, the products of the reaction are DNA segments
carryin~ polymeric linker sequences at their ends.
These DNA segments are then cleaved with the
, ~ ~
. ,,
:; , - -:::
~ r~ 7 ~
~.,
24
appropriate restriction enzyme and ligated to an
expression vector that has been cleaved with an
enzyme that produces termini compatible with those of
the DNA segment.
Synthetic linkers containing a variety of
restriction endonuclease sites are commercially
available from a number of sources including
International Biotechnologies, Inc., New Haven, CT.
Also contemplated by the present invention
are RNA equivalents of the above described
recombinant DNA molecules. The CR2 polypeptides are
thus readily synthesized and expressed by an
appropriate host cell such as the S~odoptera
frugiperda (SF9) insect cells.
Exam~les
The following examples are given for
illustrative purposes only and do not in any way
limit the scope of the invention.
1. Preparation of CR2 and CR2 Polv~eptl des
a. Isolation and Purification of C~R2
CR2 was isolated from detergent lysates
of Raji B lymphoblastoid cells by immunoaffinity
chromatography.
Raji cells (2 x 101) were washed twice in
cold (about 4 C) phosphate buffered saline (PBS, pH
7.4) and suspended in 400 ml of 10 mM phosphate
buffer (pH 8.0) containing 3.3% Bri; 96 (Sigma
Chemical Co., St. Louis, MO), 3 mM sodium azide, 50 ;
mM iodoacetamide, 5 mM phenylmethylsulfonyl fluoride,
5 mMiEDTA and 0.2 trypsin inhibitory units of
Aprotinin/ml (Sigma Chemical Co.). ` ;
The suspension was thoroughly mixed at 4 C
for 30 min followed by high speed centrifugation to
remove the nuclei and cell debris.
Sodium deoxycholate (DOC) in 10 mM Tris
hydrochloride (pH 8.0) was added to the supernatant
: ~ ' ." -
.
~237~
to obtain a final concentration of 1%. This mixture
was stirred at 4 c for 60 minutes, and then
maintained, with constant mixing, for 18 hours with 2
ml of packed fixed Staphvlococcus aureus (Pansorbin,
Calbiochem-Behring, La Jolla, CA). The mixture was -~
centrifuged and the supernatant was saved to be ; ;~
applied to an immunomatrix column having anti-CR2 ~ ~
monoclonal antibody HB-5 attached (HB-5 immunomatrix -
column).
The immunomatrix was batch-washed four times
with 200 ml of 10 mM Tris hydrochloride (pH 8.0)
containing 0.5% DOC and four times with 200 ml of 10
mM Tris hydrochloride (pH 8.0) containing 150 mM NaCl
and 0.5% DOC. The immunomatrix was poured into a
column.
The CR2-containing supernatant was applied to
the HB-5 immunomatrix column and eluted with 7 to 10
ml of 50 mM diethylamine (pH 11.5) (Kodak, Rochester,
NY) containing 0.5% DOC.
The pH of the CR2-containing eluate was
adjusted to pH 8.0 by the addition of solid glycine.
The protein was rechromatographed on the immunomatrix
column to remove minor contaminants. ~-~
Purification of CR2 was monitored by sodium
dodecylsulfate-polyacrylamide gel electrophoresis
under reducing conditions, and the gels were silver
stained.
The protein concentration of CR2 was
estimated by comparing densitometric scans of CR2
: ~ !
with known amounts of standard markers (Bio-Rad
Laboratories, Richmond, CA) run in adjacent lanes on
the gels.
An ELISA was used to monitor purified CR2.
Various amounts of the purified CR2 (0.25 to 4 ng) in
100 uL aliquots in 0.1 M bicarbonate buffer (pH 9.5)
were coated onto 96 well microtiter plates (Immulon
~77~
II, Dynatech Laboratories Inc., Alexandria, VA) for
18 hours at 4 C. The wells were then blocked with
0.5% nonfat dry milk in PBS (pH 7.4) containing 0.05
antifoam A (Sigma Chemical Co.) as blotto buffer for
5- 1 ho-~r at 37 c. Anti-CR2 antibodies (1 ug) in loo ul
of blotto buffer were added to the wells and
maintained for one hour at 22 c.
The wells were then washed three times with
blotto buffer, 100 ul of a 1:500 dilution of
biotinlyated anti-mouse immunoglobulin (Vector
Laboratories, Burlingame, CA) in blotto buffer was
added, and the plates incubated at 22 C for 1 hour.
The wells were again washed and then incubated for 30 ;
minutes at 22 C with 100 ul of a 1:250 dilution of
avidin-glucose oxidase (Vector Laboratories). The
plates were washed and the reaction was developed by
the addition of chromogenic substrate (140 ug/ml
2,2'-azino-bis-3-ethylbenzthiazoline sulfonate, 7 -~
ug/ml peroxidase, 19 mg/ml glucose in 0.1 M phosphage
buffer (pH 6.0)). The reaction was read at 405 nm ~ -
wavelength in a Titertak Multiskan MC ELISA plate
reader (Flow Laboratories, Inc., McLean, VA). ;~
b. Isolation of cDNA for `
Expression of CR2 Polypeptides
A cDNA clone of about 4.2 kilobases
(Kb), repres~nting the entire CR2 coding sequence was
prepared as described by Moore et al., Proc. Natl.
Acad. Sci. USA, 84:9194-98, ~1987). A cDNA library
was first constructed in lambda gtll (Stratagene, San
Diego, CA) using mRNA isolated from Raji B
lymphoblastoid cells. Complementary DNA was
synthesized with a commercially available synthesis
kit (cDNA synthesis system, Amersham, Arlington
Heights, IL). Packaging of recombinant cDNA clones
with phage coat proteins was done as recommended by
, . . . .
.
'.. ' '~, ' ~,
,' " ' :; '
~ J
~237~
27 :
: , ~,,.
the manufacturer (Gigapack, Stratagene). The library
containisi 2.5 x lo6 recombinants.
Candidate CR2 cDNA clones were isolated from
85 mm plate lysates using affinity chromatography
with anti-lambda phage antibody (LambdaSorb, Promega
Biotec, Madison, WI). Large scale preparation of
lambda phage was carried out, digested with EcoR I
and the cDNA inserts were isolated by preparative gel
electrophoresis.
Approximately 5 x 105 recombinant lambda phage
were screened with a CR2 oligonucleotide probe in
order to isolate a clone containing a 1.2 kilobase
insert encoding CR2. The CR2 cDNA insert isolated
from the clone was nick-translated and used to
reprobe the Raji library to obtain additional cDNA
inserts encoding CR2.
The isolated CR2 inserts were ligated into
the EcoRI site of plasmid Bluescript (Stratagene),
designated plasmid Bluescript/CR2.
c. Construction of CR2/Baculovirus
Expression Vectors and Production
of CR2 Polv~e~tides
The preparation of CR2 polypeptide
expression vectors is described hereinbelow and is
` 25 represented æchematically in Figure 10.
;~ ~5$~: CR2 polypeptide rCR2 has an amino acid
residue sequence that corresponds to the sequence ~ ;
shown in Figure 9 from residue 1 to residue 1005.
rCR2 is expressed by t;he baculovirus expression
vector pAC373 CR2(1-16) shown in the middle portion ~
;~ of Figure 10. For the preparation of pAC373 CR2(1- ~;
16) plasmid Bluescript/CR2 (pBS/CR2) was digested
with Bgl 1 and Pst 1 to release the cDNA insert
encoding the entire extracellular domain of CR2.
Following subcloning of the CR2 fragment into pBS, a
synthetic oligonucleotide BamH I linker (5'-
:
: : ~
2 ~ 7 ~
28
dCGGATCCG-3'; Pharmacia/LKB Biotechnology, La Jolla,
California) was ligated to the 5' end of the CR2
insert that had been previously digested with B~ll
and blunt-ended with T4 DNA polymerase.
The construct was further modified by two
successive ~ligonucleotide-based site-directed
mutagenesis reactions according to the method of
Kunkel, Proc. Natl. Acad. Sci., USA, 82:488-492
(1985). The first mutagenesis step involved ablation
of the BamH I site at nucleotide position No. 2017
(using the nucleotide base pair numbering system
shown in Figure 9) by substitution of a single
nucleotide (GGATCC to GGACCC) that preserved the
: . :
amino acid coding sequence of CR2. Verification of
the mutation was carried out by sequence analysis and
restriction mapping.
The second mutagenesis step was then carried ;~
out by incorporating a termination codon and BamiH I
site (TGAGGATCC) following nucleotide position No
3137, immediately preceding the transmembrane domain
of CR2.
Subsequently, the truncated CR2 fragment
encoding the extracellular domain of CR2 was inserted ;~
into the BamH I cloning site of pAC373. Recombinant
plasmids containing a single copy of truncated CR2 in
the correct orientation was identified by restriction
mapping. Transfer of the rCR2 se~uence from the
plasmid to the Autoqra~ha californica nuclear
polyhedrosis virus (AcNPV) genome was carried out by
cotransfection of Spodoptera fruiqiperda (SF9) cells
with 10 ug of rCR2 plasmid and 1 ug of AcNPV DNA
using calcium phosphate. Recombinant viruses were ;;
~elected and characterized. ;
The transfected SF9 cells were cultured and
the cell supernatant was subjected to
immunoprecipitation with anti-CR2 monoclonal
' -'~' .: ':
. , .
:
F,? ..
- 2~2~77~
29
antibodies (HB5) attached to a resin. The resin was
washed and the rCR2 was eluted with 50 mM
diethylamine (pH 11.5) containing 0.5~ sodium
deoxycholate, a mild chaotropic agent, to produce
purified rCR2 which was greater than 90% homogenous.
rCR2-4: CR2 polypeptide rCR2-4 has an amino
acid residue sequence that corresponds to the
sequence shown in Figure 9 from residue 1 to residue
756. rCR2-4 is expressed by the baculovirus
expression vector pAC373 CR2(1-4) shown in the left
portion of Figure 10. The preparation of pAC373
CR2(1-4) differed from the preparation of pAC373
CR2(1-16) in that the first mutagenesis was omitted,
and the second mutagenesis step was conducted so as
to incorporate a termination codon and BamH I site
(TGAGGATCC) following nucleotide position No. 887. ~ ~
Subsequently the mutagenized and truncated ~ ~-
CR2 polypeptide encoding fragment was inserted into
the BamH I cloning site of pAC373 as shown for pAC373
;~ 20 CR2(1-16), the vector was manipulated as before to
produce a recombinant virus. The virus was
introduced into SF9 cells as before, and the
expressed rCR2-4 protein was harvested from the ~ ~;
transfected SF9 cell culture to and purified as
~ 25 before to produce purified rCR2-4 which was greater
;~ than 90~ homogenous.
rCR2-10: CR2 polypeptide rCR2-lO is a fusion
protein that includes at its carboxy terminus a ~
porkion of the polyhedrin protein, and has at its ~;
amino terminus an amino acid residue sequence that
corresponds to the sequence shown in Figure 9 from ~
residue 1 to residue 632. rCR2-10 is expressed by ~ ~-
the baculovirus expression vector pAC373 CR2(1-10)
shown in the right portion of Figure 10. The
~ 35 preparation of pAC373 CR2(1-16) in that no
}~ mutagenesis was conducted. Rather, the BamH I linker
~' ~
::
: ~
~23~7~-
modified plasmid pBS/CR2 was digested with BamH I and
inserted into the BamH I cloning site of pAC373 that -
contained the polyhedrin structural gene and
promoter. Thus, the polyhedrin gene was fused to the
carboxyl terminus of the CR2 polypeptide coding gene.
The resulting vector pAC373 CR2(1-10) was
manipulated as before to produce recombinant virus.
The virus was transfected into SF9 cells and rCR2-10
protein was expressed therefrom, harvested and
purified as before. ~;~
2. Preparation of qp350/220 and C3da
The Epstein-Barr virus membrane glycoprotein -~
gp350/220 was isolated from GH3 19 rat pituitary
tumor cells transfected with gp350/220 cDNA as
described by Whang et al., J. Virol. 61:1795-1807
(1987) and purified by immunoaffinity chromatography
using anti-gp350/220 monoclonal antibodies.
The C3dg fragment of the third component of -~
complement was purified from Mg+2EGTA-activated aged
human serum as described by Vik et al., J. Immunol.,
138:254-258 (1987). ~ -
3. Physical Characterization of rCR2
Soluble recombinant CR2 polypeptide (rCR2)
corresponding to the extracellular region of CR2 was ;~ ;~
produced and purified as described in Example 1. The
molecular weight of rCR2 was determined by SDS gel
electrophoresis to be 125 kDa. This value is higher
than the molecular weight of 110 kDa predicted from
the cDNA sequence for rCR2. The difference is
. .
presumed to be due to post-translational
modification, such as glycosylation, of the soluble
rCR2.
Soluble rCR2 (2 milliliters (ml)) was applied
to a 2.6 x 95 cm column of Sephacryl S-300
; 35 (Pharmacia, Piscataway, NJ) that had been
equilibrated with 300 mM NaCl, 20 mM imidazole-HCl pH
2377~,
7.3 at 4 c. Fractions t2.5 ml each) were collected
at a flow rate of 17 ml/hour. [125I]-labelled
proteir.s were detected by gamma counting in a
Micromedic Systems 4/600 gamma counter, and
unlabelled protein in the fractions were detected by
absorbance at 280 nm. The Sephacryl S-300 column was
calibrated with the following standards, where the
diffusion coefficient (D2ow) is listed in parenthesis:
thyroglobulin (2.54 x 107cm2/s), Factor H
(2.54 x 10~7cm2/s), C3 (4.53 x 10~7cm2/s), ;~
bovine serum albumin (5.94 x 10~7cm2/s), and
myoglobulin (1.13 xlO~6cm2/s). The partition
coefficients were determined as Kd = (V, - VO)/(Vt -
VO)I where V~ is the elution volume of the sample, VO
is the void volume for Blue Dextan and Vt is the -
included volume plus the void volume.
The diffusion coefficient (D2ow) of rCR2 was
determined to be 3.1 x 10~7cm2/s. ;~
The Stokes radius for rCR2 was calculated
~ 20 from the D2o~w to be 69.3 Angstrom, and the frictional
`;~ coefficient (f/fo) was determined as 2.1.
~ Solukle rCR2 (100 ul) was centrifuged through
; a 5-16% linear sucrose gradient (4 ml) prepared in
phosphate-buffered saline (PBS) at 45,000 rpm in an
SW-60 rotor (Beckman Instruments, Palo Alto, CA) for ;~-
18-24 hours at 4 C. Gradient fractions (160 ul) were ;
collected and the sedimentation constant (s20~w) for
rCR2 was determined to be 4.5S.
The values of ftfo and S20~w obtained for rCR2 ~
are donsistent with it being an extended molecule. ` `
Circular dichroism (CD) spectral analysis was
carried out in an Aviv CD spectropolarimeter model 61 ;~
DS, standardized with d-10-camphosulfonic acid. A
sample of rCR2 (0.12 mg/ml) in 20 mM Tris HCl, pH 8.2
was scanned for CD between 187 and 260 nm in a 0.1 cm
path length cuvette. The CD spectrum for rCR2
:.
2~2377~
32
(Figure 1) is unlike that of most globular proteins
because it is dominated by a maximum positive ;
ellipticity at 229 nm and a maximum negative
ellipticity between 200 and 205 nm. The CD spectrum
for rCR2 also indicates the absence of alpha-helix ~; ;
secondary structure. This spectrum is similar to
that for the complement regulatory protein, factor H,
that also contains repeating regions.
Transmission electron microscopic analysis of
negatively stained samples of rCR2 were prepared by
the "pleated sheet" technique using 2~ uranyl acetate
according to the method described in Smith et al., J.
Ultrastructure Res. 89:111-122 (1983). The samples
were examined in Hitachi 12A and Hitachi 600
transmission electron microscopes, calibrated using a
diffraction grating for low magnification (2-48,000
x) and a catalase crystal for high magnifications
(28,000 - 200,000 x) with an objective aperature of ;
50 um and accelerated voltage of 75 kilovolts.
The anchorless form of rCR2 was determined to ~;
be an elongated flexible molecule with an estimated
contour length of 386+35 angstroms. When rCR2 was ;
imaged at a high primary magnification of 150,000x, ;
the ultrastructure of the polypeptide chain was - ;
discernable to be composed of a chain of ringlets,
; each of which presumably represents a single
repeating region. Since there are 16 tandem
repeating regions in the rCR2 used in this study,
each region is calculated to be 24.1+2 angstroms.
TABLE 1 summarizes the physical data
obtained.
,:
~ '- ': . '.,':
^` 2~2377~
33
TABLE 1
Molecular Weight rCR2
Apoprotein, predicted from sequence 110,200
Calculated from D20W and S20w 30~000
SDS gel electrophoresis125,000
Partial specific volume, v (cm31g) 0.71
Diffusion coefficient,D20w(cm2/sec)
Strokes radius (angstroms)69.3
S20w(S) 4.5
Frictional ratio, f/fO 2.1
:
a Assuming the difference in molecular weight between ; ;~
the apoprotein and the mature glycoprotein is
entirely due to carbohydrate, and without knowing
the carbohydrate composition a range was calculated
for the most and least dense sugars for rCR2. The
values for both ends of the range were the same for ~;
CR2 to two significant digits.
b Values given are average of six runs.
4. Binding of CR2 Polypeptide -~
to qp350/220 or C3dg ~ -
Purified gp350/220 and C3dg obtained as
described in Example 1, and ovalbumin were -~
independently coated and immobilized onto separate
wells of 96 well plates. Aliquots of rCR2 prepared
in Example 1 were then added to each well at varied
concentrations, maintained for 2 hrs to allow binding
of rCR2 to the immobilized ligand, followed by
rinsing to remove any unbound rCR2. The presence of
bound rCR2 was determined by the CR2-specific ELISA
described in Example la. The results are illustrated
in Figure 2. The rCR2 bound to both gp350/220 and
C3dg in a dose-dependent manner, while only
background binding was observed with ovalbumin.
Similarly, CR2 polypeptides rCR2-4 and rCR2-
10, prepared in Example 1, were analyzed by ELISA for
.~ : ..
~ 2~2~7~ : ~
34 ;~
their ability to bind ligand. rCR2-4 and rCR2-10
both exhibited binding to immobilized ligands
comprised of EBV virus particles purified gp350/220
or complement C3dg.
Therefore the exemplary CR2 polypeptides
rCR2, rCR2-4 and rCR2-10 were shown to have the
capacity to specifically bind to the target ligands
for CR2.
5. Liqand Bindinq to rCR2
The binding of rCR2 to ligands was
measured by mobility shift of the receptor in sucrose
density gradients in which rCR2 was distributed
throughout the gradient prior to centrifugation. The
apparent increase in sedimentation rate of rCR2-
ligand complexes was measured, and the fractional ;;
occupancy of the binding sites was calculated from
the relative increase in sedimentation rate of the
complex as a function of increasing rCR2. The
sedimentation rate of the complex was calculated as
described by Ziccardi et al., J. Biol. Chem.
259:13674-13679 (1984) as Sc = So(MC/Mo)o68 where Sc i6
the S20w of the complex, Mc is the molecular weight of
the complex, SO is the S20 W of the rCR2 and No is the
molecular weight of rCR2. A value r represents the
increase in SzO,w normalized to the S20 W Of a 1:1
complex of rCR2 to ligand where r=(S~pp-SO)/(S~-SO)
where S4pp i8 the measured value for a given sample
and is equivalent to the number of ligands bound per
rCR2.
,
For the binding of gp350/220 to rCR2,
gp350/220 was centrifuged through a sucrose gradient
in which rCR2 was evenly distributed throughout. ~;
Saturable binding of gp350/220 to rCR2 was exhibited ~;
at physiological pH and ionic strength that is
univalent, forming a 1:1 complex with rCR2. Results
are shown in Figures 3A, 4A, 4B and 4C. A Scatchard
~ ""'"'.'
: ".
7 7 ~
plot of the binding data (Figure 4B) indicated a
single high affinity (KD = 3.2 x 10-9M) binding site
on gp350/220 for rCR2. Linear regression analysis of
the data gives a slope of 0.311, and a linear
correlation coefficient of -0.95. A further
examination of the data by a Hill plot (Figure 4C)
revealed a binding slope of 0.75 with a linear
correlation coefficient of 0.~8, indicating that
multiple binding sites involved in cooperative rCR2
gp350/220 binding are not present. `
For the binding of C3dg to rCR2, C3dg
prepared as described in Example 1 was used in a
sucrose gradient, as described above. Under
conditions of physiologic pH and ionic strength, no
binding of monomeric C3dg to rCR2 was detected ~ ~
(Figure 3B). -
In order to maximize the potential binding of
the low affinity interaction of C3dg with rCR2,
studies were carried out at reduced ionic strength
using two concentrations of C3dg (1 or 2 uM), as
illustrated in Figure 5.
Ultracentrifugation of rCR2 in 5-15% sucrose
gradients was carried out with 2 uM [125I-]-C3dg in
10, 25 and 150 mM NaCl, respectively. Unlabeled CR2
(50mg) was detected in gradient factors by an ELISA.
A dose-dependent increase in C3dg binding to
rCR2 was observed with decreasing ionic strength
(Figure 5A). The binding data obtained indicated
that the C3dg intersection with rCR2 is univalent. A
dissociation constant of 2.75 x 10-5 M for rCR2
binding to C3dg at physiologic ionic strength and pH
was obtained by extrapolation of the binding data
(Figure 5B). KD was calculated for each of the
binding experiments from the fractional saturation
~l-r/r): KD = [CR2] x ((1 - r)/r). The negative log
KD was plotted against the salt concentration and
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used to extrapolate KD for 150 mM NaCl, indicated by
the arrow. Least squares analysis was used to
determine the best fit of the data to a line, from
this the predicted value for KD at 150 mM NaCl is 27.5
uM. The linear correlation coefficient for this line
is 0.91. The slight increase of sedimentation rates
with 2 uM C3dg in 10 mM NaCl indicates that small
amounts of C3dg aggregation may have occurred. -
6. Inhibition of aP350/220 Bindinq to Cells
Fluorescent microspheres coated with
purified gp350/220 were preincubated with rCR2
purified as described in Example 1, or with buffer
alone prior to addition to cells to determine the
ability of rCR2 to inhibit the binding of gp350/220
lS to the cells. The binding of gp350/220 was
determined by fluorescent activated cell sorting
(FACS). The results of the FACS analysis are
illustrated in Figure 6. The gp350/220 coated
microspheres that were preincubated in buffer alone
were reacted with CR2 negative SF9 cells (Panel A)
and CR2 positive Raji cells (Panel B). Micropheres
preincubated with 2 ug rCR2 (Panel C) or 10 ug rCR2
~Panel D) were reacted with Raji cells, and show that
rCR2 produced a dose-dependent inhibition of
gp350/220 fluorescent microsphere binding to Raji
cells, while no inhibition was observed with
microspheres incubated with buffer alone.
7. Inhibition of Epstein-Barr
Virus Infection
The ability of rCR2 to altér EBV
infection of peripheral blood B cells was measured by
outgrowth of transformed B cells and by virus-induced ~
stimulation of DNA synthesis. --
EBV was preincubated with varying amounts of
purified rCR2 prior to addition of unseparated
peripheral blood mononuclear cells (6 x 105 cells).
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The mixture of cells with EBV and rCR2 were
maintained for 1 hour at 4 C, washed and the cells (2
x 105/plate, in triplicate) were then cultured for 14 ~ ~;
to 21 days in complete RPMI medium with o.1 ug/ml
cyclosporine A and then assessed for transformation.
As illustrated in Figure 7, a preincubation
of EBV with rCR2 resulted in dose-dependent
inhibition of EBV infection as measured by both
outgrowth of transformed colonies and by 3H-thymidine -~
incorporation. The ability of rCR2 to block
infection was not due to toxic effects of rCR2 upon
the cells because the highest dose of rCR2 used (7.5
ug) did not abolish infection of B cells if EBV was
allowed to bind to B cells prior to exposure to rCR2.
When varying amounts of Esv were preincubated
with a constant amount of CR2 (10 ug) prior to
addition of B cells, EBV induction of B cells
transformation was reduced by nearly 90% as
illustrated in Figure 8.
These results show that rCR2 blocks EBV
infection in vitro and indicates that CR2 is the
primary EBV receptor on B cells.
The foregoing specification, including the
specific embodiments and EXAMPLES, is intended to be
illustrative of the present invention and is not to
be taken as limiting. Numerous other variations and
modifications can be effected without departing from
the true spirit and scope of the present invention.
