Note: Descriptions are shown in the official language in which they were submitted.
t ~
wos6/10415 pcT~sssll2s7s
PREPARATION OF MHC-PEPTIDE COMPLEX~
FIELD OF THE INVENTION
The present invention relates to methods of
preparing MHC-peptide complexes in which essentially all of
the complexes comprise the same peptide. In particular the
methods comprise incubating an MHC component with a large
molar excess of a desired antigenic peptide. Alternatively
the methods comprise incubation of the desired peptide with
the MHC component under optimized pH conditions.
BACKGROUND OF THE INVENTION
The major histocompatibility complex (M~C) class II
antigens are heterodimeric cell surface (glycoproteins and are
crucial in p.ese.t---g a.lt.genic peptides to CD4 positive T
rells (Yewdell and Bennick Cell 62: 203-206 (1990)). Several
in vltro studies have showed that the percent of MHC class II
antigens occupied with antigenic peptide varied significantIy
~nd in many cases the antigen occupied fraction comprised only
a very small portion of the total MHC preparation. This can
]De explained due to one or a combination of the following
reasons: (i) the presence of various prebound endogenous
peptides in affinity-purified MHC class II antigens (Chicz et
. Nature 358:764-768 (1993) (ii) the presence of associated
invariant chain polypeptides with purified MHC class II
~olecules (Riberdy et al., Nature 360 474-477 (1992) or (iii)
lthe existence of multiple conformational states of these
molecules in solution (Dornmair et al. Cold Spring Harbor
~ymp. 54:409-416 (1989)).
Due to the low percentage of synthetic peptide-
occupied MHC antiger~ aS well as limited yield of purified MHC
class II antigens various attempts to prepare homogeneous
pure complexes of MHC class II and antigenic peptides with a
good recovery from uncomplexed or endogenously-bound complexes
have proven to be difficult or inefficient over the last
~ ~ ~ 1 5 I q PCT/US9~/12575
WO 96/10415
several years. The first method described to purify class
II-peptide complexes of defined composition involves a
biotin-avidin system where the antigenic peptide contains a
long chain thiol cleavable biotin moiety (Demotz et al. Proc.
Natl . Acad. sci. USA 88:8730-8734 (1991)). This method has
limitations in the sense that it involves several steps and
the recovery of defined complexes is significantly low
(usually 0.4-4% of the starting samples).
Thus, there remains a need for preparation methods
that are easy to carry out, readily scalable and wherein the
recovery of the desried MHC-peptide complexes is relatively
high.
SUMMARY OF THE INVENTION
The present invention provides methods for the
preparation of MHC-peptide complexes useful in ameliorating
immunological disorders, such as, for example, autoimmune
diseases, allergic responses and iransplant responses. These
complexes consist essentially of (1) an effective portion of
the MHC-encoded antigen-presenting glycoprotein; and (2) a
peptide representing a fragment of an autoantigen or other
antigenic sequence associated with the disease state to be
treated (i.e., an antigenic peptide).
The invention provide methods include contacting an
MHC component with about a 75 fold to about a 2000 fold molar
excess of the peptide, thereby forming an MHC Class II-peptide
complex. The MHC component can be derived from any MHC
allele, such as HLA-DR2. The antigenic peptide can be derived
from any antigen, for example, myelin basic protein (MBP).
Preferred peptides include MBP(83-102)Y83. The methods may
further comprise the step of ~;~ing the MHC Class II-peptide
complex with the pharmaceutically acceptable excipient in a
ratio suitable for therapeutic or diagnostic administration of
the complex.
The invention also provides methods which include
contacting the MHC component with the peptide under optimal pH
conditions, thereby forming an MHC Class II-peptide complex.
PCT~S95/12575
~096/10415
If the MHC component is DR2 and the peptide is MBP(83-102)Y83,
the optimal pH conditions are about pH 6. If the MHC molecule
is DR2 and the peptide is MBP(124-143), the optimal pH
conditions are about pH 8. If the MHC molecule is DR2 and the
peptide is MBP(143-168), the optimal pH conditions are about
pH 7.
Additionally, the present invention provides a
composition comprising a plurality of MHC-peptide complexes of
defined or homogenous composition. These compositions are
designed to target T helper cells which recognize a particular
antigen in association with a glycoprotein encoded by the MHC.
The complexes bind T cell receptors and cause non-
responsiveness in target T-lymphocytes and other cells of the
immune system.
Other advantages, objects, features and embodiments
of the present invention will become apparent from the
detailed description which follows.
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WO 96/10415 PCT/US95/12575
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA-lC show optimum pH for maximum bin~g of
various MBP peptides to purified HLA- DR2. Affinity purified
HLA-DR2 at a concentration of 20 ~g/ml was incubated with
50-fold molar excess of biotinylated MBP(83-102)y83 peptide
(Figure lA), MBP(124-143) peptide (Figure lB) and MBP(143-168)
peptide (Figure lC) at various pH. 100 mM citrate buffer was
used for pH 5.0 and 6.0, 100 mM phosphate buffer was used for
pH 7.0 and 8.0, and 10mM Tris buffer was used for pH 9 and 10.
The open circles represent the control peptide MBP (1-14).
Each data point represent an average of two determinants.
Figures 2A-2B show characterization of
DR2.MBP(83-102) complexes made at acidic pH. Complex
preparations were captured by anti-DR2 dimer specific
polyclonal antibody and the presence of heterodimer was
detected by L243 coupled peroxidase in an ELISA (Figure 2B).
Native HLA-DR2 at neutral pH was used as positive control in
this zssay ~F y~re 2A). Each data point represents an average
of triplicate determinants.
Figures 3A-3C show a time course of various MBP
peptide binding to HLA-DR2. Purified HLA-DR2 at a
concentration of 20 ~g/ml was incubated with 50-fold molar
excess of biotinylated MBP(83-102)y83 peptide (Fiugure 3A),
biotinylated MBP(124-143) peptide (Figure 3B) and biotinylated
MBP(143-168) peptide (Figure 3C) at pre optimized pH values as
described above at 37C. At indicated times aliquots were
removed and stored at - 20C and analyzed by antibody capture
plate assay. The open circles represent the binding of MBP
(1-14) peptide. Arrows in panels represent the optimum time
required for maximum binding.
Figures 4A-4C show the effect of increasing peptide
concentrations on binding of MBP peptides to DR2. Purified
HLA-DR2 at a concentration of 20 ~g/ml was incubated with
increasing mol~. excess of biotinylated-MBP(83- 102)Y83
peptide (Figure 4A), biotinylated-MBP (124-143) peptide
(Figure 4B) and biotinylated-MBP(143-168) peptide (Figure 4C)
at pre optimized pH for 72 hours at 37C. The open circles
represent the binding of MBP (1-14) peptide.
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~1V0 96/10415 PCTIUS95/12575
Figures 5A-5C show competitive binding of
biotinylated MBP peptides in presence of non-biotinylated
peptides. Figure 5A represents the binding of biotinylated
MBP(83-102)Y83 peptide with increasing concentration of
non-biotinylated MBP(83-102)Y(closed circles) and MBP(124-143)
peptide (open circles). Figure 5B represents the binding of
biotinylated MBP(124-143) in presence of non-biotinylated
MBP(83-102)Y83 (closed circles) and MBP(124-143) (open
circle). Figure 5C represents the binding biotinylated
MBP(143-168) with increasing concentrations of
non-biotinylated MBP(83-102)Y83 (closed circles) and
MBP(143-168) (open circles) at fully optimized conditions.
Figures 6A-6C show Sephadex G-75 gel filtration
purification of various DR2-peptide complexes. One mg of
complexes of DR2 and various MBP peptides were prepared and
applied on Sephadex G-75 (20ml bed volume). Fractions of
500ul was collected and measured for absorbency at 280nm.
Figures 7A-7C sho-w stabiiity of DR2.MBP peptide
complexes at various temperatures. Purified complexes were
incubated at 4C (open circles) 25C (closed circles) and 37C
(open square) and at various time, samples aliquotes were
removed as described below.
Figures 8A-8B show binding of peptide to
affinity-purified HLA-DR2. Figure 8A represents the kinetics
of biotinylated- MBP(83-102)Y33 peptide binding to HLA-DR2.
Purified HLA-DR2 at a concentration of 2 ~g/ml was incubated
with 50-fold molar excess of either biotinylated-MBP
(83-102)Y33 peptide (closed circles) or biotinylated-MBP(1-14)
peptide (open circle) at 37C and at neutral pH. At various
times aliquots were removed and frozen at -20C. At the end
of the experiment, samples were analyzed as described below.
Figure 8B represents the quantitation of biotinylated-MBP
(83-102)Y33 peptide (closed circles) and biotinylated-MBP
(1-14) peptide (open circles) bound to HLA-DR2 at various
molar excess peptide concentrations.
Figures 9A-9B show competitive binding of
biotinylated-MBP (83-102)Y33 peptide in the presence of either
MBP (83-102)Y83 or MBP (124-143) peptide. Purified HLA-DR2 at
q
~ 0 96/10415 PCT~US95/12575
a concentration of 2 ~g/ml was incubated with 300-fold molzr
excess of biotinylated-MBP (83-102)Y83 peptide and in the
presence of 0-10,000 fold molar excess of either MBP
(83-102)Y83 peptide (Figure 9A) or MBP (124-143) peptide
(Figure 9B) at 37C for 96 hours. The amount of
biotinylated-MBP (83-102)Y83 peptide associated with HLA-DR2
was then quantitated as described below.
Figures 10A-lOB show narrowbore HPLC analysis of
acid-eluted peptides. One mg of purified DR2 or MBP
(83-102)Y83-bound DR2 complexes were subjected to acetic acid
extraction. The acid-eluted peptides were analyzed on a
Waters (Millipore) HPLC system using narrowbore C-18 reverse
phase column as described below. Figure 10A shows standard
MBP (83-102)Y83 peptide; Figure 10B, endogenous peptides
eluted from purified HLA-DR2; Figure 10C, blank buffer
profile; and Figure 10D, peptide eluted from fully occupied
HLA-DR2.MBP (83-1o2)Y83 complexes.
~ ~ 0 ~ ~ ~ 9
WO96/10415 PCT~S95/12575
DETAILED DESCRIPTION OF THE INVENTION
AND PREFERRED EMBODIMENTS
Unwanted T cell activation is known to be associated
with a number of pathological, immunological disorders such
as, for example, autoimmune diseases, allergic responses and
transplant rejections. Autoimmune diseases are a particularly
important class of the diseases involving deleterious or
unwanted immune responses. In autoimmune diseases, self-
tolerance is lost and thus, the immune system attacks "self"tissue as if it were a foreign target. More than 30
autoimmune diseases are presently known to exist; myasthenia
gravis (MG) rheumatoid arthritis (RA) and multiple sclerosis
(MS), for example, are three autoimmune diseases which have
received wide-spread public attention.
Moreover, a number of allergic diseases have been
found to be associated with particular MHC alleles or have
been suspected of having an autoimmune component.
Add i ti ona 11 y, oth.er deleterious T cell-mediated responses
include the destruction of foreign cells that are purposely
introduced into the body as grafts or transplants from
allogeneic hosts. This process, known as "allograft
rejection," involves the interaction of host T cells with
foreign MHC molecules. Quite frequently, a broad range of MHC
alleles are involved in the response of the host T cell to an
allograft.
The present invention provides methods for preparing
a composition comprising a plurality of MHC-peptide complexes
of defined composition. Once formed, this composition of
homogenous MHC-peptide complexes can be used to modulate T
cell function in the treatment of immunological disorders such
as, for example, autoimmune diseases, allergic responses and
transplant rejections. In addition, the purified complexes of
the present invention can be used as vaccines to promote
immune respon.ses. When used in this embodiment, the MHC
component (either Class I or Class II) is typically modified
to allow attachment to a competent antigen presenting cell
bearing ligands involved in the co-stimulatory signal.
Alternatively, the complex may be linked to isolated co-
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~7V0 96/10415 PCTIUS95/12575
stimulatory ligands such that T cell proliferation is induced.Thus, T cells will respond to the antigenic peptide presented
by the complexes and an immune response will be initiated.
Homogenous complexes are also useful in understanding the
kinetics of MHC-peptide interaction, crystallographic
analysis, and in generating antibodies specific for a given
complex.
Complexes and methods have been described that are
useful for identifying and inhibiting those aspects of the
immune system that are responsible for undesirable immune
responses, such as, for example, autoimmunity. See, U.S.
Patent Nos. 5,130,297, 5,194,425, 5,284,935 and 5,260,422.
These complexes and methods are designed to target T helper
cells which recognize a particular antigen in association with
a glycoprotein encoded by the MHC. The complexes effectively
bind T cell receptors and cause non-responsiveness in target
T-lymphocytes and other cells of the immune system.
The complexes mad by the metnods of the present
invention contain at least two components: (1) a peptide
representing a fragment of an autoantigen or other antigenic
sequence associated with the disease state to be treated
(i.e., an antigenic peptide); and (2) an effective portion of
an MHC-encoded glycoprotein involved in antigen presentation.
An effective portion of an MHC glycoprotein is one which
comprises an antigen binding site and the regions necessary
for recognition of the MHC-peptide complex by the appropriate
T cell receptor. The MHC component can be either a Class I
or a Class II molecule. The association between the peptide
antigen and the antigen bin~ing site of the MHC protein can bQ
by covalent or noncovalent bonding. Additionally, the MHC-
peptide complex may contain an effector component which is
generally a toxin or a label. The effector portion may be
conjugated to either the MHC-encoded glycoprotein or to the
autoantigenic peptide. Complexes containing an effector
component are disclosed and claimed in U.S. Patent No.
5,194,425, supra.
Each aspect of the presently disclosed method for
the purification and characterization of MHC-peptide complexes
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'WO 96/10415 PCT/US95112S75
useful in ameliorating immunological disorders (such as, for
example, autoimmune diseases, allergic responses and
transplant rejections) will be described in great detail
below.
Isolation Of The MHC-Derived ComPonent:
As previously stated, the present invention provides
a method for preparing a composition comprising a plurality of
MHC-peptide complexes of defined composition. As used herein,
the term "of defined composition" refers to a plurality of
MHC-peptide complexes wherein at least 60 percent, usually
above 70 percent, preferably about 75 percent, and more
preferably about 95 percent or more of the complexes are
identical and free from endogenous MHC-peptide complexes. An
endogenous MHC-peptide complex is one comprising a peptide
which is associated with the MHC molecule when the molecule is
isolated from a cell that expresses the MHC molecule. In the
initial step of this method, an MHC component, having an
antigen binding site or sites, is isolated from a cell which
produces such components. The MHC component can be readily
isolated using the methods and procedures set forth herein.
Usually, the MHC component is isolated from a
natural antigen presenting cell (e.g., a B cell, a dendritic
cell, or a macrophage) or an immortalized cell line derived
from such a cell. Thus, the MHC molecule will be loaded with
endogenous peptide.
The glycoproteins encoded by the major
histocompatibility complex have been extensively studied in
both the human and murine systems. In general, they have been
classified as Class I glycoproteins, which are found on the
surfaces of all cells and primarily recognized by cytotoxic
T cells; and Class II glycoproteins, which are found on the
surface of several cells, including accessory cells such as
macrophages, and which are involved in the presentation of
antigens to T helper cells. Some of the histocompatibility
proteins have been isolated and characterized. For a general
review of MHC glycoprotein structure and function, see, e.g.,
Fundamental Immunology (3d Ed., W.E. Paul, (ed.), Ravens
q
WO96i10415 PCT~S95/12575
Press, N.Y. (1993)). The term "isolated MHC component" as
used herein refers to an MHC glycoprotein or an effective
portion of an MHC glycoprotein (i.e., one comprising an
antigen binding site or sites and the sequences necessary for
recognition by the appropriate T cell receptor) which is in
other than its native state (i.e., not associated with the
cell membrane of the cell that normally expresses MHC). As
described in detail below, the MHC component is preferably
solubilized from an appropriate cell source. For human MHC
molecules, human lymphoblastoid cells are particularly
preferred as sources for the MHC component.
The MHC glycoprotein portions of the complexes of
the invention, then, can be obtained by isolation from
lymphocytes and screened for their ability to bind the desired
peptide antigen. The lymphocytes are from the species of
individual which will be treated with the complexes once
formed. They may be isolated, for example, from the human B
cc''s ~, al. individual suffering from the targeted autoimmune
disease, which have been immortalized by transformation with a
replication deficient Epstein-Barr virus, utilizing techniques
known to those in the art.
MHC glycoproteins have been isolated from a
multiplicity of cells using a variety of tP~hniques including,
for example, solubilization by treatment with papain, by
treatment with 3M KCl and by treatment with detergent. In a
preferred method, detergent extraction of Class II protein
from lymphocytes followed by affinity purification is used.
The detergent can subsequently be removed by dialysis or
through the use of selective binding beads, ~.g., Bio Beads.
Methods for purifying the murine I-A (Class II)
histocompatibility proteins have been disclosed by Turkewitz,
et al., Molecular Immunology (1983~ 20:1139-1147. These
methods, which are also suitable for Class I molecules,
invGlvc .he preparation of a soluble membrane extract from
cells containing the desired MHC molecule using nonionic
detergents, such as, for example, NP-40, TWEEN~ 80 and the
like. The MHC molecules are then purified by affinity
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WO96/10415 PCT~Sg~/12~75
11
chromatography, using a column containing antibodies raised
against the desired MHC molecule.
The isolated antigens encoded by the I-A and I-E
subregions have been shown to consist of two noncovalently
bonded peptide chains: an alpha chain of 32-38 kD and a beta
chain of 26-29 kD. A third, invariant, 31 kD peptide is
noncovalently associated with these two peptides, but it is
not polymorphic and does not appear to be a component of the
antigens on the cell surface (Sekaly, J. Exp. Med. (1986)
164:1490-1504). The alpha and beta chains of seven allelic
variants of the I-A region have been cloned and sequenced.
The human Class I histocompatibility proteins have
also been studied. The MHC of humans (HLA) on chromosome 6
has three loci, HLA-A, HLA-B, and HLA-C, the first two of
which have a large number of alleles encoding alloantigens.
These are found to consist of a 44 kD subunit and a 12 kD
beta2-microglobulin subunit which is common to all antigenic
specificities. Isoiation or ~nese detergent-soluble HLA
antigens was described by Springer, et al., Proc . Natl . Acad .
Sci . USA ( 1976) 73:2481-2485; Clementson, et al., in "Membrane
Proteins" (Azzi, A., ed.); Bjorkman, P., Ph.D. Thesis Harvard
(1984).
The Antiqenic PePtide:
Antigenic proteins or tissues for a number of
autoimmune diseases are known. In experimentally induced
autoimmune diseases, for example, the following antigens
involved in pathogenesis have been characterized: native
txpe-II collagen has been identified in collagen-induced
arthritis in rat and mouse, and mycobacterial heat shock
protein in adjuvant arthritis (Stuart, et al., (1984), Ann.
Rev. Immunol. 2:199-218; van Eden, et al., (1988), Nature
331:171-173.); thyroglobulin has been identified in
lexperimental allergic thyroiditis (EAT) in mouse (Maron, et
~l., (1988), J. Exp. Med. 152:1115-1120); acetyl choline
receptor (AChR) has been identified in experimental allergic
;myasthenia gravis (EAMG) (Lindstrom, et al. (1988), Adv.
.Immunol. 42:233-284); and myelin basic protein (MBP) and
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WO 96/10415 PCT/US95/12~75
12
proteolipid protein (PLP) have been identified in experimental
allergic encephalomyelitis (EAE) in mouse and rat (See Acha-
Orbea, et al., supra). In addition, target antigens have been
identified in humans: type-II collagen has been identified in
human rheumatoid arthritis (Holoshitz, et al., (1986), Lanc~t
ii:305-309); and acetyl choline receptor in myasthenia gravis
(Lindstrom, et al ., ( 19 88), su pra ) .
It is believed that the presentation of antigen by
the MHC glycoprotein on the surface of antigen-presenting
cells (APCs) occurs subsequent to the hydrolysis of the
antigenic proteins into smaller peptide units. The location
of these smaller segments within the antigenic protein can be
determined empirically. These segments are thought to be
about 8 to about 18 residues in length and to contain both the
agretope (recognized by the MHC molecule) and the epitope
(recognized by the T cell receptor on the T-helper cell). The
length of peptides capable of binding an MHC molecule,
however, can vary. Thus, peptides or greater length, e.g., up
to 100 residues can also be used in the complexes. Usually,
the peptides will be less than about 50 residues in length,
preferably less than about 30.
Using standard procedures one of skill in the art
can readily determine the relevant antigenic peptide for the
antigens associated with many immune disorders. For instance,
in multiple sclerosis (MS), which results in the destruction
of the myelin sheath in the central nervous system, it is
known that myelin basic protein (MBP) (i.e., the major protein
component of myelin) is the principal autoantigen. Pertinent
segments of the MBP protein can also be determined empirically
using the procedure described above and a strain of mice which
develops experimental allergic encephalitis (EAG) when
immunized with bovine myelin basic protein.
Moreover, although systemic lupus erythematosus
(SLE) has a complex systemology, it is known to result from an
autoimmune response to red blood cells. Peptides which are
the antigenic effectors of this disease are found in the
proteins on the surface of red blood cells. Rheumatoid
arthritis (RA), a chronic inflammatory disease, results from
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WO 96/10415 PCT/US95/12575
13
an immune response to proteins found in the synovial fluid.
Insulin-dependent diabetes mellitus (IDDM) results from
autoimmune attack on the beta cells within the Islets of
Langerhans which are responsible for the secretion of insulin.
Circulating antibodies to Islets cells surface antigens and to
insulin are known to precede IDDM. Critical peptides in
eliciting the immune response in IDDM are believed to be
portions of the insulin sequence and the beta cell membrane
surface proteins.
Once determined, the relevant antigenic peptide
subunits can be readily synthesized using standard automated
methods for peptide synthesis being that they are relatively
short in length. Alternatively, they can be made
recombinantly using isolated or synthetic DNA sequences, but
this is not the most efficient approach for peptides of this
length.
mh2 Ef2c.0r Com~onent:
Additionally, the complexes of the invention can be
designed to destroy the immune response to the peptide in
question. In this instance, the MHC-peptide complex will
contain an effector component. The effector portion of the
MHC-peptide molecule can be, for example, a toxin, a
chemotherapeutic agent, an antibody to a cytotoxic T-cell
surface molecule, a lipase, or a radioisotope emitting "hard"
radiation (e.g., beta radiation). A number of protein toxins
are well known in the art and include, for example, ricin,
diphtheria, gelonin, Pseudomonas toxin, and abrin.
Chemotherapeutic agents include, but are not limited to,
doxorubicin, daunorubicin, methotrexate, cytotoxin, and anti-
sense RNA. Moreover, antibiotics can also be used as the
effector component. Antibodies have been isolated to
cytotoxic T-cell surface molecules and these may thus operate
as toxins. In addition, radioisotopes such as yttrium-90,
phosphorus-32, lead-212, iodine-131, or palladium-109 can be
used. The emitted radiation effects the destruction of the
target T-cells.
WO96/10415 PCT~S95/12575
14
In some cases the active portion of the effector
component is entrapped in a delivery system such as a liposome
or dextran carrier; in these cases, either the active
component or the carrier may be bound in the complex.
If the effector molecule is intended to be a label,
a gamma-emitting radioisotope such as technetium-gg or indium-
lll can be used. In addition, other types of labeling such as
fluorescence labeling by, for example, fluorescein can be
used.
The effector component can be attached to the MHC
glycoprotein or, if its nature is suitable, to the peptide
portion. Iodine 131 or other radioactive labels, for example,
can often be included in the peptide determinant sequence.
Complexes containing an effector component are disclosed and
claimed in U.S. Patent No. 5,194,425, supra.
Formation of the MHC-PePtide ComDlex:
~ nce ~he ~nC component has been isolated and the
antigenic peptide has been synthesized, these two elements can
be associated with one another to form an MHC-peptide complex
using the methods of the invention. The antigenic peptides
are preferably associated noncovalently with the pocket
portion of the MHC protein by, for example, mixing the two
components together. Excess peptide can be removed using a
2S number of st~n~Ard procedures, such as, for example, by
ultrafiltration or by dialysis.
The present invention is based in part on the
discovery that large molar excess of peptide can be used to
produce 100% loaded, homogenous MHC-peptide complexes, that is
complexes of defined composition. Typically, about a 50 to
about a lO0-fold molar excess of peptide is used. About 75-
fold molar excess is usual. However, higher levels of between
about a 200 and about a 300-fold excess can be used. No more
than about a 2000-foid excess of peptide is used, usually less
than about lO00-fold excess and preferably less than about
500-fold excess.
Alternatively, homogenous compositions of MHC-
peptide complexes can be prepared by optimizing the pH
WO 96110415 PCT/US95/1257
conditions in which the MHC component and peptide are
incubated. As shown below, such an approach has provided for
increased peptide loading using three different MHC-peptide
complexes.
s
Formulation and Administration of the MHC-Peptide Complex:
Administration of the complexes made the methods of
the invention is usually systemic and is effected by
injection, preferably intravenous. Formulations compatible
with the injection route of administration may, therefore, be
used. Suitable formulations are found in ~emington~s
Pharmaceutical Sciences, (Mack Publishing Company,
Philadelphia, PA, 17th ed. (1985)). A variety of
pharmaceutical compositions comprising complexes of the
present invention and pharmaceutically effective carriers can
be prepared. The pharmaceutical compositions are suitable in
a variety of drug delivery systems. For a brief review of
present methods of drug delivery, see, e.g., Langer, Science
249:1527-1533 (1990).
In preparing pharmaceutical compositions comprising
MHC-peptide complexes, it is frequently desirable to modify
the complexes to alter their pharmacokinetics and
biodistribution. For a general discussion of
pharmacokinetics, see, Remington's Pharmaceutical Sciences,
supra, Chapters 37-39. A number of methods for altering
pharmacokinetics and biodistribution are known to one of
ordinary skill in the art (see, e.g., Langer, supra).
The pharmaceutical compositions are intended for
parenteral, topical, oral or local administration, such as by
aerosol or transdermally, for prophylactic and/or therapeutic
treatment. The pharmaceutical compositions can be
administered in a variety of unit dosage forms depending upon
the method of administration. For example, unit dosage forms
suitable for oral administration include powder, tablets,
pills, and capsules.
Preferably, the pharmaceutical compositions are
administered intravenously. Thus, compositions for
intravenous administration are provided which comprise a
WO 96/10415 ~ PCT/US95/12575
16
solution of the complex dissolved or suspended in an
acceptable carrier, preferably an aqueous carrier. A variety
of aqueous carriers may be used, e.g., water, buffered water,
0.4% saline, and the like. For instance, phosphate buffered
saline (PBS) is particularly suitable for administration of
soluble complexes of the present invention. A preferred
formulation is PBS containing 0.02% TWEEN-80. These
compositions may be sterilized by conventional, well-known
sterilization techniques, or may be sterile filtered. The
resulting aqueous solutions may be packaged for use as is, or
lyophilized, the lyophilized preparation being combined with a
sterile aqueous solution prior to administration. The
compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological
conditions, such as pH adjusting and buffering agents,
tonicity adjusting agents, wetting agents and the like, for
example, sodium acetate, sodium lactate, sodium chloride,
pu.as~ium chloride, calcium chloride, sorbitan monolaurate,
triethanolamine oleate, etc.
The concentration of the complex can vary widely,
i.e., from less than about 0.05%, usually at or at least
about 1% to as much as lO to 30% by weight and will be
selected primarily by fluid volumes, viscosities, etc., in
accordance with the particular mode of administration
selected. Preferred concentrations for intravenous
administration are about 0.02% to about 0.1% or more in PBS.
For solid compositions, conventional nontoxic solid
carriers may be used which include, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharin, talcum, cellulose, glucose,
sucrose, magnesium carbonate, and the like. For oral
administration, a pharmaceutically acceptable nontoxic
composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed,
and generally 10-95% of active ingredient.
For aerosol administration, the complexes are
preferably supplied in finely divided form along with a
surfactant and propellant. The surfactant must, of course, be
r q
~0 96/10415 PCT/US95/12575
17
nontoxic, and preferably soluble in the propellant.
Representative of such agents are the esters or partial esters
of fatty acids containing from 6 to 22 carbon atoms, such as,
for example, caproic, octanoic, lauric, palmitic, stearic,
linoleic, linolenic, olesteric and oleic acids with an
aliphatic polyhydric alcohol or its cyclic anhydride such as,
for example, ethylene glycol, glycerol, erythritol, arabitol,
mannitol, sorbitol, the hexitol anhydrides derived from
sorbitol, and the polyoxyethylene and polyoxypropylene
derivatives of these esters. Mixed esters, such as mixed or
natural glycerides may be employed. The surfactant may
constitute 0.1%-20% by weight of the composition, preferably
0.25-5%. The balance of the composition is ordinarily
propellant. Liquefied propellants are typically gases at
ambient conditions, and are condensed under pressure. Among
suitable liquefied propellants are the lower alkanes
containing up to 5 carbons, such as butane and propane; and
prefer~ly fiuorinated or fluorochlorinated alkanes. Mixtures
of the above may also be employed. In producing the aerosol,
a container equipped with a suitable valve is filled with the
appropriate propellant, containing the finely divided
compounds and surfactant. The ingredients are thus maint~i n~
at an elevated pressure until released by action of the valve.
The compositions containing the complexes can be
administered for therapeutic, prophylactic, or diagnostic
applications. In therapeutic applications, compositions are
administered to a patient already suffering from a diseasQ, as
described above, in an amount sufficient to cure or at least
partially arrest the symptoms of the disease and its
complications. An amount adequate to accomplish this is
defined as "therapeutically effective dose." Amounts
effective for this use will depend on the severity of the
disease and the weight and general state of the patient. As
discussed above, this will typically be between about 0.5
mg/kg and about 25 mg/kg, preferably about 3 to about 15
mglkg.
In prophylactic applications, compositions
containing the complexes of the invention are administered to
/ q
WO96/10415 PCT~S95/12575
18
a patient susceptible to or otherwise at risk of a particular
disease. Such an amount is defined to be a "prophylactically
effective dose." In this use, the precise amounts again
depend on the patient's state of health and weight. The doses
will generally be in the ranges set forth above.
In diagnostic applications, compositions containing
the appropriately complexes or a cocktail thereof are
administered to a patient suspected of having an autoi~llnP
disease state to determine the presence of autoreactive T
cells associated with the disease. Alternatively, the
efficacy of a particular treatment can be monitored. An
amount sufficient to accomplish this is defined to be a
"diagnostically effective dose." In this use, the precise
amounts will depend upon the patient's state of health and the
like, but generally range from O.Ol to lO00 mg per dose, espe-
cially about lO to about lO0 mg per patient.
This invention will be described in greater detail
by way of specific examples. The rollowing examples are
offered for illustrative purposes, and is intended neither to
limit or define the invention in any manner.
5 1 q
WO 96/10415 PCT/US9~/12~7
19
EXAMPLES
Example 1
In this example, high and low affinity
immunodominant peptide epitopes from MBP were selected and
shown to have pH dependent binding characteristics to purified
HLA-DR2.
Materials and Methods
Cell lines, antibodies and chemicals
The hybridoma cell line L243, producing monoclonal
antibodies against monomorphic human HLA-DR molecules was
obtained from American Type Culture Collection, Bethesda, MD.
Homozygous lymphoblastoid cell line GM 03107 expressing
HLA-DR2 (DRB1*1501 and DRB5*0101) was obtained from the
National Institute of General Medical Sciences (NIGMS) human
genetic mutant cell repository (Coriell Institute of Medical
Research, NJ). Rabbit polyclonal antibody against HLA-DR2
heterodimer was obtained from Zymogenetics, WA.
Para-nitrophenyl phosphate disodium hexahydraie was purcnased
from Sigma Chemicals, MO. Immunopure biotinylated bovine
serum albumin conta_-.ing known amount of biotin molecules was
purchased from Pierce Chemicals. Streptavidin conjugated
purified alkaline phosphatase was obtained from Tropix, Inc.
MA.
Purification of human HLA-DR2 from lymphoblastoid cells
Purification of HLA-DR2 from EBV-transformed
lymphoblastoid cells was carried out as described earlier tNag
B. et al, Proc. natn. Acad. sci. U.S.A. 90:1604-1608 (1993)
with some minor modifications. Triton X-100 cell lysate was
applied on to L243 coupled sepharose-4B column and the bound
DR2 was eluted in phosphate buffer containing 0.05% n-dodecyl
~-D-maltoside (DM) detergent at pH 11.3. Fractions were
immediately neutralized with lM acetic acid and the DR2 pool
was collected through a DEAE ion exchange column in a
phosphate buffer containing 0.5M NaCl and 0.05% DM, p~ 6Ø
Purified protein was then filtered through a 180 kD membrane
and characterized by 13.5% SDS polyacrylamide gel
electrophoresis followed by silver staining (T~hTogix silver
stain kit, Belmont, CA).
q
WO 96/10415 PCT/US9~/1257
Synthesis of various MBP peptides
Various N-acetylated myelin basic protein peptide
analogs: the MBP(83-102)Y83 peptide with the sequence
Ac-YD~N~vv~KNlv~ K~ P; the MBP(124-143) peptide with the
sequence Ac-GFGYGGRASDYKSAHKGFKGi MBP(143-168) with the
sequence Ac-FKGVDAQGTLSKIFKLGGRD and the MBP(1-14) with the
sequence Ac-ASQKRPSQRHGSKY were synthesized by the standard
solid phase method using side-chain protected Fmoc amino acids
on an Applied Biosystems 43lA automated peptide synthesizer.
The de protected, crude peptides were purified by
reverse-phase HPLC, and the homogeneity and identity of the
purif ied peptides were confirmed by mass spectrometry.
Preparation of Biotin-MBP peptide conjugates
The peptide resin (0. 25 mmoles) was suspended in 15
ml N-methylpyrrolidinone (NMP) containing 122 mg of D-biotln,
79.6 mg of l-hydroxybenzotriazole hydrate (HOBT) and 81 ~l of
6.4 M diisopropylcarbodiimide (DIPCDI) solution. The
~usper.slû,. was gen-ly mixed overnight at room temperature. A
small sample of resin was washed with NMP and subjected to
ninhydrin test to confirm the completion of the reaction. The
resin was then filtered, washed with 50 ml NMP and methanol
alternately twice and then with methanol and dichloromethane
(DC~) alternately twice. The resin was dried under vacuum.
The peptide was cleaved from the resin using trifluoroacetic
acid (TFA) containing scavengers. The crude biotinylated
peptides were isolated by precipitation with ether and dried
under vacuum. The biotinylated peptides were then purified by
reverse-phase HPLC and the identity of the purified peptides
were confirmed by mass spectrometry.
Complex preparation and peptide binding assay
For the quantitation of bound peptide, affinity-
purified HLA-DR2 at a concentration of 2 ~g/ml was incubated
with increased molar excess of biotinylated-MBP peptides at
37C for 9~ hours at pH 7Ø The resulting complex
preparations were analyzed by antibody capture plate assay
using an enzyme-conjugated avidin system as described earlier
(Jensen, J. Exp. Med 171:1779-1784(1991); Reay et al, Eur.
Molec. Biol. Org. J. 11:2829-2839 (1992)) with some
1 9
WO 96/10415 PCTIUS95/12575
21
modifications. The standard curve was generated using the
BSA-biotin conjugate ranging between 0.014-1.80 pmoles.
Equivalent amounts of biotinylated peptides in the absence of
MHC class II antigens were used as controls which showed less
than 1% non-specific binding in this assay and was subtracted
for calculating the percent peptide occupancy.
Dissociation kinetics measurements by plate assay
Complexes of HLA-DR2 and biotinylated-MBP peptides
were prepared and purified from unbound peptide by G-75 size
exclusion gel filtration chromatography. Resulting complexes
were then incubated at three different temperatures (4, 25
and 37C). At various time, complex samples were removed and
frizzed at -20C. At the end of each experiment, samples were
analyzed by antibody capture plate assay as described above.
Results
Various association parameters such as pH, peptide
concentration and the duration of peptide incubation were
tested using affinity purified HLA-DR2 ( containing DRBl*
1501/DRB5* 0101) and four different MBP peptides. These
peptides were selected based on their immunodominant
characteristics along with affinities toward HLA-DR2 (Valli et
al, J. clin. Invest. 91:616-628 (1993)). As shown in example
2, the MBP (1-14) peptide had almost no affinity to purified
HLA-DR2 and was used as a control peptide in all binding
assay.
To determine the optimum pH for maximum peptide
occupancy, HLA-DR2 was incubated with 50 fold molar PYce~s of
various MBP peptides in binding buffer with pH ranging from
5 to 10. As shown in Figure lA, only the MBP(83-102) peptide
showed increased binding at acidic pH. In contrast, the
MBP(124-143) peptide showed maximum binding at basic pH
(Figure lB). The maximum peptide binding of the third MBP
peptide MBP(143-168) was observed at neutral pH (Figure lC).
Since acidic conditions (pH 4 or below) are known to
dissociate MHC class II heterodimers into monomeric ~ and ~
chains (Passmore et al, J. Immunol. Meth. 155:193-200 (1992)),
we sought to evaluate the molecular characteristics of
DR2.MBP(83-102) complexes prepared at pH 5 and 6 have by non-
2 ~ ! q
WO 96/10415 PCT/US95/12575
22
reduced SDS-PAGE analysis. The gel electrophoresis result
shows that complexes prepared at pH 5 and 6 have significant
dissociation of hetrodimers into monomers. One possibility of
such dissociation could be due to the effect of
electrophoresis conditions. To distinguish the observed
dissociation at pH 5 or pH 6 in gel from the electrophoretic
condition in the presence of SDS, complex preparations made at
various pH values were analyzed by heterodimeric specific
ELISA. Ninety six well plates were coated with anti-DR2
polyclonal antibody that has been characterized to recognize
only the heterodimeric DR2. Bound complexes were then
detected by peroxidase conjugated L243 monoclonal antibody
which is also specific for DR2 heterodimers. It was found
that complexes prepared at pH 5.0 and 6.0 were fully
recognized by dimer specific antibodies suggesting that the
DR2.MBP complexes heterodimers exist prepared at pH 5.0 and
6Ø Although this result clearly confirms that the
dissociation observed in SDS-PAGE analysis is due to
electrophoretic conditions, MHC class II appears to be more
open in structure as predicted earlier (Jenson, J. Exp. Med
171:1779-1784 (1990)). The specificity of peptide binding at
various pH values was demonstrated by incubating equal amount
of the non-binding MBP(1-14) peptide with HLA-DR2 which did
not show any significant binding in all cases. Based on these
results, the optimum pH 6, 7 and 8 were selected for
MBP(83-102), MBP(143-168) and MBP(124-143) peptides,
respectively.
The time course for maximum peptide binding was
examined by incubating various biotinylated MBP peptides to
HLA-DR2 at respective optimum pH of the binding buffers.
Samples were incubated at 37C and at various time aliquots
were removed and stored at -20C. The on rate kinetic results
presented in Figurè 3 show that the binding of all three MBP
peptides was complete by 72 hours. Further increase in
incubation period did not increase the occupancy of DR2 with
peptide. In case of all MBP peptides, an increase in peptide
binding was observed initially with time followed by a small
decrease before stabilization of the MHC-peptide complexes.
~ -7 ~ 9
WO 96/1041~ PCT/US95/12575
23
This characteristics kinetics of peptide binding to purified
MHC class II molecules was consistently observed for several
murine, rat and human class II-peptide complex formation in
our laboratory. The explanation of such kinetics is not
clearly understood well at this time. One explanation could
be due to the existence of two conformational states of
purified MHC class II molecules so called "floppy" and
"compact" (Dornmair et al, Cold Spring Harbor Symp. 54:409-416
(1989)). The floppy form is considered as an open structure
molecule based on SDS PAGE analysis and may have weak peptide
binding affinity.
In order to ~Y~ine the effect of peptide
concentrations on percent occupancy of MHC class II, HLA-DR2
was incubated with increasing amount of three MBP peptides at
their optimum pH for 72 hours. An increase in peptide
concentration showed increase in binding in case of all three
MBP peptides tested. Results presented in Figure 4 show that
approximateiy 50-100 fold molar excess of each MBP peptide
over DR2 concentration was sufficient enough for complete
saturation of DR2. In case of both high affinity MBP peptides
tMBP(83-102) and MBP(124-143)], 50 fold molar excess peptide
concentration lead to almost 100% occupancy of DR2 at their
optimum pH (Figure 4A and 4B). However, in the case of low
affinity MBP(143-168) peptide, such increase in peptide
concentration did not show 100% occupancy of DR2. In this
case the binding was saturated at 100 fold molar excess
peptide concentration with 35% occupancy of DR2 (Figure 4C).
In a separate experiment when the binding of M3P(124-143)
peptide to DR2 was examined at pH 6 instead of the optimized
pH 8, percent peptide occupancy did not reached to 100% even
at 1000 fold molar excess peptide suggesting that the pre
selection of optimum pH is a critical step for in vitro
peptide loading. The specificity of peptide binding at
incre~sed peptide concentrations was demonstrated by
incubating equivalent amounts of MBP (1-14) peptide as shown
in each panel of Figure 4.
Further specificity of the binding of all three MBP
peptides to HLA-DR2 at optimized binding conditions was
5 t ~
'WO96/10415 PCT~Sg~/12575
24
demonstrated by competitive binding assay (Figure 5).
Purified HLA-DR2 was incubated with biotinylated peptides in
the presence of increasing concentrations of either non
biotinylated same peptide or next high affinity MBP peptide
under optimized binding conditions. As shown in Figure 5A,
the binding of biotinylated MBP(83-102) was completely
inhibited by 10 fold excess concentration of non biotinylated
MBP(83-102) peptide. Similarly, approximately 40% inhibition
of the binding of biotinylated MBP(83-102) peptide was
observed with MBP(124-143). The partial inhibition of
biotinylated MBP (83-102) peptide binding to DR2 by
MBP(124-143) can be explained due to the existence of two
different conformational states of purified DR2 with different
affinities towards MBP(83-102) peptide. The binding of
biotinylated MBP(124-143) peptide was completely inhibited by
both MBP(83-102) as well as MBP(124-143) non biotinylated
peptides (Figure 5B). As expected the MBP(83-102) being more
effective than ~P t;~4-i~3; baced on their affinities to DR2.
Similarly, the binding of the biotinylated MBP (143-168)
peptide to DR2 was drastically inhibited by equimolar
concentrations of both MBP (83-102) as well as MBP (124-143)
peptides. Thus, the competitive binding results correlate~
well with the affinity of three MBP peptides to DR2.
Finally, the stability of various complexes of DR2
and MBP peptides prepared at fully optimized binding
conditions were eYA~ined at three different temperatures (4,
25O and 37C) for 45 days. Prior to the stability studies,
complexes were purified from free unbound peptides by SephAA~Y
G-75 gel filtration size-exclusion chromatography (Figure 6).
The dissociation kinetics data presented in Figure 7 show that
all three complexes of DR2 and MBP peptides are stable at 4C.
At 25C, only the complexes of DR2 and MBPt83-102) peptide
showed stable association for a longer period of time and at
37C, all three complexes show increased dissociation of the
bound peptide with time. The off-rate kinetics was clearly
different for the DR2.MBP(83-102) peptide complexes as
compared to other two complexes at 37C and the dissociation
~ ~ o ~ ~ ~ q
WO 96/10415 PCT/US9~/1257S
kinetics data correlate well with the binding affinity of
three MBP peptides to purified DR2.
In conclusion, results presented here clearly
demonstrate that the optimization of the in vitro binding
conditions can ~X;m; ze the loading of antigenic peptides to
purified MHC class II molecules. Among various binding
parameters tested in this study, pH of the binding buffer
appears to be the most critical in peptide loading. The
optimum pH for maximum peptide binding differs for each
peptide and MHC class II molecule based on the net charge of
the peptide and the binding groove of MHC class II molecule.
In the case of high affinity peptides, changing pH of the
binding buffer can result in 100% occupancy of MHC class II
molecules. Such binding of peptides at altered pH appears to
be specific as demonstrated by both competitive assay in this
report. Good correlation of high affinity peptide and
immunodominant epitopes suggest that binding at a pH spectrum
of antigenic epitopes can be utilizeu in sc~eening nigh
affinity immunodominant epitopes of an antigen. Since
purified complexes of MHC class II and antigenic peptides can
be utilized in antigen specific therapy of auto immune
diseases, pH dependent preparation of MHC class II-peptide
complexes of defined composition has significant clinical
relevance.
Example 2
This example describes an alternative method of
loading purified MHC class II antigens with synthetic peptide
with 100% recovery by co-incubating MHC class II and antigenic
peptide at higher peptide concentrations at neutral pH.
Materials and Methods
C~ll lines, antibodies and chemicals
The hybridoma cell line L243, producing monoclonal
antibodies against monomorphic human HLA-DR ~olecules was
obtained from American Type Culture Collection, Bethesda, MD.
Homozygous lymphoblastoid cell line GM 03107 expressing
HLA-DR2 (DRB1*1501 and DRB5*0101) was obtained from the
National Institute of General Medical Sciences (NIGMS) human
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WO 96/10415 PCT/US95/12575
26
genetic mutant cell repository (Coriell Institute of Medical
Research, NJ). Ampholines and various isoelectric point
markers for two-dimensional electrophoresis were purchased
from Bio-Rad Laboratories, Inc.
Purification of human HLA-DR2 from lympho~lastoid cells
EBV-transformed lymphoblastoid cells were cultured
in RPMT 1640 medium containing 2 mM L-glutamine and 10% heat
inactivated FBS, and were harvested at a density of 1x106
cells/ml. Purification of monoclonal antibody and coupling to
CNBr-activated Sepharose 4B was carried out as described
earlier (Nag et al, J. ~mmun. 148:3483-3491 (1992)). HLA-DR2
molecules were purified from Triton X-100 membrane extracts of
cultured GM 03107 lymphoblastoid cells on L243 monoclonal
antibody-coupled Sepharose 4B column as described earlier (Nag
et al, ~. Immunol. 150:1358-1364 (1993)) with some
modifications. Detergent extracted cell lysate was applied on
an antibody affinity column and washed with 10 column volumes
GL PDS containing 0.5% Triton X-100 followed by 5 column
volumes of PBS containing 0.01% Tween-80. The bound DR2 was
eluted in 20 mM phosphate buffer containing 0.01% Tween-80, pH
11.3. Fractions were immediately neutralized with acetic acid
and the DR2 pool was collected through a DEAE ion exchange
column in a phosphate buffer containing 0.5M NaCl and 0.01%
Tween-80, pH 6Ø Purified protein was then filtered through
2S a 180 kD membrane. Affinity-purified DR2 characterized by
13.5% SDS polyacrylamide gel electrophoresis followed by
silver staining (LabLogix silver stain kit, Belmont, CA).
Synthesis of peptides
Various N-acetylated myelin basic protein peptide
analogs: the MBP(83-102)Y83 peptide with the sequence
Ac-YDE~vvn~KNlv~ P; the MBP(124-143) peptide with the
sequence Ac-GFGYGGRASDYKSAHKGFKG and the MBP(1-14) with the
sequence Ac-ASQKRPSQRHGSKY were synthesized by the standard
s~iid phase method using side-chain protected Fmoc amino acids
on an Applied Biosystems 431A automated peptide synthesizer.
The deprotected, crude peptides were purified by reverse-phase
HPLC, and the homogeneity and identity of the purified
peptides were confirmed by mass spectrometry.
~ ~ o ~
WO96/10415 pcT~s9~ll2s7s
27
Pre para ti on o f ~3i o tin -MBP pe pti d e con j u ga tes
The peptide resin (0.25 mmoles) was suspended in 15
ml N-methylpyrrolidinone (NMP) containing 122 mg of D-biotin,
79.6 mg of l-hydroxybenzotriazole hydrate (HOBT) and 81 ~l of
6.4 M diisopropylcarbodiimide (DIPCDI) solution. The
suspension was gently mixed overnight at room temperature. A
small sample of resin was washed with NMP and subjected to
ninhydrin test to confirm the completion of the reaction. The
resin was then filtered, washed with 50 ml NMP and methanol
alternately twice and then with methanol and dichloromethane
(DCM) alternately twice. The resin was dried under vacuum.
The peptide was cleaved from the resin using trifluoroacetic
acid (TFA) containing scavengers. The crude biotinylated
peptides were isolated by precipitation with ether and dried
under vacuum. The biotinylated peptides were then purified by
reverse-phase HPLC and the identity of the purified peptides
were confirmed by mass spectrometry.
Complex preparation and peptid~ binding assay
For the quantitation of bound peptide, affinity-
purified HLA-DR2 at a concentration of 2 ~g/ml was incubated
with increased molar excess of biotinylated-MBP peptides at
37C for 96 hours at pH 7Ø The resulting complex
preparations were analyzed a plate assay using an
enzyme-conjugated avidin system as described earlier (Reay et
al, E~BO J. 11:2829-2839 (1992)) with some modifications. One
~g per 50 ~g affinity-purified L243 monoclonal antibody was
coated per well of a 96-well plate in PBS. The plate was
incubated at for 18 hours at 4C and wells were blocked with
1% fish gelatin at 25C for 30 minutes. Preformed complexea
(0.78-100 ng) at a concentration of 15.6 ng/ml-2.0 ~c/ml
(0.013 - 1.66 pmoles) were applied to each well in a PBS
buffer containing 0.1~ fish gelatin, 0.01% Tween-80 and 0.02%
azide. The plate was incubated at 25C for 2 hours and washed
with PBS containing 0.1~ Tween-20. The bound biotinylated
peptide was detected by incubating the plate at 25OC for 30
minutes in the presence of streptavidin-alkaline phosphatase
conjugate (Tropix, MA) diluted to 5000 fold in PBS containing
0.1~ fish gelatin. Wells were washed with 50 mM Tris HCl, pH
` I q
WO 96/10415 PCT/US95/12575
28
7.0 containing 0.1% Tween-20 and developed with 200 ~l/well of
1 mg/ml of p-Nitrophenyl phosphate disodium (Sigma Chemicals)
dissolved in o.lM Diethanolamine pH 10. The percent of DR2
antigens with bound peptide was then calculated from the
standard curve. The st~n~rd curve was generated using the
BSA-biotin conjugate ranging between 0.014-1.80 pmoles.
Equivalent amounts of biotinylated peptides in the absence of
MHC class II antigens were used as controls which showed less
than 1% non-specific binding and was subtracted for
calculating the percent peptide occupancy.
Acid extraction of bound peptides and narrowbore HPLC analys~s
For the reverse phase HPLC analysis, milligram
quantity of DR2 at a concentration of 0.1 mg,/ml was incubated
with non-biotinylated MBP(83-102)y83 peptide and the unbound
peptide was removed by passing the complex mixture through
L243-coupled Sepharose 4B column. Tween-80 detergent was then
removed by ethanol : chloroform (1:4) phase separation prior
to the acid extraction of endogenously-bound peptides.
Extraction of bound peptides from complexes of HLA-DR2 and MBP
peptide was carried out as described earlier. (Chicz et al,
Nature 358:764-768 (1992)) with some minor modifications.
Precipitated complex preparation was incubated at 70C for 15
minutes in the presence of 10% acetic acid. The reaction
mixture was centrifuged and the peptide pool supernatant was
collected, frozen to -80C and lyophilized. Reverse-phase
high performance liquid chromatography (HPLC) was performed on
a Waters (Millipore) 590 model using C-18 (0.21 x 15 cm) Vydac
218TP5215 narrowbore column with a linear gradient of
increasing acetonitrile from 10-60% in 0.1% trifluoroacetic
acid (TFA). The acid-eluted HPLC peptide peak from purified
complexes was collected and lyophilized, and the identity was
confirmed by mass spectrometry.
Two-dimensional gel electrophoresis
The method described earlier by O'Farrell (O'Farrell
and Goodman, Proc. Natl. Acad. Sci. USA 90:8797-8801 (1976))
was used with some modifications to separate the polypeptides
according to their charge in the first dimension (IEF) and
then according to their size in the second dimension.
q
WO 96/10415 PCT/US95/12575
29
Briefly, the first dimensional gels were poured to a héight of
6.5 cm in a glass tube (1 mm inner diameter x 7.5 cm length).
The gel solution contained 9.2 M urea, 5.5% acrylamide/bis, 2%
Triton X-100 and a mixture of 1.5% ampholines pH 5-7 and 0.5%
pH 3-10 which was degassed and polymerized by adding 50 ~l of
10~ ammonium persulfate and 18 ~l of
N,N,N',N'-tetramethylethy-lenediamine (TEMED) per 10 ml of gel
mixture. Purified HLA-DR2 and complexes of DR2 with
MBP(83-102)Y83 peptide were concentrated by acetone
precipitation, resuspended in sample buffer (9.5 M urea, 2.0%
Triton X-100, 150 mM DTT and a mixture of 1.5% ampholines pH
5-7 and 0.5% pH 3-10) and incubated at 37C for 18 hours.
Five ~f of each sample was loaded in the tube (gels and
overlaid with 8 ~l of 2D standards (Bio-Rad Laboratories,
Inc.). 30 ~l of sample overlay solution (9 M urea, 1%
ampholine pH 5-7, 0.5% ampholine pH 3-10 and 0.05% bromophenol
blue) was used to overlay the sample solution. The IEF
eiectrophoresis was carried out at 900 V for 3.5 hours. The
lower rhA~her buffer contained 10 mM phosphoric acid and the
upper chamber contained 20 mM sodium hydroxide. After
completion of the first dimensional run, the gels were removed
and placed on top of a 13.5% polyacrylamide-SDS gel cont~ining
a 4.5% stacking gel in a Bio-Rad minigel assembly. The IEF
tube gels were incubated for 5 minutes with reducing sample
buffer (62.5 mM Tris HCl, pH 6.8, 10% glycerol, 2% SDS and 25
mM DTT and 0.05% bromophenol blue), preheated to 95C for 5
minutes and then electrophoresis was performed at a constant
current of 50 mA for 2 minutes followed by 25 mA for 4S
minutes. GQ1S were stained for analysis using LabLogix silver
stain kit.
Results
HLA-DR2 containing both DRBl*1501 and DRB5*0101
class II molecules were purified from lymphoblastoid cells on
an antibody-coupled affinity column followed by ion-exchange
chromatography. Silver staining of the purified proteins
showed purity greater than 98%. Peptide binding was measured
by plate assay using biotinylated-MBP peptides and quantitated
by colorimetric method using alkaline-phosphatase coupled
1 q
VVO 96/1041S PCT/US9~/1257
streptavidin. An equivalent amount of biotinylated peptide
incubated under identical conditions but in the absence of
HLA-DR2 was used as a control. The time course for maximum
peptide occupancy of HLA-DR2 with MBP (83-102) y83 peptide at
neutral pH was optimized in the presence of 50-fold molar
excess peptide concentration and found to be 72-96 hours at
37C (Figure 7A). The specificity of the M~3P (83-102)Y83
peptide binding was demonstrated by incubating MBP (1-14)
peptide under identical conditions which showed no significant
binding to HLA-DR2. These results were consistent with recent
observations where the MBP (1-14) peptide showed no binding to
different DR2 alleles and isotypes (Valli et al, (1993),
supra). Following the optimized condition, purified HLA-DR2
was incubated with increasing concentrations of
biotinylated-MBP (83-102)y83 peptide. As shown in Figure 7B,
the percent of DR2 occupied with bound peptide increased with
increasing peptide concentration. The complete saturation of
HLA-DR~ Wi-~l rlor (~ 23Y83 peptide was observed at 300 to
500-fold molar excess peptide concentration. Further increase
in peptide concentration up to 2000-fold molar excess did not
show additional binding. This suggests that the saturation of
HLA-DR2 with MBP (83-102) y83 peptide is not due to the
aggregation of the peptide at higher concentrations. The
specificity of the peptide binding was demonstrated by
incubating purified DR2 with equivalent amounts of
biotinylated-MBP (1-14) peptide. The biotinylated-M~3P
(83-102)Y83 peptide alone did not show any significant binding
in our plate assay. This was confirmed by incubating
equivalent amount of biotinylated-MBP (83-102) y83 peptide
alone in the absence of HLA-DR2 which showed less than 1%
binding and was subtracted for c~lculating percent peptide
occupancy. Slightly increased binding of MBP (83-102) y83
peptide over 100~ was observed consistently in several
experiments. Previous results from our laboratory showed that
purified isolated ~ and ~ polypeptide chains of MHC class II
antigens are equally capable of binding, antigenic peptides
like the intact heterodimer (Passmore et al, J. Immunol. Meth.
155:193-200 (1992); Nag et al, Proc. Natl. Acad. Sci. USA
5 l 9
WO 96/10415 PCT/US95/12575
31
90:8797-8801 (1993)). It has also been reported that MHC
class II antigens exists in two conformational states known as
'floppy' and 'compact' (Dornmair et al, (1990), supra) and the
floppy form is capable of binding two peptides per molecule
(Tampe et al, Science 254:87-89 (1991); Kroon and McConnell,
Proc. Natl. Acad. sci. USA 90:8797-8801 (1993)). The
observed increased binding of MBP (83-102)Y33 peptide to
HLA-DR2 over 100% may be explained either due to the presence
of a small fraction of dissociated (~ and ~ chains in purified
DR2 preparation or the conformational state of the
heterodimers.
Further specificity of the bitinylated-MBP
(83-102) y83 peptide binding with DR2 at higher peptide
concentration was demonstrated in a competition assay. In
this experiment, purified HLA-DR2 was co-incubated with 300
fold molar excess of biotinylated-MBP (83-102) y83 peptide in
the presence of increasing concentrations of non-
biotinylated-MBP (83-102~ y83 2ep~ide. A~ shown in Figure 8A,
the biotinylated-MBP (83-102) y83 binding was competed out with
increasing concentrations of nonbiotinylated MBP (83-102)Y83
peptide and was completely inhibited at a concentration of
33-fold over biotinylated-MBP (83-102)Y33 peptide. Similarly,
another epitope from the same human myelin basic protein MBP
(124-143) which has higher binding affinity to HLA-DR2 (Valli
et al, (1993), supra) was able to compete for the binding of
biotinylated-MBP (83-102)Y33 peptide (Figure 8B).
In order to demonstrate that the purified complexes
of HLA-DR2 and MBP (83-102)Y83 contain a single MBP
(83-102) y83 peptide, milligram quantities of complexes with
non-biotinylated-MBP (83-102) y83 peptide were prepared and
used to characterize bound peptides. This was achieved by
comparing the acid-eluted profile of bound peptides from
preloaded and postloaded DR2 molecules. Prior to the acid
extraction, complete removal of unbound free peptide from the
complex preparation was accomplished by binding the DR2.MBP
(83-102) y83 complexes to L243 coupled Sepharose 4B column,
followed by extensive washing. Purified complexes were then
eluted from the resin and subjected to acetic acid extraction.
~o ~ ~ l q
WO 96/10415 PCT/US95/12575
32
The eluted peptides were characterized by narrowbore HPLC
analysis (Figure 9). Reverse-phase HPLC analysis of peptide
extracted from 100% loaded complexes showed a single peak with
a retention time identical to that of pure MBP (83-102)Y83
peptide (Figure 9D). In contrast, HPLC analysis of
acid-eluted peptides from purified HLA-DR2 showed a
significant amount of endogenous peptides (Figure 9B). The
identity of the peptide peak eluted from 100% loaded complexes
was confirmed by mass spectrometry.
lo Beside endogenously bound peptides, a significant
portion of purified MHC class II molecules are often known to
be associated with invariant chain polypeptides. The
association of the invariant chain in the endoplasmic
reticulum serves two important functions. First it prevents
class II molecules from binding peptides in the early stage of
transport (Roche and Cresswell, Proc. Natl. Acad. sci. USA
88:8730-8734 tl991); Lotteau et al, Nature 348:600-605 (1990);
Roche and Cresswell, Nature 345:615-618 (1990)). Secondly, it
contains a cytoplasmic signal that targets the class
II-invariant chain complexes to an acidic endosomal
compartment (Bakke and Dobberstein, Cell 63:707-716 (1990);
Lotteau et al, Nature 348:600-605 (1990)) where proteolysis
and subsequent dissociation of the invariant chain takes place
allowing antigenic peptides to bind prior to their transport
to the cell surface. It has been shown that purified MHC
II-invariant complexes are unable to bind antigenic peptides
in vitro (Roche and Cresswell, (1990), supra; Newcomb and
Cresswell, J. Immunol. 150:1358-1364 (1993)) and that both
soluble and membrane associated invariant chains can block
binding of peptides to MHC class II heterodimers (Lotteau et
al, (1990), supra; Roche et al, EMBO J. 11:2829-2839 (1992)).
To demonstrate the complete absence of invariant chain
polypeptides in 100% loaded HLA-DR2 and MBP (83-102)Y83
complexes, two-dimensional gel electrophoresis was performed.
In this gel system, IEF (pH 5-7) was carried out in the first
dimension followed by 13.5% polyacrylamide-SDS in the second
dimension. From the gel results, no invariant chain
polypeptides were detected in purified complexes. In
~ ~ o ~
~W096i10415 PCT~S95/12~75
33
contrast, purified HLA-DR2 showed multiple bands of invariant
chain polypeptides with varying molecular sizes.
Theses results demonstrate that high concentration
of MBP (83-lO2)Y83 peptide can be used for complete loading of
HLA-DR2 antigens. The plate-binding assay described here
facilitates study of this phenomenon. Using the
biotinylated-peptide where the sensitivity and the affinity of
streptavidin is enormously high, we were able to conduct these
experiments with as low as 0.025 pmoles of complexes where
lO,OOO to 20,000-fold molar excess peptide concentration
represents only 400-800 ~g of biotinylated peptide. In
addition, the plate assay with biotinylated peptide has the
advantage that many samples can be analyzed at a time and the
removal of unbound peptide is not required.
The above examples are provided to illustrate the
invention but not to limit its scope. Other variants of the
invention will be readily apparent to one of ordinary skill in
the art and are encompassed by the appended claims. All
publications, patents, and patent applications cited herein
are hereby incorporated by reference.
