Note: Descriptions are shown in the official language in which they were submitted.
WO 92/07952 2 0 9 ~ 3 2 3 PCl/US91/08080
PEPTIDE BINDING ASSAYS WITH ~C ANTIGENS
~ .
Technical Field
The invention is related to methods to
determine the ability of candidate moieties to bind to
15 specific major histocompatibility complex glycoproteins. ,
Successful candidates are useful as therapeutics in
conditions mediated by particular MHC glycoproteins.
Back~Eou~,d ~f the Invention
Because the immune system is such an essential
part of the well-being.of mammalian species, there has "
been intense research in attempting to understand how the ?
immune system works. Only recently was it determined
that the major histocompatibility complex (MHC)
25 glycoproteins play an essential role in B-lymphocyte and
T-lymphocyte responses. The MHC glycoproteins are
divided into two types, Class I and Class II, where each
of these classes appears to play a substantially
different role. In addition, the structure of the Class
30 I and Class II glycoproteins is different. Despite the
differences, in each case it is found that an antigen to
- which the immune system will respond is degraded
intracellularly and a fragment of the antigen is 7
expressed on the cell surface associated with an MHC
35 glycoprotein. Each MHC glycoprotein has polymorphi_
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W092/07952 PCT/US91tO8080
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regions, which are assoclated with a groove, and have
some degree of specificity as to the peptide which binds
in the groove. Thus, each MHC glycoprotein has only a
specific repertoire of relatively low molecular weight
moieties to which it is capable of binding. It is the
identification of this repertoire of low molecular weight
moieties to which the invention described below is
direcced. I
The MHC glycoprotein-peptide complex is
presented to a T-cell receptor which specifically
recognizes the fragment in conjunction with the MHC
glycoprotein to which it is bound. The T-cell becomes
stimulated and secretes lymphokines, with resulting
expansion of the lymphocytes.
Since these fragments are critical to the
activation of lymphocytes, peptides or small molecular
weight organic molecules may be devised or discovered
which will play a role in enhancing or diminishing the
activation of specific lymphocytes. In this way, ~- and
T-lymphocyte activation may be controlled to enhance or
suppress a particular immune response. It is therefore
of substantial interest to be able to identify successful
candidate moieties which activate or inhibit lymphocyte
response, where these identified moieties may find use in
the treatment of autoimmune disease, infection,
allergies, and the like. Thus, determination of moieties
capable of bi~ding a specific MHC glycoprotein of
interest provides potential therapeutic compounds for use
in conditions mediated by presentation of antigen
moieties by the relevant MHC glycoprotein.
Ordinary competitive binding assays do not
appear to be appropriate for identifying suitable
candidate moieties. Unlike more conventional
ligand/receptor binding, the rate of forma~ion of the
moiety/MHC glycoprotein complex is very slow, bu. the
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W092/07952 PCT/US91/08080
2 ~ 2 3 ' ,
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-3-
resulting complex is stable. As a consequence, MHC
glycoproteins obtained from native materials are
complexed with a heterogeneous array of endogenous
peptides and are thus prevented from binding candidate
alternate moieties, except at a very slow and varlable
rate. It is estimated that only about 1% of isolated MHC
glycoproteins are in an "open form" that is capable of
directly binding alternate moietiesi about 99% of the
preparation is already complexed with endogenous peptide.
~0 Thus, there is a need to devise assays for
successful ligand binding which allow screening of large
numbers of candidate moieties with respect to a
particular MHC glycoprotein in a rapid and efficient
manner. The invention provides several protocols to
achieve this result.
Relevant Literature
The following references are believed to be
relevant. Babbitt et al. ~1985) "The Binding of
Immunogenic Peptides to Ia Histocompatibility Molecules"
Nature 317:359-61; Buus. (1986) "Isolation and
Characterization of Antigen-Ia Complexes in T-cell
Recognitionll Cell 47:1071-77; Watts and McConnell (1986)
"High Affinity Fluorescent Peptide Binding to I-Ad in
Lipid Membranes" Proc Natl Acad Sci USA 83:9660-4; Busch
et al. (1990) "Degenerate Binding of Immunogenic Peptides
to HLA-DR Proteins on B-cell Surfaces" Int Immunol 2:443-
1; Roche and Cresswell (1990) "High Affinity Binding of
an Influenza Hemagglutinin-derived Peptide to Purified
HLA-DR" J Immunol 144:1849-56; Chen and Parham (1989)
nDirect Binding of Influenza Peptides to Class I HLA
Molecules" Nature 337:743-5; and Bouillot et al. ~1989)
"Physical Association Between MHC Class I Molecules and
Immunogenic Peptides" Nature 339:473-5.
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w092/07952 2 0 9 ~ ~ 2 3 PCT/US91/08080
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Disclosure of the Invention
In one protocol provided by the invention, .
dispersed soluble MXC glycoproteins are treated with a
detectable agonist in the presence of a competitor
candidate moiecy under conditions wherein the agonist is
known to form a complex with the MHC glycoprotein. The
resulting complex is separated from the reaction mixture,
and the effect of the candidate moiety on the agonist
included in the complex is determined.
In a second protocol, especially appropriate
for instances where purified MHC glycoprotein is not
readily available, MHC glycoprotein is captured on an
assay plate which plate is, for example, derivatized to
anti-MHC glycoprotein antibody or other reagent with
affinity for the MHC glycoprotein and used in place of
the soluble MHC in the competitive assay. Again, the
effect of the candidate moiety on the binding of the
immobilized MHC to detectable agonist is determined.
In an additional protocol, isolated MHC
glycoprotein is preloaded with a homogeneous peptide
preparation to providej therefore, a homogeneous
population of already-coupled MHC glycoprotein. As the
dissociation of the preloaded peptide is the rate-
determining step in the association of alternate
moieties, this approach permits control of the reaction
rate to assure uniform competition between the detectable
agonist and the candidate. In addition, dilution of the
performed MHC complex results in rapid dissociation of
the preloaded peptide, thus creating an "empty pocket"
(i.e., an available domain or binding site) for binding
to the agonist or candidate. ~he homogeneous, preloaded
peptide is preferably chosen to be comparatively readily
released by the MHC glycoprotein, thus shortening the
,ime of the assay.
,
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w092/07~2 2 0 9 ~ 3 2 3 PCT/US9l/08080
In another protocol, isolated MXC glycoprotein
is preloaded with a labeled peptide whose rate of
dissociation from the MHC glycoprotein is known to be
influenced by the presence of other peptide. The ability
5 of test compounds to accelerate the dissociation o-
displacement of the labeled agonist from the MHC
glycoprotein is determined.
In addition, a useful means to detect the }
formation of complex with the agonist and/or candidate
10 moiety is to determine the effect of the complex on T-
cell stimulation.
Thus, in one aspect, the invention is directed
to a method to determine the affinity of a test compound
to an MHC glycoprotein, which method comprises combining
15 in a reaction mixture cell-free dispersed MHC
glycoprotein, a soluble agonist capable of binding to
said MHC glycoprotein to form a complex and capable of
being detected when in said complex, and said test
compound, under conditions wherein the test compound and
20 the a,gonist compete for binding to the MHC glycoprotein
separating MHC glycoprotein-bound agonist from unbound
agonist; and detecting the amount of agonist bound in the
complex as a function of the concentration of test
compound in the reaction mixture.
In a second aspect, the invention is directed
to a method tO determine the affinity of a test compound
- for a specific MHC glycoprotein, which method comprises
treating a solid support to which the MHC glycopro~ein is
coupled with a reaction mixture containing a solubie '
agonist capable of binding to the MHC glycoprotein to
form a complex and capable of being detected when in said
complex and said test compound, under conditions wherein
the test compourd and said agonist compete for binding to
the MHC glycoprotein; removing said reaction mixtu-e from
33 the solid suppor~; and detec~ing the amour._ of ago.ist .
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w092/07952 PCT/USgl/08080
2n9~323 ~;
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bound to the solid support as a function of the
concentration of th~ test compound in the reac~lon
mixture.
In a third aspect, the invention is directed to
a method for determining the affinity of a test compound
to an MHC glycoprotein, which method comprises treating
an MHC glycoprotein preloaded with a first agonist with
reaction mixture containing the test compound and a
second agonist capable of binding to said MHC
glycoprotein to form a complex and capable of being
detected when in said complex under conditions wherein
the test compound and second agonist compete for binding
to said MHC glycoprotein; wherein said MHC glycoprotein
is preloaded with a preloading agonist and diluted prior
to the addition of the reaction mixture; and detecting
the amount of second agonist bound in the complex as a
function of the concentration of test compound in the
reaction mixture.
In a fourth aspect (a dissociation rate assay),
the invention is directed to a method for determining the
affinity of a test compound to an MHC glycoprotein, which
method comprises combining in a reaction mixture the test
compound and said MHC glycoprotein which has been
preloaded with a labeled agonist, whose dissociation rate
has been shown to be affected by the presence of other
peptides and comparing the dissociation rate of the
preloaded complexes in the presence and absence of the
test compound to detect any accelerated off rate by the
test compound.
In a fifth aspect, the invention is directed to
a method to detect the presence of a moiety in an MHC
glycoprotein complex which method comprises contacting
said complex with a culture of T-cells primed with said
moiety and detecting the presence, absence or amount of
prolifera~ion of said T-cells.
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W092/07952 PCT/U~92 SP)~R~
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B~ief Description of the Drawinqs
Figure 1 shows a typical inhibition curve
wherein unlabeled HA 307-319 competes with labeled HA
307-319 for binding to DR4Dw4.
Figure 2 shows a binding curve for labeled HA
307-319 using DR4Dw4 captured from a crude lysate by
antibody-coated microtiter plates.
Figures 3A, 3B and 3C show l~off rates~
determined for various test peptides after preloading
onto DR4Dw4 MHC glycoprotein.
Figures 4A and 4B show a binding curve and an
inhibition curve, respectively, for ~3P 90-102 peptide
. with respect to preloaded and control MHC glycoprotein.
Figures 5A and 5B show dissociation curves for,
respectively, biotinylated R~3P 90-102 and biotinylated
HSP 3-14 in the absence and presence of HA 307-319.
Figure 6 shows a displacement curve of
biotinylated HSP 3-14 by RMBP 90-102.
Figure 7 shows a binding curve for HA 307-319
peptide using sensitized T-cell proliferation as an assay
for bound peptide.
:
Modes of Carrying Out the Invention
Methods and compositions are provided for
determining the binding affinity of a candidate moiety
for a specific MHC glycoprotein using competition between
detectable agonist and the candidate of interest using
several efficient assay strategies.
In all of the strategies of the invention,
except the dissociation rate assay, competition is
effected between a detectable agonist and the candidate.
The effect of at least one concentration, and preferably
varying concentrations, of the candidate moiety on
binding of the detectable agonist is then determined.
-
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WO 92/07952 2 ~ 9 S 3 2 3 PCr/US91/08080
- 8 --
In the dissoclation rate assay, the effect of
a least one concentration, and preferably varying
concentrations of the candidate moiety, on the
dissociation rate of the preloaded MHC/labeled a~onist
comDlex is determined.
By detectable "agonist" is meant a peptide or
other low molecular weight compound known to be capable
of binding to the specific MHC glycoprotein being tested.
Useful agonists for DR alleles, for example, are peptides
de~ived from hemagglutinin such as that represented by
positions 307-319 (HA 307-319) (Int Immunol (1990)
2: 443 ) . Other useful agonists that have been found to
bind MHC glycoprotein products of DR alleles include, but
. are not limited to, HADP 3.2, RRFAAAYAARAAAi HADP 3.6,
RRFAAQYAARAAA; R~3P 90-102, HFFKNIVTPRTPA; HSP 3-14 (R5
K13), RVRRG~TVAVKG; HSP 3-14 (K5 K13), RVKRGLTVAVKG; HSP
3-14 (K5 A13), RVKRGLTV~VAG and shortened versions
thereof. Useful agonists may include the peptides shown
above and equivalents in which the amino and carboxyl
charges eliminated. Some agonists are allele specific
such as pertussis toxin 31-43, ragweed 3 50-62 and flu
matrix 18-30.
The agonist is generally labeled in a manner so
as to permit its convenient detection when complexed to
MHC. However, if detection is effected in a manner which
is dependent on the nature only of the agonist, as
described with respect to the T-cell proliferation assay
for determination of complexed peptide, no extraneous
labeling is needed. Thus, the agonist need only be
detectable when in the complex.
However, if the agonist is detected by means of
a label, the label may be in various forms. Thus,
various labels may be employed, such as radioisotopes,
biotin, fluorescers, chemiluminescers and the like. The
-:~oice o- the label will be primarily directed t^
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w092/07952 ~-; PCT/US91/0808~
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c:onvenience, sensi. vity, minimizing background,
minimizing interference with binding of the agonist to
t:he MHC glycoprotei~, and the like. Generally, the
agonists will be at a concentration of about 0.1-50 times
the concentration of the MHC glycoprotein.
A particularly preferred label is biotin. When
biotin is used as "label," streptavidin which is in turn
labeled with a wide variety of labels may be used for
detection. Thus, the streptavidin may be labeled with
radioisotopes, fluorescers, chemiluminescers, enzymes,
colloidal particles, or the like. As indicated above, a
variety of considerations will dictate which label will
be employed.
The concentration of candidate moiety of
interest will vary depending upon the concentration of
agonist present in the medium, and the relative
affinities of the candidate and agonist. Usually, the
amount of the candidate will not differ by more than
about 100-fold from the amount of agonist present in the
medium.
~ - According to the invention method, the various
components (e.g., MHC glycoprotein, agonist and test
compound) are combined and allowed to stand for
sufficient time for the mixture to come to equilibrium.
Generally the temperature is at about 37C. Usually, the
time for reaching equilibrium will be at least about 0.5
hours, more usually about 12 hours, and will generally
not exceed about 48 hours. While a rate determination
can be used, where a plurality of samples are employed
and each sample analyzed for the amount of complex
formation, it will usually be ~ufficient to do a single
determination at varying concentrations of candidate.
Suitable candidate moieties include any small
molecular weight material which is thought to block the
MHC glycoprotein of interest. Typically, these
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W092/07952 2 0 9 S 3 2 3 PCT/US91/08080
' ' - 10 -
caLndidates are peptides of on the order of 5 or more
amino acids or are small organic molecules which mimic
the conformation of such peptides. A wide variety of
candidates is generally known in the currently active
field of rational drug design. There is no theoretical
limitation on the range of candidates, and any putatively
active compound may be employed.
In one strategy a soluble MHC glycoprotein in
solution is used in the complex formation reaction. The
labeled agonist complexed with the MHC glycoprotein is
separated from free agonist and the amount of complexed
agonist determined as a measure of the affinity of the
candidate to the MHC glycoprotein. The method provides
for a rapid, simple and accurate technique for screening
large numbers of candidates and obtaining relevant values
for the affinity of the candidate to MXC solubilized
glycoproteins which is thought to correlate to the
affinity found when said MHC glycopro~eins are in their
natural locations in cellular membranes.
In carrying out the method, a solution is
prepared of the MHC glycoprotein, where the glycoprotein
may be the naturally occurring dimeric glycoprotein freed
of the cell membrane or a soluble glycoprotein which
lacks the transmembrane region. The latter can be
prepared in a variety of ways, using recombinant
techniques, where the genes encoding the alpha and beta
chains of a Class II MHC glycoprotein or the alpha chain
of the Class I MHC glycoprotein may be truncated by
removal of all or a portion of the transmembrane region.
Conveniently, the transmembrane sequence may be replaced
with a region capable of linking to a lipid. See, for
example, Caras et al., Science (1987) 238:1280;
Tykocinski et al., Proc Natl Acad Sci USA (1988) 85:3555.
The lipid may then be removed by an appropriate esterase,
e.g., phosphatidyl inositol-specific phospholipase C.
- .: .
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W092/079S2 2 0 9 5 3 2 3 PCT/U~n~
"','','J
The concentration of the MHC glycoprotein will generally
be in the range of about 0.01 to 50 ~M, more usually
about 0.1 to 1 ~M. This range is convenient and is not
critical, since in some experiments it may be desirable
tO use either higher or lower concentrations, depending
upon the affinity of the mixtures to be bound, the
concentration of moiety employed, and the like.
The medium will generally be buffered at abou~
physlologic pH, pH 4.5-8, preferably about 5-6.5, with a
buffer concentration of about 10 to 200 mM. Other
additi~es may include salt, to a concentration of abou~ :
10 to 20 mM, or nonionic surfactants. The nonionic
surfactants will generally be present in a concentration
of about 0.1 to 2~. If a peptide agonist is used, the
peptide will generally be at least about 3 amino acids
and not more than about 30 amino acids, preferably being
from about 3 to 16 amino acids, more preferably from
about 5 to 15 amino acids.
Once sufficient M~C-agonist complex has been
forme,d, the complex may be separated in a variety of
ways. The complex may be separated from free agonis~ by
gel filtration, gel electrophoresis in a nonreducing SDS
polyacrylamide gel, or by binding the complex to a plate
coated with antibodies or other affinity reagent (ligand)
specific for the M~C glycoprotein. For gel
fractionation, Sephadex G-50 is found to be useful. For
gel electrophoresis, a 12.5~ SDS-PAGE gel under
nonreducing conditions is found to be satisfactory.
Preferably, however, affinity separation,
particularly antibody separation is used, preferably on a
plate, more particularly on a multi-well plate. For
example, by employing excess antibody or fragment thereof
specific for the constant region of one of the subunits
of the MHC glycoprotein, one can capture the complex
between the agonist and the MHC glycoprotein. One can
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W092/07952 2 0 9 ~ ~ 2 3 PCT/US91/08080
,- .
then quantitate the amount Oc agonist bound to th~
surface by washing away any nonspecifically bound or
unbound labeled agonist and then detecting the agonist
present bound to the surface. Washing may be performed
S with any convenient buffered medium, including the medium
employed for complex formation. Where the agonist is
labeled with biotin, by employing labeled avidin one can
obtain a plurality of labels bound to a single agonist-
MHC glycoprotein complex. Of particular interest with
O the avidin is the use of fluorescent labels, particularly
lanthanide chelates, more par~icularly europium chelates,
or enzymes, particularly horseradish peroxidase. These
labels may be quantitated in accordance with conventional
procedures, there being numerous fluorimeters for
detecting fluorescence from lanthanide chelates and
numerous spectrophotometers for detecting peroxidase
substrates which result in chromophores.
In a second strategy, particularly useful for
determination of the ability of a candidate to form a
complex with an MHC glycoprotein present in a crude
lysate, the MHC glycoprotein of interest is first
captured on a solid support and the remaining components
of the lysate washed free of the adsorbed MHC
glycoprotein on the support. The coupled support is then
25 treated with a reaction mixture containing the agonist -
capable of detection when complexed with the MHC
glycoprotein and the competing candidate moiety. The
nature of the labeling of the agonist, if needed, and of
the concentrations of the competing substances in the
reaction mixture is similar to that described above with
respect to the use of solubilized MHC glycoprotein.
The derivatized solid support is prepared by
passive adsorption or by covalent coupling of an affinity
reagent specific for the MHC glycoprotein of intcrest by
~5 standard techniques well known in the art. Typically,
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Wos2~079s2 2 0 9 ~ 3 2 3 PCT/US91/08080
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microtlter plates or other multiwell reaction matrices
are used as solid support. This method has the advantage
of removing contaminants from the crude lysate which
might interfere with the binding of the candidate and/or
agonist to the MHC glycoprotein. The activity of such
contaminants must be minimized since binding of agonists
to MHC glycoproteins occurs only at elevated temperatures
where proteases and the like present in the lysates would
exhibit activity.
Thus, in this method, a reaction mixture
containing the relevant amounts of agonist and candidate
is incubated for a suitable time period, usually about 3-
48 hours, in the presence of the MHC complex coupled to
solid support. At the end of the incubation time the
solid support is removed from the reaction mixture,
washed, and the agonist bound to MXC glycoprotein
determined according to the nature of the label as
described above.
In a third strategy, the first competition
protocol may be used, but may be further optimized by
preloading the MHC glycoprotein with a homogeneous,
usually peptide agonist, which has a suitable off rate to
permit the binding of the agonist/competitor combination.
In this method, the purified MHC glycoprotein or the
crude lysate is incubated with a preloading agonist,
generally overnight in a suitable bu~fer, generall~
containing octylglucoside, for a time sufficient to
replace the heterogeneous endogenous peptides contained
in the native MHC glycoprotein with the preloading
homogeneous moiety. The preloaded MHC com~lexes are then
diluted and used in the assay systems described above.
This approach provides a uniform provision of "empty" MHC
glycoprotein binding domains for binding or coupling with
the agonist or candidate. Generally, the acceleration of
what is otherwise the rate-limiting step i-. the
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W092/07952 PCT/US91/08~80
209~323 ~' !
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association of the candidate or agonist provides a
shorter assay time and provides a more accurate
determination of the association rate and affinity of the
candidate. ~y providing a preloading condition where lt
is unnecessary for the incoming agonist or candidate to
dislodge the preloaded moiety, the affinities of the
competitors for the unloaded MHC glycoprotein can be
compared more readily. Because the MHC molecule is
preloaded with a peptide with a defined dissociation
rate, the assay can be run for a shorter period of time,
preferably three hours.
In a fourth strategy, the MHC glycoprotein is
preloaded with a homogeneous, labeled peptide agonist
which has been demonstrated to be displaced in the
presence of other peptides. In this method, the purified
MHC glycoprotein is incubated with the labeled agonist,
generally overnight in a suitable buffer, generally
containing octylglucoside, for a time sufficient to
replace the heterogeneous endogenous peptides with the
preloading moiety. The preloaded MHC complexes are then
diluted into a solution of a displacing candidate moiety
for about 0.5 hour and loss of counts with and without
the candidate moiety is monitored as described above.
An additional aspect of the invention obviates
the necessity to label the agonist used in the
competition assays. In this approach, T-cells which have
been primed with the agonist are used to detect the
presence of the agonist in the complex. In this method,
the complex is used in a T-cell proliferation assay with
the primed T-celis. Enhanced proliferation of the primed
T-cells is a measure of the binding of the agonist to the
complex. This method can also be used to detect any test
moiety in the complex by priming the T-cells with test
moiety.
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wo 92/07952 2 0 9 ~ 3 2 3 P~tUS9~ 8~ 1
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The following examples are offered by way of
illustration and not by way of limitation.
Example 1 ' -
Purification of HLA-DR and DO Proteins
Anti-DR (LB3.1) and anti-DQ (IVD12) affinity
columns were prepared using spherical cellulose resin I
(Amicon) and 40 mg of each of the antibodies. The ~ --
maximum amount of solubilized Class II that combined to ~ -
- 10 the columns is twice the molar concentration of the bound
antibody. Therefore, 40 mg of immunoglobulin can bind a~
most 32 mg of Class II ~C glycoprotein. Practically,
only between 10-30~ of the theoretical capacity of an
affinity column is allowed, which corresponds to between
3 and 9 mg of DR and DQ. A comparison of the L243 and
LB3.1 columns was performed with an NP-40 detergent
extract from 1.8 x 101 cellg which was equally divided,
loaded on each column, and eluted with either 5 or 15
minute exposure to pH 11.5 buffer. The yields are as
follow,s:
5'exposure 15'exposure total yield
(mg)* (mg) (mg)(mg)
L243 1.8 0.886 2.69
LB3.1 2.10 2.02 4.126.81
IVD12 1.37 3.21 4.584.58
*measured by BCA assay using BSA as a standard.
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W092/079522 0 9 S ~ 2 3 PCT/US91/08080
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Example 2
Separation of Radioactive Pep~ide-MHC Compl~
From Fr~e Peptide by Gel Filtration
An iodinated HA 307-319 analog (2 ~M) was
incubated with HLA-DR4Dw4 (2 ~M) overnight in PBS/l~
octylglucoside (50 ~L total volume). The reaction
mixture was applied to a 25 ml Sephadex G-50 column.
Fractions (0.5 ml) were eluted in PBS/1~ octylglucoside
and counted. The elution profile of the radiolabeled
10 peptide was also determined. A peak was observed at from ~ -
about 40 to 50 relative elution volume percent which
could be attributed to the complex free of the peptide.
This peak was not observed when the peptide control was
performed.
Example 3
Separation of Bound From Free Peptide
Usin~ c~y_9g~ted Plates
a) A radioactive iodinated analog of HA 307-
319 at 1 ~M was incubated overnight with DR4Dw4 at 2 ~M
in PBS/1~ octylglycoside with or without HA 307-319 as a
competitor in a volume of 50 ~l. At the same time,
microwell plates were coated with LB3.1 overnight at 4C,
washed with PBS/0.05% Tween/0.1~ BSA and blocked with
PBS/5~ FCS at 4C. Fifty ~l of PBS/1% octylglucoside/5%
FBS was then added to each well followed by 50 ~l of the
incubation mixture of peptide and Class II MHC
glycoprotein. After an hour of incubation the place was
washed and counted.
b) An agonist corresponding to residues 307-
319 of influenza hemagglutinin ;HA 307-319), biotinylated
at the amino terminus, was incubated with affinity-
purified DR4Dw4 (2 ~M) PBS/1~ octylglucoside). Peptide
DR complexes were separated from free peptide as w~th the
iodinated HA except 25 ~l o. P~3S/1~ octylglucoside !5~
,, . ,. . , , . .
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W092/~795~ Z 0 9 ~ 3 2 3 PCT/U591/0808~
FCS) and 10 ~l of the incubation mixture were added to
the wells of the antibody-coated ELISA plates. After
incubation for an hour the plate was washed with
PB~/0.05~ Tween/0.1~ BSA and the amount of HA 307-319
S bound was quantitated by incubation with 125I
streptavidin (6-30 mg/well) for an hour at 4C followed
by washing and counting.
c) Competition for binding of biotinylated HA
307-319 to DR4Dw4 was performed as in b) with or without
several HA monosubstituted peptides (e.g., lys, ser or
phe at position 309) (200 ~M) in PBS/1% octylglucoside.
With antibody LB3.1, the counts observed in the presence
of DR antigen as compared to background were about twice
that of background, while in the presence of a
nonradioactive competitor, the counts were somewhat lower
than background. With antibody IVD12, the observed
counts in the presence of DR were lower than background.
Typical results for inhibition curves are shown in Figure
1.
For experiment b), except for the combination
of the radioactive peptide and the DR antigen,
substantially no signal was observed.
In the competition experiment c), with peptide
competitors corresponding to analogs containing point
substitutions (i.e., lysine or serine at 309), the counts
were about the same as background, while in the presence
of nonradioactive peptide 307-319 or phenylalanine at
position 309 there was substantially no radioactivity
observed.
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w092/0~9s2 PCT/US9~/O~
209~323 ~
Example 4
Comparison of Nonradioactive and Radioactive
Detection Systems for the i3indinq of a
Biotinylated Hemaggl~inin Analo~ to DR4Dw4
5The assay was formed as described in Example 3b
using a biotinylated alanine backbone analog of ~A
(AAFKAAEAAAARA) at 2 ~M and DR4Dw4 at 0.5 ~M. The
peptide DR complexes were quantitated using either a
fluorescent europium-conjugated streptavidin, a
horseradish peroxidase-conjugated streptavidin or 125I-
conjugated streptavidin. Each staining reagent was
titrated to obtain the maximum signal over background
possible.
With streptavidin labeled with the fluorescent
europium chelate, signal-to-noise ratios as great as seen
with 125I streptavidin (between 80-100:1) were observed.
Good signal-to-noise ratios (10-20:1) were also observed
with horseradish peroxidase-streptavidin conjugates.
20~ Example 5
Assay of Specifi-c Binding in a Crude Lysate
The monoclonal antibody LB3.1, specific for DR
Class II molecules at 2 ~g/ml, 200 ~l/well, was coated
onto a Costar EIA-RIA plate in 50 mM Tris-HCl, p~ 9.O,
either overnight at 4C or for 1 hour at 37C. The plate
was washed and blocked for 1 hour at room temperature
with 5~ FCSlP~S and then washed 3-4 times with 0.05~
Tween 20/0.01~ azide/PBS (wash buffer) using a Titertek
plate washer. Cell lysates assessed to contain
approximately 20 nM DR MHC glycoprotein was then
incubated for 4 hours at 4C on the coated plate. The
plate was washed 3-4 times with 0.05~ Tween 20/0.01
azide/PBS. Biotinylated HA peptide 307-319 was then
added to the plate in 5~ FCS/1~ octylglucoside (OG)/PBS
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a. 200 ~l/well and incubated overnight at 37C in a CO2
incubator.
The plate was washed 3-4 times with wash buffer
and incubated for 4 hours at 4C with 200 ~l of europium
chelated streptavidin (Pharmacia/LKB Nuclear) at 60
ng/ml. After an additional washing cycle, the plate was
treated for 30 min at room temperature with 200 ~l/well
of enhancement solution (Pharmacia/LKB) which releases
bound europium for detection in a 1234 Delfia research
fluorometer (Pharmacia/LKB).
Typical results are shown in Figure 2. Binding -
curves proportional to concentration of the biotinylated
HA 307-319 peptide are obtained when a Priess cell lysate
was used (solid circles) as well as when purified DR4 MHC
glycoprotein at 10 nM (open circles) or the lysate from
Cos 7 cells transfected with DR4Dw4 (open s~uares) were
used to provide the MHC glycoprotein. Mock transfected
Cos cells used as a control (closed squares) showed no
uptake of the labeled peptide.
Example 6
Alternate Plate Assay Protocol
The assay was conducted in a manner similar to
that set forth in Example 5 except that the antibody-
coupled plates were prepared as follows:
Avid-HZ~ plates (Bioprobe International) which
covalently couple oxidized antibody to their surfaces
were used. LB3.1 monoclonal antibody was first oxidized
by diluting to 10 ~g/ml in 50 mM acetate buffer pH 5,
followed by addition of a 1/10 ~olume freshly prepared 10
mM sodium metaperiodate. After 30 minutes at room
temperature, the reaction w~s stopped by addition of 1/lO
volume of 20 mM ethylene glycol in acetate buffer. 115
~i of the oxidized antibody solution were added to the
wells ol an Avid-HZ~ plate and incubated overnigh- a~
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4C. The plates were then washed 4 times with wash
solution (PBS/0.05~ Tween-20/0.01~ sodium azide) and then
blocked for 1 hour at 4C with PBS/5~ FCS/0.01~ sodium
azide.
125 ~l of 20 nm DR4Dw4 in PBS/0.75%
octylglucoside/0.01~ sodium azide (binding buffer) were
added to each well and incubated for 4 hours at 4C. The
plates were washed 4 times with wash buffer and treated
with 125 ~l of biotinylated HA 307-319 peptide contained
0 in binding buffer and incubated overnight at 37c. The
plates were then washed 4 times with wash buffer and
incubated overnight at 4C with 125 ~l of europium
chelated streptavidin ~Pharmacia/LKB) at 60 ng/ml. After
an additional wash cycle, the plates were treated for 1
hour at room temperature with 125 ~l/well enhancement
solution (Pharmacia/LKB) and read in a 1234 Delfia
research fluorometer.
Results similar to those in Example 5 were
obtained.
Example 7
Use of Preloaded MHC Glycoprotein
To determine a suitable preloading peptide, off
rates were determined for several candidate preloading
peptides: rat myelin basic protein 90-102 (RMBP90-102)
which has the sequence HFFKNIVTPRTPA; HA derived protein
3.2 (HADP 3.2) which has the sequence RRFAAAYAARAAA; and
HA derived protein 3.6 (HADP 3.6) which has the sequence
RRFAAQYAARAAA. 50 nM of labeled preloading peptide was
incubated for 48 hours with 400 nM DR4Dw4 in PBS, pH 7.0
binding buffer. Off rates were determined by diluting
the complexes 1:40 into PBS and various concentrations Or
unlabeled HA 309-319 at various times, followed by
capturing the complex on antibody plates coupled to LB3.1
as described above. As shown in Figures 3A-3C, nonQ or
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the tested peptides showed off rates that were affected
by the presence of HA 307-319 and the dlssociation rate
for RMBP 90-102 was the most rapid of the three.
Therefore, preloading with RMBP 90-102 was selected for
use in the assays.
In the assay, 2 ~M purified DR4Dw4 and 1 ~M
RMBP 90-102 were incubated overnight in PBS/O . 75%
octylglucoside/O.O1~ sodium azide binding buffer as
described above.
0 Antibody capture plates were prepared using 115
~1 LB3.1 at 2 ~g/ml overnight at 4C and washed and
blocked as described above.
The preloaded complexes (i.e., DR4Dw4/RMBP
90-102) were diluted 1:100 and incubated with varying
concentrations of biotinylated RMBP 90-102 or with 8.8 nM
biotinylated R~3P 90-102 in the presence of varying
amounts of the same peptide unlabeled. Controls using
diluted nonpreloaded DR MHC glycoprotein were also run.
Aliquots (50 ~1) of the reaction mixtures were
removed to the capture plate and incubated at 4C for 4-
24 hours. The plates were washed as described above and
treated with 125 ~1 europium-streptavidin at 60 ng/ml in
assay buffer (calcium and magnesium-free PBS + 0.5% BSA,
20 ~M diethylenetriaminepentacetic acid (DTPA) in 0.1%
sodium azide) and incubated 2-24 hours at 4C. After
additional washing, the plates were enhanced and read as
described above.
Typical results are shown in Figures 4A and 4B.
Figure 4A shows a binding curve for biotinylated R~3P for
control and preloaded DR. Figure 4B shows the percent
inhibition obtained with varying concentrations of
unlabeled R~3P. As shown above, the results are
comparable for preloaded and nonpreloaded DR MHC
glycoprotein. After titratiny reagents, _ has been
determined that the screen will be run by preloading 2 ~M
w092/07952 PCT/US91/08Q80
209~323
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DR4Dw4 with 250 nM RM3P 90-102 by incubating in calcium,
ma~nesium free PBS adjusted to pH 6.5 with 0.1 M KH2P04
overnigh~ at 37C. The preformed complexes will be
diluted 1/40 and reacted with the labeled agonist and the
test compounds for 3 hours. The complexes will'then be
captured as described above.
~ . .
Example 8
Detection of Ligand/Receptor Dissociation Rates
Ligand/receptor dissociation rates should be
first order, depending only on the concentration of the
complex present. In studying class II/peptide
dissociation rates, rates are first order in some cases
but not all.
To determine the dissociation rates, 400-500 nM
DR4Ew4 was incubated with 50 nM biotinylated RMBP or 25
~m biotinylated HSP (19 kD heat shock protein 3-14 form
My~obacterium tuberculosis) overnight at 37C as
described previously. These preformed complexes were
then diluted 100-fold into buffer with or without varying
concentrations of HA 307-319. As can be seen in Figure
5A, the dissociation rate of RMBP is not affected by the ;~
second peptide. However, as seen in Figure 5B, the
dissociation of HSP was accelerated by the presence of
the other peptide. Since the dissociation rate increases
with the concentration of the other peptide, this is
clearly not a first order reaction.
This suggested a displacement model, where the
second peptide pushes off the first peptide. Therefore,
in a second preloaded screening assay, 800 nM DR4Dw4 and
6 ~M biotinylated HSP 3-14 were incubated as described
above. Complexes were diluted 40-fold into a buffer
solution with or without the competitor candidate moiety
and the displacement monitored by a decrease in counts at
thirty minutes. Figure 6 shows displacement by peptide
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competitor (RMBP), but any small molecule could
potentially have the same effect.
Example 9
Detection by Sensitized T-Cell Proliferation
T-cell clones responsive specifically to HA
307-319 were prepared from an individual with HLA Class
II phenotype DR4Dw4, DR7, DRw53, DQw8, DQw9 (as described
in Example 1). Briefly, peripheral blood mononuclear
10 cells were primed ln vitro with 1/100 dilution of ~ -
influenza virus vaccine (Parke Davis) in RPMI
supplemented with streptomycin (100 ~g/ml) penicillin
(100 U/ml) and 5~ pooled A~ serum (Whittaker,
Fredricksburg, MD). After incubation at 37C in 5~ C02
for 5 days, T-cell blasts were cloned by limiting
dilution at 1 cell per 3 wells in the presence of a
feeder mixture consisting of 1o6 allogeneic PBMC per ml
(30 Gy irradiated), 105 cells of autologous EBV
transformed B cells (50 Gy irradiated), 1/100 dilution of
20 influenza virus vaccine and 1 ~g/ml leucoagglutinin-A .-
(Pharmacia). The suspe~sion was plated in 96 well fla~-
bottom microtiter plates and incubated as described
above. Growing cultures were transferred to a 24-well
tissue culture plate and restimulated with the same
feeder mixture. Three days later, 10% IL-2 was added and
the cells were frozen or further expanded by
restimulation. Specificity was determined with HA 307-
319.
DR4Dw4 glycoprotein was affinity purified from
Priess EBV-B cells as described in Example 1. Briefly,
the cells were grown in RPMI medium supplemented with
fetal calf serum and collected by centrifugation, washed
with PBS and lysed with 1~ Nonidet P-40. The cellular
debris was removed by centrifugation and the lysate
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loaded dlrectly to a Sepharose CL-4B column connected in
series with a monoclonal antlbody LB3.1 cellulose column
prepared by coupling 40 mg of antibody to 10 ml Matrex
Cellufine Formyl (Amicon) according to the manufacturer's
instructions. The columns were washed with 20 volumes of
10 mM Tris HCl, pH 7 . 5, 150 mM NaCl, o.l~ deoxycholate.
The LB3.1 cellulose column was then washed with 5 column
volumes of 10 mM Tris HCl, pH 7.5, 1~ OG and eluted with
50 mM glycine, pH 11.5, 1~ OG. The fractions were
adjusted to pH 7. 5 with 2 M glycine, pH 2. The full
fractlons were dialyzed against 10 mM Tris HCl, pH 7 . 5,
1~ OG and stored at 4C.
To prepare the MHC glycoprotein/peptide
complex, purified DR4Dw4 was incubated overnight at 37C
15 with HA 307-319 at varying concentrations in 1% OG/PBS.
50 ~l of the mixture were added to 96-well flat-bottom
E~ISA plates which had been coated with L~3.1 by
incubation overnight at 4C in 5 ~g/ml ~3.1 in 50 mM
Tris HCl, pH 9. The mixture was incubated with the
coated plates for 6 hours at 4C and then washed twice
with PBS and once with complete medium.
For T-cell proliferation assays, 3 x 104 T-
cells were added in 200 ~l of complete medium to each
well (the T-cells were used 10-11 days after
restimulation). After incubation for 24 hours, 1.0 ~Ci
per well of tritiated thymidine were added, and the
plates were incubated for another 13 hours. The samples
were harvested on glass fiber filters using a
semiautomatic harvester and thymidine incorporation was
assessed by counting in a scintillation counter. The
results of triplicate determina~ions are shown in Figure
7.
As shown in Figure 7, T-cell proliferation can
be used to determine a binding curve for the test HA 307-
3~ 319 peptide.
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It is evident from the above results that
rapid, efficient assays are provided for screening
candidate moieties for binding affinity to particular MHC
glycoproteins. Thus, the methodology allows for
evaluation of a wide variety of candidates and their
ability to interact with MHC glycoprotein and ultimately
to modulate the immune response in a host having such MHC
glycoprotein.
All publlcations and patent applications
mentioned in the specification are herein incorporated by
reference to the same extent as if each individual
publication or patent application were specifically and
individually indicated to be incorporated by reference.
The invention now being fully described, it ,-
will be apparent to one of ordinary skill in the art that
many changes and modifications can be made thereto
without departing from the spirit or scope of the
appended claims.
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