CA2201519A1 - Preparation of mhc-peptide complexes - Google Patents

Abstract

The present invention provides methods for preparing MHC-peptide complexes of defined composition. The methods use large molar excesses of peptide.
Alternatively, the methods use optimized pH conditions.

Description

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 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.

q 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.

~Q1~ / q ~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-' I q ~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 ~ 2 ~ ~ ~ I Y
'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 l q WO96/10415 PCT~Sg~/12~75 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 5 1 q WO 96/10415 PCT/US95/12~75 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 ~ ~ Q ~ ~ l q 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 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

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 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 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 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 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 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 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 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 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 q 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 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 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 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 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 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 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.

Claims (16)

WHAT IS CLAIMED IS:

1. A method of preparing a complex comprising an antigenic peptide and an MHC component, the method comprising:
contacting the 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.

2. The method of claim 1, wherein the MHC
component is an HLA-DR2 molecule.

3. The method of claim 2, wherein the MHC
component contains a DRB*1501 molecule.

4. The method of claim 2, wherein the MHC
component contains a DRB*0101 molecule.

5. The method of claim 1, wherein the antigenic peptide is derived from myelin basic protein.

6. The method of claim 1, wherein the antigenic peptide is MBP(83-102)y83.

7. The method of claim 1, wherein the step of contacting is carried out using about a 100 fold to about a 300 fold molar excess of peptide.

8. The method of claim 1, further comprising the step of mixing the MHC Class II-peptide complex with the pharmaceutically acceptable excipient in a ratio suitable for therapeutic or diagnostic administration of the complex.

9. A method of preparing a complex comprising an antigenic peptide and an MHC component, the method comprising:
contacting the MHC component with the peptide under optimal pH conditions, thereby forming an MHC Class II-peptide complex.

10. The method of claim 9, wherein the MHC
component is DR2.

11. The method of claim 10, wherein the MHC
component contains a DRB*1501 molecule.

12. The method of claim 10, wherein the MHC
component contains a DRB*0101 molecule.

13. The method of claim 10, wherein the peptide is MBP(83-102)y83 and the optimal pH conditions are about pH 6.

14. The method of claim 10, wherein the peptide is MBP(124-143) and the optimal pH conditions are about pH 8.

15. The method of claim 10, wherein the peptide is MBP(143-168) and the optimal pH conditions are about pH 7.

16. The method of claim 9, further comprising the step of mixing the MHC Class II-peptide complex with the pharmaceutically acceptable excipient in a ratio suitable for therapeutic or diagnostic administration of the complex.