Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70539—MHC-molecules, e.g. HLA-molecules
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the body's ability to discriminate between self and nonself becomes impaired in a variety of conditions.
• Such conditions include autoimmune diseases and may also include immunodeficiency diseases such as AIDS, cancer and ageing.
• the ability of the immune system to discriminate between self and non-self deteriorates with age, as is evidenced by an increase in the prevalence of autoimmune phenomena and cancers in aged individuals.
• the strength of immune responses to foreign antigens decreases and the amount of autoantibodies produced during such responses to foreign antigens increases.
• a failure of the immune system to respond adequately to tumor tissue is a basis of the "immune surveillance" theory of oncogenesis.
• the idiotypic network model provides a basis for understanding a wide range of immunological phenomena. It includes roles for B cells, T cells and non-specific accessory cells (A cells) , antibodies, specific T cell factors and non ⁇ specific "second signal” lymphokines, and provides explanations for numerous aspects of immunoregulation. See generally. Hoffmann, Eur. J. Immunol. 5: 638-647 (1975) ; Hoffmann, in "Theoretical Immunology", Bell et al., (eds.) Marcel Dekker, NY, pp.
• the model is based on three kinds of symmetrical interactions between X x and X 2 lymphocytes, namely symmetrical stimulation, symmetrical inhibition and symmetrical killing.
• Symmetrical stimulation involves the cross-linking of receptors and specific inhibition is ascribed to specific T cell factors.
• Specific T cell factors are believed to have a molecular weight of roughly half (or less) that of an IgG antibody molecule and are therefore assumed to be monovalent. Hence free specific T cell factors are assumed to be unable to cross-link specific lymphocyte receptors.
• Symmetrical killing may involve antibody-dependent cellular cytotoxicity (ADCC) , antibody plus complement mediated lysis and/or cytotoxic T cells (CTL) .
• ADCC antibody-dependent cellular cytotoxicity
• CTL cytotoxic T cells
• a mathematical model that reflects the idiotypic network theory has been formulated (see, e.g., Gunther and Hoffmann, supra) .
• the same variables are used for the concentrations of B cells and T cells of the same (or similar) specificities.
• the dynamic variables refer primarily to cell concentrations, with antibodies and specific T cell factors implicitly included in the model, that is, their effects are included without the addition of any more variables.
• the concentration of specific molecules secreted by cells of a particular specificity is related to the concentration of the cells that secrete the molecules, and in some cases also to the amount of stimulation that the cells receive.
• the concentration of antibodies is treated as being proportional to the concentration of cells that produce them.
• the concentration of specific T cell factors is set proportional to the product of the concentration of the cells that produce them times the concentration of cells that specifically stimulate those cells.
• the concentration of X x cells is denoted by x and the concentration of X 2 cells by x 2 .
• the two-dimensional model has the form:
• Interactions between X ⁇ and X 2 cells and antibodies are assumed to be inhibitable by both idiotypic (X ⁇ and antiidiotypic (X 2 ) specific T cell factors. This is a model of the stable states of the system, and is not a model of all the events that occur during antigen-triggered induction of an immune response.
• Such switching involves non-specific lymphokines and accessory cells in addition to the components that are included in this model.
• Suitably chosen values of the parameters lead to four stable steady states.
• the virgin state there are low levels of both X x and X 2 clones for a given specificity, with a balance between influx of cells and a linear killing mechanism, for example IgM plus complement mediated killing.
• the immune state there is an elevated level of X ⁇ cells and a low level of X 2 cells.
• the suppressed (tolerant, unresponsive) state there are elevated levels of both X- j ⁇ and X 2 clones, and mutual stimulation between X ⁇ and X 2 T cells leads to inhibition by specific T cell factors.
• an anti-immune state which is the converse of the immune state. It has elevated X 2 and low X ⁇ population levels.
• Trajectories in the x ⁇ /x 2 phase plane are obtained by integrating the above pair of differential equations.
• Fig. 1 four stable states of the system, where the trajectories converge to a point, can be seen. They are labeled VS, SS, IS and AIS for Virgin State, Suppressed State, Immune State and Anti-Immune State, respectively.
• the two-dimensional model has been be generalized to N dimensions by Hoffmann and his collaborators (D.J. Mathewson, M.J. Lyons and K.G. Soga) as follows.
• the strength of interaction (affinity) between the V regions of clones i and j be K j .
• dx 1 S + X i e li uli - k, *2i ⁇ 2i - k- x i »3i U 3i dt
• ⁇ is a measure of (proportional to) the concentration of specific T cell factor that inhibits interactions with clone i.
• N ⁇ K i. k
• This model is an approximation in that one is not attempting to model inhibition of stimulation of clone k to produce factor k. This is a good approximation if the level of factors needed to specifically inhibit factor production is high relative to C l t C 2 and C 3 .
• ⁇ ir i is computed by the above equation, then normalized such that the average value of the ⁇ if i 's is mid-way between C 2 and C 3 .
• This global regulation aspect of the model simulates a possible mechanism for keeping the total concentration of specific T cell factors approximately constant, and ensures that not all the clones go to the suppressed state.
• Fig. 2 shows the results of a numerical experiment done with this model.
• a sparse 20 x 20 interaction matrix K ⁇ and initial values x ⁇ for 20 clones were generated using a random number generator.
• the matrix connectance (number of non-zero K ⁇ as a fraction of the total number of K ⁇ ) is 0.1 in this experiment.
• a set of values for the parameters of the model was chosen based on the analysis of the two-dimensional model (Gunther and Hoffmann, supra) .
• the 20 simultaneous equations are integrated until the system reaches a stable steady state. This experiment was done three times for the same K ⁇ matrix and three different sets of values of the initial conditions.
• the immune system normally discriminates between self and foreign antigens, in that it does not respond to immunizations with self antigens as readily as it does to immunizations with foreign antigens. Thus the immune system is naturally in a suppressed state (or other unresponsive state) for self antigens.
• the "induction of specific immunological tolerance” refers to injecting antigen in a way such that subsequent challenge with the antigen (in a way that would induce immunity in a naive animal) results in no immune response, or a response that is smaller than the response that would be observed in a naive animal.
• Such immunological tolerance is seen mainly at the humoral immunity or cellular immunity levels (Parish, . Exp. Med.
• Non-specific accessory cells namely macrophages, monocytes and dendritic cells (A cells) are known to play an important role in immune responses.
• the symmetrical network theory includes a postulated role for these cells in antigen- triggered switching from the virgin state to the immune state.
• Antigen-specific T cell factors bind to the surface of macrophages, so in the model the A cell is assumed to have a receptor for the constant part of specific T cell factors. It is postulated that the cross-linking of that receptor by the antigen via the specific T cell factor activates the A cell to secrete a non-specific factor.
• This non-specific factor provides B cells with a second signal, which permits them to differentiate from a form secreting very little antibody, to a plasma cell, which secretes large amounts of antibody.
• An immune response occurs when a larger dose of the antigen more effectively arms the A cell (and/or the antigen) with antigen-specific T cell factors, leading to A cell activation by the antigen via the specific factors.
• the activated A cell secretes a non-specific factor, which constitutes a differentiation signal for B cells to become antibody-secreting plasma cells. Only those B cells that have been activated by the cross-linking of their receptors express the receptor for the non-specific factor.
• a very high dose of antigen would stimulate too many T cells, in the sense that the available sites on both the antigen and the A cell would be saturated with specific T cell factor. The activation of the A cell is then inhibited, and the B cells do not receive the "second signal". The armed A cells would nevertheless induce low affinity T cells (that are not so effectively blocked by antigen-specific factors) to proliferate, leading again to the suppressed state with elevated levels of antigen-specific and antiidiotypic cells. This would explain the phenomenon of the induction of unresponsiveness with doses of antigen that are higher than immunogenic doses ("high zone tolerance”) .
• T lymphocytes are positively selected to recognize MHC molecules, and others are believed to be selected to be similar to ("images of") MHC molecules. (Hoffmann, 1988, supra) .
• Helper T cells are selected on the basis of their receptors having weak complementarity to class II MHC molecules
• cytotoxic T cells are selected on the basis of their receptors having weak complementarity to class I MHC
• suppressor T cells are selected on the basis of their receptors having similarity to class II MHC (Hoffmann (1988) , supra) .
• lymphocytes from the graft recipient's immune system infiltrating the grafted tissue.
• the infiltrating T lymphocytes recognize MHC antigens of the donor as foreign and act through several effector mechanisms to eliminate the foreign tissue.
• Immunosuppressive agents such as cyclosporin A and corticosteroids inhibit the elimination of the transplanted foreign tissue, but also cause generalized immunodeficiency in the recipient, leaving the graft recipient dangerously susceptible to infection. Long term administration of immunosuppressive drugs, as is necessary with mis-matched transplants, is also associated with other clinical problems such as kidney failure, hypertension and neoplasia.
• MHC genes are highly polymorphic within the outbred human population, histocompatibility typing to find appropriate MHC matches may require screening of large numbers of potential donors. This is costly, extremely time consuming, technically difficult. and many times appropriate matching with recipients is impossible. Many potential transplant recipients die before acceptably MHC-matched organs become available.
• lymphocytes contained within the graft (“passenger lymphocytes”) and the lymphocytes of the graft reacting to the host are believed to play a role in the rejection process, since graft survival can be improved by culturing the graft tissue for a period prior to grafting (Lafferty, Science 188: 259-261 (1975)). During this culture period, the passenger lymphocytes within the graft tissue presumably die, and can no longer participate in the interactions that lead to graft rejection.
• MHC antigens HLA alleles
• a class I MHC molecule is protective, namely H-2D d (Makino et al., J. Immunol. 144: 4347-4355 (1990)), although the effect depends also on the presence of other MHC molecules.
• MHC molecules can influence the rate of progression to AIDS (Steel et al.. Lancet i: 1185-1188 (1988)), consistent with the proposition that AIDS may be an autoimmune disease.
• AIDS is an immunodeficiency disease that results in the development of both autoimmunity (Ziegler et al., Clin. Immunol. Immunopat . 41: 305-313 (1986); Andrieu et al., AIDS Research 2: 163-174 (1986); Grant et al., J. Immunol. 144: 1241-1250 (1990); Martinez-A, Lancet, i, 8583, 454 (1988); Shearer, Mt. Yale J. Med. 53: 609-615 (1986); Siliciano et al., Cell 54: 561 (1988); Zarling et al., J. Immunol.
• MRL-lpr/lpr mice make both MHC-image and anti-MHC- image antibodies (Kion and Hoffmann, supra) .
• MI-anti-MI MHC image anti-MHC-image
• the two kinds of immunity invoked in the MHC image anti-MHC-image (MI-anti-MI") model of AIDS pathogenesis, namely MHC image and anti MHC image are present in these autoimmune mice, and the MI-anti-MI pathogenesis model may have applicability to other autoimmune diseases besides AIDS.
• Autoimmunity may in general occur when B cells make antibodies with shapes that are specific for the T cell V regions along the "major axis" of shape space, namely MHC-image (specific for anti-MHC T cell V regions) and anti-MHC-image (specific for MHC-image T cell V regions) . It is therefore important to devise strategies for the inhibition or suppression of such B cell clones. Chimpanzees can be infected with HIV, but the virus does not cause AIDS in chimpanzees. It has been reported that cytotoxic T lymphocytes (CTL) exist in HIV infected humans that kill normal human T cells, while HIV infected chimpanzees do not contain CTL specific for chimpanzee T cells. (Zarling et al., J. Immunol. 144: 2992-2998 (1990)).
• CTL cytotoxic T lymphocytes
• Allergies are a common health problem in which an individual makes an immune response of the IgE class to (typically) some environmental antigen. Allergies are typically treated clinically with a desensitization procedure, consisting of injections with increasing levels of antigen. This technique is only partially successful, and it would be very useful to have a more reliable method.
• the present invention fulfills these and other related needs.
• the present invention provides methods and compositions for modifying the T cell repertoire of an individual to antigens of interest as needed.
• an adverse immune response may be suppressed, or a desired response may be enhanced.
• subsequent additional doses of the TCR4 and/or TCR8 binding substances may be necessary. In this situation the subsequent doses are typically equal to or escalating in amount compared to a previous dose.
• the TCR4 and TCR8 binding substances are MHC class II and MHC class I molecules, respectively, and are alloantigens or xenogeneic antigens, depending on the source of the tissue, cells or organ to be transplanted.
• MHC class II molecule associated with the absence of the autoimmune disease and a TCR8 binding antigen, such as a class I MHC molecule.
• the MHC class I and class II antigens can be allogeneic or xenogeneic MHC molecules, and can be covalently linked to one another. If the MHC molecules are xenogeneic, they are derived from a species which does not suffer from the autoimmune disease.
• the invention further provides methods and compositions for inhibiting immunodeficiency disease in a subject, which comprises administering to a subject susceptible to developing the disease a subimmunogenic amount of one or more MHC antigens and a physiologically acceptable carrier.
• additional doses of MHC antigens may be given in amounts which individually are equal to or greater than a previous dose, where said subsequent doses may reach or exceed doses which, if given initially to an immunologically naive subject, would cause a humoral immune response.
• the MHC antigens which are given are typically alloantigens of class I and/or class II, or are obtained from a xenogeneic species which is not prone to developing the immunodeficiency disease.
• the MHC antigens can be covalently linked to each other for optimal activity.
• the invention also provides methods and compositions for inducing immunological tolerance to a particular antigen, as is important in the treatment of allergies.
• Tolerance to the antigen comprises administering subimmunogenic amount(s) of the antigen and one or more MHC molecules.
• MHC molecules typically the antigen and the MHC molecule will be covalently linked.
• additional doses of the MHC- antigen composition in amounts which individually are equal to or greater than a previous dose, can be used to induce or maintain tolerance to the antigen.
• the MHC molecule is selected from class I, class II, or a combination of MHC class I and II molecules, and can be allogeneic or xenogeneic.
• Another aspect of the invention comprises administration of a subimmunogenic amount (that is, sub ⁇ immunogenic for elicitation of a humoral immune response) of a subject's own MHC molecules of one class (class I or II) in combination with self or foreign MHC of the other class.
• the self MHC molecules can be given in a more immunogenic form than that to which the individual is usually exposed.
• self MHC molecules can be aggregated or covalently cross-linked to each other, and class II MHC molecules can be covalently coupled to class II MHC molecules.
• Fig. 2 shows representative stable steady states of an immune network consisting of twenty clones.
• a network of twenty clones was modelled by generating a random K ⁇ matrix of clonal interaction strengths, and the dynamics of the system was investigated using the N-dimensional mathematical model described above.
• the system started in different randomly set initial conditions and the twenty- dimensional equation was integrated until a stable steady state was reached.
• Fig. 3 illustrates a model of the interactions between CD4 and CD8 T cells, that are selected to have affinity for class II MHC and class I MHC respectively.
• this model there is a network of idiotypic interactions between CD8 T cell V regions and CD4 T cell V regions.
• the present invention provides a means for inducing tolerance to a variety of antigens. This is useful in a variety of circumstances, including the induction of transplantation tolerance to permit allografts or xenografts, the prevention of acquired immune deficiency syndrome (AIDS) that is associated with HIV infection in humans, treatment and prevention of autoimmune diseases and other conditions associated with the dysfunction of the immune system, such as allergies.
• AIDS acquired immune deficiency syndrome
• the invention relates to the induction of tolerance by the administration of low doses of compounds which binds preferentially to T cell receptors on a subset of CD4 cells ("TCR4") , as exemplified by MHC class II molecules, or to T cell receptors on a subset of CD8 cells (“TCR8”) , as exemplified by MHC class I molecules.
• TCR4 and/or TCR8 binding substances are administered to an individual susceptible to or suffering from a disease that is treatable or preventable by achieving a suppressed state as to particular antigen(s) , such as autoimmune diseases, allergies, transplantation of allogeneic or xenogeneic tissues, etc.
• the class I MHC in humans is located on chromosome 6 and has three loci, HLA-, HLA-B, and HLA-C.
• the first two loci have a large number of alleles encoding alloantigens. These consist of a 44 Kd heavy chain subunit and a 12 Kd ⁇ 2 - icroglobulin subunit which is common to all antigenic specificities. Isolation of these detergent-soluble HLA antigens has been described by, e.g.. Springer et al., Proc. Natl. Acad. Sci. USA 73: 2481-2485 (1976), and Clementson et al., in "Membrane Proteins: A Laboratory Manual," A.
• MHC glycoproteins can be isolated from appropriate cells or can be recombinantly produced. Methods for purifying the murine I-A (Class II) histocompatibility proteins are well known and have been disclosed by Turkewitz et al., Molecular- T ⁇ mn ⁇ no1 nq ⁇ (1983) 20:1139-1147, which is incorporated herein by reference.
• MHC genes e.g., as described by Estess et al., infra. or Steinmetz et al.. Nature 300: 35-42 (1982) , incorporated herein by reference
• the and ⁇ chains of seven allelic variants of the I-A region have been cloned and sequenced (Estess et al., in "Regulation of Immune Gene Expression,” Feldman et al., eds.. The Humana Press, Inc., 1985, pp. 3-19).
• isolated MHC subunit component refers to an MHC glycoprotein subunit (i.e., an a or ⁇ chain of MHC II or a heavy chain of MHC I) , which is in other than its native state, for example, not associated with the cell membrane of a cell that normally expresses MHC.
• MHC glycoprotein subunit i.e., an a or ⁇ chain of MHC II or a heavy chain of MHC I
• This term embraces a full length subunit chain, as well as an effective portion of the MHC subunit.
• MHC molecule or fragment thereof is meant a histocompatibility molecule that is recognized as foreign by the recipient host.
• An MHC fragment will typically comprise an antigen binding site and sequences necessary for recognition by the appropriate T cell receptor.
• MHC proteins suitable for use in the present invention have been isolated from a multiplicity of cells using a variety of techniques, including solubilizatio.n by treatment with papain, by treatment with 3M KC1, and by treatment with detergent.
• detergent extraction of Class II protein from lymphocytes followed by affinity purification is used.
• Detergent can then be removed by dialysis or selective binding beads.
• the molecules can be obtained by isolation from any MHC bearing cell, such as B lymphocytes, e.g., from an individual or species naturally tolerant to the particular disease or condition being treated. Isolation of individual fragments from the isolated MHC proteins is easily achieved using standard techniques known to those skilled in the art.
• the heavy chain in the case of Class I molecules, can be separated using SDS/PAGE and electroelution of the heavy chain from the gel (see, e.g., Dornmair et al.. Cold Spring Harbor Svmp. Quant. Biol. 54:409-416 (1989), and Hunkapiller et al., Meths.
• the MHC molecules and fragments can also be readily modified and manufactured utilizing various recombinant DNA techniques well known to those skilled in the art.
• the amino acid sequences of a number of Class II MHC proteins are known, and the genes have been cloned, therefore, the proteins and polypeptide fragments can be made using recombinant methods.
• the synthetic gene encoding the MHC molecule will typically include restriction sites to aid insertion into expression vectors and manipulation of the gene sequence.
• the genes encoding the appropriate molecule or fragment thereof are then inserted into expression vectors, expressed in an appropriate procaryotic or eucaryotic host, such as E. coli. yeast, or other suitable cells, and the recombinant proteins are obtained.
• recombinant techniques allow a number of modifications of the MHC molecules.
• recombinant techniques provide methods for carboxy terminal truncation which deletes the hydrophobic transmembrane domain.
• the carboxy termini can also be arbitrarily chosen to facilitate conjugation to various antigens, for example, by introducing cysteine and/or lysine residues into the molecule.
• the molecules can vary from the naturally occurring MHC protein sequence by amino acid substitutions, additions, deletions, and the like.
• modifications of the genes encoding the MHC proteins or fragments may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis.
• Amino acid sequence variants of MHC molecules can be prepared to increase the affinity of the molecule for T cell receptors, facilitating the stability, purification and preparation of the molecules, modifying plasma half life, improving therapeutic efficacy, and lessening the severity or occurrence of side effects during therapeutic or prophylactic use of MHC molecules in the present invention.
• low dose an amount that is small compared to the dose typically used to induce humoral immune responses to foreign proteins, with the proviso that the initial low dose or doses may be followed by additional injections of higher doses of the same MHC antigens to reinforce the suppressed state when it has been induced or partially induced. Induced tolerance to a foreign protein in not necessarily long-lasting, hence the “stable state” is an approximation. A series of increasing doses, beginning at low doses, will most effectively and most reliably induce the suppressed state. In an "increasing dose tolerogenic immunization regimen", or simply “increasing dose regimen,” the subject is exposed to a dose that induces the suppressed state, without the possibility for the immune state to be induced.
• increasing dose regimen is defined herein as doses that begin with lower doses of the antigen than are typically used to induce immunity, and allowing enough time between injections to ensure that the immune system goes to a stable (or quasi-stable) suppressed state for the antigen, followed by occasional additional infections of the antigen as needed to ensure long-term maintenance of the suppressed state.
• an antigen Several factors play a role in determining whether the administration of an antigen is immunogenic or not.
• the factors include route of injection, dose, and the physical form of the antigen, namely whether or not the antigen is polymeric or contains aggregates that would be efficient in cross-linking receptors.
• intravenous or oral administrations of antigen are non-immunogenic and favor induction of tolerance.
• a low dose for example, a dose that is small compared with the doses typically used to induce humoral immune responses to proteins such as bovine serum albumin, keyhole limpet hemocyanin, tetanus toxoid or diphtheria toxoid
• repeated low doses can be tolerogenic, and low doses of antigen can induce suppressor T cells.
• a preferred mode of this invention is to use intravenous injections of an escalating dose of antigen, beginning with a low dose (as defined above) .
• the initial low doses "push" the system towards the suppressed state for that antigen.
• Subsequent higher doses will then consolidate the suppressed state for that antigen.
• the MHC molecules may be administered in aggregate-free form or (particularly in the case of self MHC molecules) in an aggregated form or attached to a polymeric carrier to enhance the extent to which they cross ⁇ link receptors on T cells.
• the combination of MHC class I and MHC class II molecules to induce tolerance to both classes is useful in a variety of settings, as described in more detail below.
• the envisaged mechanism relates to the fact that within the idiotypic network model, an immunological perturbation has to be above a certain threshold for switching between states to occur.
• the stable steady states of the mathematical model have a range (or "basin") of attraction. If an antigen perturbs the system only a small amount and only transiently, the model suggests the system can then relax back to its initial state. A larger perturbation is necessary to move the system to a new stable or quasi-stable steady state.
• Very low doses of antigen have been shown to be capable of inducing immunological tolerance, but this normally requires that the antigen is injected repeatedly, so that there is a long- lasting perturbation to the system (Parish and Ada, Proc. Natl. Acad. Sci.
• the substances are chosen such that one of them binds preferentially to T cell receptors on a subset of CD4 cells ("TCR4") , while the other binds preferentially to T cell receptors on a subset of CD8 cells (“TCR8”) .
• TCR4 T cell receptors on a subset of CD4 cells
• TCR8 T cell receptors on a subset of CD8 cells
• Fig. 4 shows an example in which a foreign class II MHC molecule is used to stimulate the CD4 clones 2, 4 and 6, while a foreign MHC class I molecule stimulates CD8 clones b, e and g.
• the TCR4 clone "6" is not idiotypically connected to any of the CD8 clones that are stimulated by the foreign class I MHC, so it is only transiently perturbed. The same is true for the TCR8 clone b.
• clones 2 and 4 are connected to clones g and e respectively, which are being stimulated by the foreign class I MHC molecule. It follows that 2 and 4 are synergistically stimulated in these circumstances, and together with e and g may switch to being in the suppressed state.
• the low dose tolerance can be used to modify the T cell repertoire of a host to induce transplantation tolerance to foreign (transplant) antigens.
• Graft rejection can occur following replacement of inadequate organs, tissues or cells with allogeneic or xenogeneic transplants.
• Rejection involves the generation of an immune response against donor histocompatibility antigens expressed on the surface of cells within the transplant.
• the method according to the present invention involves modifying the transplant recipient's functional lymphocyte repertoire by administering, sufficiently prior to the transplantation, soluble MHC molecules or fragments thereof which are identical to or closely matched to the MHC molecules encoded by the transplant donor's histocompatibility genes.
• Allogeneic histocompatibility molecules for an allograft
• xenogeneic histocompatibility molecules for an xenograft
• the invention includes administering low doses of histocompatibility molecules of the recipient's haplotype to the donor prior to the transplant to effectively tolerize the donor's cells to the recipient's MHC antigens.
• Cells from a transplant donor can serve as a source of MHC molecules for the immunizations, assuming the donor is identified sufficiently in advance to permit the preparation of the purified low dose immunization(s) .
• a library of various MHC molecules can be developed and stored so that the particular alleles needed in a particular circumstance are available.
• the MHC antigens of the donor are preferably administered to the intended transplant recipient, or the MHC antigens of the recipient are administered to the donor, as desired.
• the MHC molecules are administered in intervals, typically in multiple intravenous injections, with 1 to 10 immunizations at intervals in the range of about 3 days to 8 weeks.
• the MHC molecule will be given in the range of 1 ng to 100 ⁇ g per injection per kg body weight, with the preferred dosage being in the range of 10 ng to 10 ⁇ g per kg body weight.
• a preferred regimen will be increasing doses. For example, this can involve a series of injections beginning at an ultra low dose in the 0.1 ng/kg to 100 ng/kg range, and increasing by factors of 3 to 10, up to a maximum of 100 ⁇ g/kg given at intervals in the range of 3 days to 8 weeks.
• Representative tolerizing regimens for four species is given in Table l, in which the immunization strategies combine escalating doses with very low initial doses of antigen.
• Attaining a level of tolerance sufficient to allow transplantation can readily be determined by a mixed lymphocyte reaction or induction of cytotoxic T cells. Detailed methodology for CTL assays is given by L. Hudson and F.C. Hay in "Practical Immunology", 3rd edition, Blackwell, 1989, p. 160.
• Another embodiment of the invention for inducing transplantation tolerance involves injecting into a potential graft recipient an effective amount of both the foreign polymorphic class I MHC and class II MHC antigens expressed by a potential graft donor that are not expressed by the recipient.
• a synergistic stimulation of the subset of CD8 cells that recognizes both foreign class I MHC and the idiotypes of stimulated CD4 cells will reduce the amount of antigen needed than if either MHC I or MHC II antigens were used alone. Moreover, the duration of the injections needed with the MHC I and II combination therapy will be shorter. If the recipient and donor have the same class II antigens but different class I antigens, another TCR-4-binding substance (e.g., a class II MHC molecule of another allele) should be injected together with donor class I MHC antigen.
• TCR-4-binding substance e.g., a class II MHC molecule of another allele
• MHC molecules typically from about 1 ng up to about 1 ⁇ g or more per kg body weight of the TCR4-binding substance together with 1 ng to 1 ⁇ g per kg body weight of the TCR8-binding substance are administered at each interval, with intervals in the range of 3 days to 8 weeks.
• Increasing doses of the combination of the two classes of antigens can also be used.
• the dose will vary according to, e.g., the particular disease being treated or prevented and its severity, the particular TCR4 and/or TCR8 binding molecule and/or antigen, the manner of administration, the overall health and condition of the patient, and the judgment of the prescribing physician.
• An effective regimen is most simply determined by performing a mixed lymphocyte reaction (MLR) with the prospective donor cells as stimulators, and the prospective (putatively tolerized) recipient cells as responders.
• Pre-immunization cells (stored frozen) may be used as controls, to determine the extent of the decrease in immunological reaction.
• production of lymphokines such as IL-2 in such cultures can be used as a measure of the strength of the immune activation by the stimulators, and hence (as measured by the absence or paucity thereof) the degree of tolerance that has been induced.
• MLR assays is given by Meo, in T ⁇ m ⁇ u- ⁇ noiogical Methods. Lefkovits and Pernis, eds., 1: 227, and also in Practical Immunology. Hudson and Hay, 3rd ed. , Blackwell, 1989, p. 160, which are incorporated herein by reference.
• immunological tolerance to xenogeneic MHC molecules can also be induced according to the present invention to prevent rejection of xenografts (grafts from individuals of a different species) .
• Low doses (“sub ⁇ immunogenic,” that is, doses lower than those typically required to elicit a humoral immune response to protein antigens) of the xenogeneic MHC molecule(s) to which immune tolerance is desired are administered.
• immune system network theory such an approach works by activating both T cells that are specific for the antigens and T cells with receptors that are complementary to the receptors of lymphocytes specific for the tolerizing antigen.
• Allogeneic or xenogeneic class I MHC molecules will stimulate mainly CD8 clones, whereas allogeneic or xenogeneic class II MHC molecules will stimulate mainly CD4 cells. If class I and class II MHC molecules are given together, then there will be simultaneous direct stimulation of both CD4 and CD8 clones.
• the idiotypic connectance between CD4 and CD8 clones is sufficiently high that some of the CD4 clones that are being stimulated will be stimulated by idiotypes of some of the CD8 clones being stimulated and vice versa.
• the invention further provides methods for modifying the immune system of an individual to protect the individual from autoimmune diseases for which protective alleles are identified.
• a combination of MHC class I and MHC class II molecules is administered in a way that efficiently induces tolerance to both class I and class II MHC molecules.
• the method is designed to cause a long- lived modification of the immune system that is functionally similar to that caused by the permanent presence of a naturally-expressed protective MHC antigen.
• autoimmune dysfunctions are correlated with specific MHC types.
• Methods for identifying which alleles, and subsequently which MHC encoded polypeptides, are associated with an autoimmune disease are known in the art.
• a method described in Todd et al., EP 286,447 (which is incorporated herein by reference) is suitable.
• autoimmune diseases including myasthenia gravis (MG) , multiple sclerosis (MS) , systemic lupus erythematosus (SLE) , rheumatoid arthritis (RA) , insulin- dependent diabetes mellitus (IDDM) , etc.
• HLA-associated autoimmune diseases examples include DR4(Dw4), DR4(Dwl4) or DR1.
• SLE in both the human and murine forms of the disease, a strong association with MHC gene products has been shown for HLA-DR2 and HLA-DR3 individuals (Reinertsen, et al., N. Engl. J. Med (1970) 299:515), while in mice (H-2 d u ) , a gene linked to the H-2 U haplotype contributes to the development of lupus- like nephritis.
• Myasthenia gravis is one of several human autoimmune diseases linked to HLA-D.
• IDDM populations have a high frequency of HLA-DR3 and -DR4 alleles, and the NOD mouse strain (H-2K d D b ) is a murine model for autoimmune IDDM.
• Experimental allergic encephalomyelitis is an induced autoimmune disease of the central nervous system which mimics in many respects the human disease of multiple sclerosis (MS) , linked to DR2. The disease can be induced in many species. including mice and rats. Methods of inducing the disease, as well as symptomology, are reviewed in Aranson (1985) in The Autoimmune Diseases (eds. Rose and Mackay, Academic Press, Inc.) pp.
• an individual having (or susceptible to) an autoimmune disease is identified, and the autoimmune dysfunction is identified. Identification may be by symptomology and/or an examination of family histories.
• the individual's MHC type is determined by one or more of several methods known in the art, including, for example, cell typing by MLR, by serologic assay, and by DNA analysis (including RFLP and PCR techniques) .
• the individual is then treated with MHC class I and/or class II molecules together with administration of the autoantigen, which is able to suppress the immune response to the specific autoantigen.
• an individual having (or susceptible to) an autoimmune disease is treated with a sub-immunogenic amount (sub-immunogenic for elicitation of a humoral immune response) of his own class II MHC molecules, in combination with self or foreign class I MHC molecules, or of his own class I MHC molecules in combination with self or foreign class II MHC molecules.
• a sub-immunogenic amount sub-immunogenic for elicitation of a humoral immune response
• the self MHC molecules are given in a form that is more immunogenic than that to which the individual is usually exposed.
• the immunogenicity of the self MHC molecules can be enhanced by non-covalent or covalent aggregation.
• MHC molecules of a given class may be covalently coupled or aggregated either to molecules of the same class or to molecules of the other class.
• the combinations are used to preferentially stimulate T cells with self anti-MHC and MHC-image specificity, and thus inhibit any B cells that have MHC-image or anti-MHC-image specificity.
• This method is intended for both prevention and therapy of autoimmune disease. It permits the immune system of the subject to become more firmly established in a suppressed state for self MHC specificities, and thus inhibits the development of any autoimmune disease that involves the production of MHC-image and/or anti-MHC-image antibodies.
• the method also permits the suppressed state for self MHC antigens (and for self-MHC-image V regions) to be regained in the case of a patient that is already suffering from an autoimmune disease involving the presence of MHC-image and/or anti-MHC- image antibodies. Therapy is monitored by observing a decrease in the production of MHC-image, anti-MHC-image and other disease associated autoantibodies.
• an increasing dose tolerogenic immunization regimen of xenogeneic MHC molecules is used to modify the T cell repertoire in a way that prevents or inhibits immunodeficiency diseases, including AIDS and other autoimmune diseases.
• the increasing dose tolerogenic immunization regimen involved doses similar to or slightly higher (up to about 10- to 100-fold) than those for allogeneic MHC molecules.
• Diseases that are particularly susceptible to this treatment are those that do not exist in the species of origin of the xenogeneic MHC molecules. For example, certain species such as the chimpanzee and rabbit can be infected with HIV but do not get AIDS. According to the idiotypic network analysis of HIV pathogenesis, this is related to differences between human and chimpanzee (or rabbit) MHC molecules.
• the disease AIDS can be ameliorated or development thereof inhibited by administering to an HIV infected individual, or to an individual susceptible to HIV infection, TCR4 and/or TCR8 binding substances in an increasing dose tolerogenic regimen.
• TCR4 and TCR8 binding substances in this method are MHC class I and/or class II molecules obtained from a xenogeneic species (or allogeneic individuals) not typically susceptible to developing the disease, e.g., chimpanzees.
• An HIV infected individual in a pre-AIDS condition can be given an increasing dose tolerogenic regimen of MHC class I and class II molecules obtained from chimpanzee cells in a dosage regimen as described herein, and susceptibility to developing the disease is prevented or inhibited.
• the invention provides a method for inducing immunological tolerance to an antigen. This method is useful in preventing or treating allergies, for example.
• a tolerogenic vaccine is provided that includes, as active components, an antigen together with one or more TCR- binding substances, the preferred embodiments of the TCR- binding substances being either a TCR8-binding substance or a combination of TCR8-binding and TCR4-binding substances.
• a second antigen that is recognized by many T cells, such as MHC molecules, which may be either xenogeneic or, more preferably, allogeneic.
• the second antigen may be covalently coupled to the primary antigen. Coupling to an antigen for which an animal is already in a suppressed state will enhance the tolerogenicity of that antigen.
• Protein antigens can be conjugated to the MHC molecule or peptide by standard dehydration reactions using carbodiimides. Heterobifunctional linkers such as SPDP, glutaraldehyde and the like can also be used. Alternatively, the entire complex may be made directly from the appropriate encoding DNA using recombinant methods.
• the invention provides a method for sensitizing an individual for subsequent immune response.
• the administration of soluble allogeneic or xenogeneic class I MHC molecules in certain doses activates the immune system in a way that results in a very substantial increase in the immune response to a subsequently administered antigen.
• MHC-I is thus a novel activator that is free of the side effects that typically characterize adjuvants. It has also been shown that the immune system is activated in a way such that a subsequently administered antigen evokes a strong immune response with a low ratio of IgE to IgG. It is unexpected (and counterintuitive) that xenogeneic or allogeneic MHC-I, which stimulates CD8 cells rather than CD4 (helper) T cells, causes an enhanced humoral immune response.
• Administration of the increasing dose tolerogenic regimen is typically systemic and is effected by injection, preferably intravenous, and thus formulations compatible with the injection route of administration may be used. Suitable • formulations are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985), which is incorporated herein by reference.
• a variety of pharmaceutical compositions comprising low dose TCR4 and/or TCR8 binding substances, together with selected antigen as noted herein, and pharmaceutically effective carriers can be prepared, including incorporation into liposome formulations, e.g., as described in U.S. Patent No. 4,837,028, which is incorporated herein by reference.
• compositions are suitable in a variety of drug delivery systems, a brief review of which appears in Langer, Science 249: 1527-1533 (1990) which is incorporated herein by reference.
• 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, as discussed in more detail above.
• compositions for intravenous administration which comprise a solution of the complex dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
• an acceptable carrier preferably an aqueous carrier.
• aqueous carriers may be used, e.g. , water, buffered water, 0.4% saline, and the like.
• PBS phosphate buffered saline
• 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.
• 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, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
• auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
• compositions can be administered according to the methods of the present invention for therapeutic or prophylactic applications.
• therapeutic applications compositions are administered to a patient already suffering from a disease, 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 and are discussed generally above.
• compositions containing the MHC or other molecules as indicated above are administered to 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, and doses will generally be in the ranges set forth above.
• This Example illustrates the induction of tolerance to prevent graft rejection by administering sub-immunogenic amounts of class I MHC antigens and, separately, class II MHC antigens. Soluble MHC molecules and fragments are prepared for administration to an intended graft recipient.
• To purify class II antigens from B cell lines the procedure of Jensen et al., J. Ex . Med. 174:1111, incorporated herein by reference, is used.
• cells are grown in bulk (i.e., roller bottles), and 10 g of cells (approximately 10 10 cells) are washed in HBSS and resuspended in 50 ml 25 mM Tris, pH 8.0 containing freshly added protease inhibitors (2 mM iodoacetamide, 1% (vol/vol) aprotinin, 1 mM PMSF) , and incubated for 60 min. at 4°C, gently stirring with a magnetic stir bar to allow hypotonic lysis. Lysed cells are then centrifuged, 2000xg for 10 min. , to remove nuclei, and then centrifuged at 100,000xg for 60 min. to pellet membranes. The membrane pellet can be stored at -70°C indefinitely at 10 9 cell equivalents/ml in 25 mM Tris, pH 8.0 containing protease inhibitors.
• the membrane pellet is thawed and the volume adjusted to 20 ml with 25 mM Tris, pH 8.0, 1% NP-40 with freshly added 2 mM iodoacetamide, 1% aprotinin, and 1 mM PMSF (the sample is not allowed to warm during thawing) .
• the sample is solubilized at 4°C overnight, then the lysate centrifuged at 2000xg for 10 min. and the pellet discarded. The supernatant is centrifuged at 100,000xg for 30 min. to clear the lysate.
• the MHC molecules are eluted from the column with 5- 6 volumes of 50 mM glycine-NaOH, pH 11.0, 0.15 M NaCl.
• Fractions are collected in borosilicate disposable glass tubes containing 100 ⁇ l 1 M Tris, pH 7.0, in -10 fractions, and those fraction with significant absorbance are pooled.
• the pH of the pooled fractions is neutralized, if necessary, and the sample concentrated to -0.5 ml using centricon 30 disposable microconcentrators.
• the sample is dialyzed against 1% N- octylglucoside/buffer.
• the class II preparations are analyzed using SDS PAGE and coomassie staining, and protein concentration determined using the BCA assay (Pierce) with BSA standards. All of the foregoing steps are performed at 4°C unless otherwise indicated. Columns must be pre-eluted.
• the class II molecule(s) may be eluted with lower pH (i.e., pH 10.7).
• Representative anti-(MHC class II) antibodies are listed in the American Type Culture Collection Catalogue, 7th ed. , and include, for example, 14-4-4 for I-E d and I-E k , MKD6 for I-A d , and 10-2-16 for I-A k .
• a mouse of a first strain is given a tolerogenic regimen of injections of the MHC class I antigens from a mouse of a second strain.
• the first mouse is then tested for the ability to develop cytotoxic T cells (CTL) against the class I MHC antigens expressed by the second strain.
• CTL responses are typically mainly to class I MHC.
• CTL experiments also referred to as "cell-mediated cytolysis" or CMC is given by Hudson et al., in Practical Immunology, 3rd edition, Blackwell, p. 160 (1989), which is incorporated herein by reference.
• a similar experiment to determine the ability of soluble class II MHC antigens to induce tolerance involves an increasing dose tolerogenic regimen of administrations of class II MHC molecules in a manner similar to that used for class I molecules.
• Class II MHC molecules from the second strain mouse are injected in an increasing dose tolerogenic regimen into a mouse of the first strain.
• Cells obtained from the injected first strain mouse are assayed for a diminished mixed lymphocyte reaction (MLR) against cells from the second strain.
• MLR mixed lymphocyte reaction
• This assay reflects the fact that an MLR response is typically mainly to class II MHC.
• MLR assays are performed according to Meo, in Immunological Methods. Lefkovits and Pernis, eds., vol. 1, p.
• intravenous or intraperitoneal (preferably intravenous) injections are performed in the range of lOOpg to 10 ⁇ g per injection per mouse, with the preferred dosage being in the range of lng to 1 ⁇ g.
• a preferred regimen includes increasing doses. For example, this involves a series of injections beginning at a dose in the lOOpg to lOng range, and increasing by factors of 3 to 10, for up to 20 injections given at intervals in the range of 3 days to 8 weeks.
• skin grafts from naive mice of both the first and second strains and a third strain are applied to representative tolerized mice as well as naive mice, where the first, second and third strains have different MHC haplotypes from each other.
• the grafting involves the recipients being anaesthetized with sodium pentobarbital given i.p. at a dose of 0.06 mg per gram of body weight.
• Each mouse receives a full thickness graft of an allogeneic donor skin and a control graft of syngeneic skin.
• the tolerized mice retain the second strain grafts longer than do naive mice, while the rate of rejection of skin grafts from mice of the third strain is similar to the naive (untolerized) mice.
• This Example illustrates the administration of the increasing tolerogenic regimen of a combination of an MHC class I antigen(s) and an MHC class II antigen(s) , in the inhibition of the development of disease by developing tolerance to one or the other or both substances.
• Transplantation tolerance in an intended graft recipient is induced using an increasing tolerogenic regimen of effective amounts of the foreign polymorphic class I MHC and class II MHC antigens expressed by the graft donor that are not expressed by the recipient. Due to the stimulation of the subset of CD8 cells that recognize both foreign class I MHC and the idiotypes of stimulated CD8 cells, the amount of antigen needed is smaller than in the case of Example 1, and the duration of the injections needed is shorter.
• the MHC class I and class II antigens are prepared as described in Example 1.
• An increasing dose regimen beginning at 0.1 ng/kg to 1 ⁇ g/kg of the TCR4-binding substance(s) and 0.1 ng/kg up to 1 ⁇ g/kg of the TCR8-binding substance, increasing by a factor of 2 to 10 for one to ten injections at intervals of from 3 days to 10 weeks (preferably one to six weeks) are given over a period.
• the transplant is performed thereafter, with a boost of the antigens being given one week to one month immediately prior to the transplant to reinforce the suppressed state.
• the size of the boost is the same as the final dose of the increasing dose regimen, or can be an order of magnitude less.
• graft survival e.g. skin graft
• class I and class II MHC molecules in preventing autoimmunity can be demonstrated in established animal systems of autoimmune disease, including the NOD mouse model for diabetes, the MRL-lpr/lpr model for systemic lupus erythematosus, and the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis.
• EAE experimental autoimmune encephalomyelitis
• female NOD mice are injected with I-E antigen together with allogeneic class I MHC molecules prior to the age when diabetes typically develops, i.e., about 3 months, with immunization regimens as given in Example 2, to determine optimum protection against development of diabetes.
• MRL-lpr/lpr mice make anti-gpl20 antibodies (gpl20 of HIV) , and a change in the T cell repertoire that suppresses the production of these antibodies can be used to suppress pathogenesis.
• gpl20 and p24 antigens of HIV have been described as MHC-class II mimicking substances (Ziegler and Stites, Clin. Immunol, and Immunopath. 41:305-313 (1986); Andrieu et al. AIDS Research 2:163-174 (1986); Hoffmann et al., Proc. Nat. Acad. Sci. fUSA) .
• TCR4-binding substances they are preferentially TCR4-binding substances.
• a combination of allogeneic MHC class I antigen and gpl20 is given in an increasing dose regimen according to the immunization schedule described in Example 2. Suppression of the production of anti-gpl20 antibodies is determined, conveniently using ELISA assays with recombinant gpl20 antigen, and pathogenesis is more effectively inhibited than if only low doses of gpl20 are injected.
• EAE Experimental autoimmune encephalomyelitis
• mice and rats by injecting myelin basic protein together with an adjuvant
• T cells that are specific for myelin basic protein have been used to vaccinate against EAE (Cohen, J. Int. Med. 230:471-477 (1991)).
• an increasing dose regimen of myelin basic protein and class I MHC is injected to change a subject that is susceptible to disease into one that is not susceptible.
• MBP myelin basic protein
• the invention can also be used for the treatment of autoimmune disease after onset of disease.
• EAE can be treated with initially small, then increasing doses of MBP together with class I MHC antigen, with an immunization regimen as specified in Example 2. This induces the immune system to switch to a suppressed state for that antigen.
• Antibodies specific for denatured collagen, DNA and RNP ribonuclear protein
• Initially small, then increasing amounts of one or more of these substances together with small and increasing amounts of allogeneic class I MHC will switch off the undesired autoimmune responses to collagen, DNA and/or RNP according to the antigenic substance which is administered.
• This Example demonstrates the administration of subimmunogenic doses of allogeneic class I MHC antigens and an allergen in therapeutic methods to stabilize an individual's immune reaction to the allergen and desensitization of IgE response to the allergen.
• Desensitization is established in an animal model, for example a rat, mouse or guinea pig model.
• An animal is made allergic to a substance (e.g., a protein such as ovalbumin (OA) , or a haptenated protein such as BPO-BGG, DNP- BGG or DNP-OA, where BPO is benzylpenicilloyl, DNP is dinitrophenyl and BGG is bovine gamma globulin (see Levine and Vaz, Int. Arch. Allergy. 39:156-171 (1970) and Lee and Sehon, J. Immunol. 114:829-836 (1975), both of which are incorporated herein by reference) .
• OA ovalbumin
• C3H mice were sensitized with antigen (hen egg lysozyme, HEL) to produce an IgE response.
• antigen hen egg lysozyme, HEL
• the allergic sensitivity of the animals to the allergen was then reduced by treatment with increasing dosages of antigen plus soluble allogeneic MHC-I.
• C3H mice were sensitized by two subcutaneous injections of 30 ⁇ g of HEL at three week intervals. One group was untreated. Mice were given i.v. injections of HEL in PBS at escalating dosages (pg-ng range) , or escalating doses of antigen (HEL) plus H-2K b (MHC-1) . Injections occurred weekly, over a 6 week period. The dose for group 3 began at 0.9 pg MHC-1 and 3pg HEL, and increased by a factor of 10 each week to final doses of 90ng and 300ng respectively. Group 4 received the same series of injections of HEL without the MHC- 1. Control mice received to treatment. On week 12 all mice were bled to determine the histamine response.
• mice treated with escalating levels of antigen were not statistically significant.
• the mean histamine response of mice that received the combination of MHC-1 and antigen was less than half that of control mice that were not desensitized.
• This Example demonstrates that xenogeneic MHC molecules can be administered in subimmunogenic amounts for inhibiting the development of AIDS.
• SIV immunodeficiency virus
• This Example describes methods to identify substances that bind preferentially TCR4 or TCR8 (e.g., anti- I-J antibodies, and the 1F7 antibody described in Wang et al., Eur. J. Immunol. 22: 1749-1755 (1992), incorporated herein by reference) , in addition to MHC class II and MHC class I molecules, which can be used as positive controls to develop and standardize the methods.
• TCR4 or TCR8 e.g., anti- I-J antibodies, and the 1F7 antibody described in Wang et al., Eur. J. Immunol. 22: 1749-1755 (1992), incorporated herein by reference
• a T cell preparation is depleted of CD4 or CD8 cells using anti-CD4 or anti-CD8 reagents, either with complement, or by the application of an immunoabsorbant method. Differences in binding of a fluorescence-dye labeled conjugate of the substance being screened to CD4 and CD8 T cells are determined, conveniently using flow cytometry.
• This Example demonstrates that self MHC molecules can be administered in sub-immunogenic doses to both prevent and provide therapy for an autoimmune disease.
• the MRL-lpr/lpr mouse which spontaneously develops fatal autoimmunity, has the H-2 k MHC haplotype. Protection from disease occurs most effectively by giving injections of increasing doses of MHC class II molecules of the H-2 k haplotype, namely I-A k and/or I-E , in combination with allogeneic class I MHC molecules.
• the self class II MHC molecules may be aggregated or covalently linked, or they may be covalently coupled to the class I MHC molecules.
• the MHC molecules are injected into three week old MRL-lpr/lpr mice beginning at very low doses, as specified in Example 2.
• Older animals that already have autoimmune disease demonstrate a remission of the various symptoms, including lymphadenopathy and the production of autoantibodies, that are typically associated with autoimmunity in these animals.
• C3H mice were given six escalating dosages (pg- ⁇ g range) of H-2K b molecules (MHC-1) , namely doses of 90pg, 900pg, 9ng, 90ng, 900ng and 9 ⁇ g. Injections occurred weekly, over a 6 week period. Two control groups received no treatment. On weeks 7 and 10 all mice (except one of the control groups) were given 30 ⁇ g of antigen (hen egg lysozyme, HEL) by subcutaneous injections, in a protocol known to induce IgE and IgGl antibody and allergic hypersensitivity. On week 13 all mice were bled to determine antibody levels and challenged with an i.p. injection of HEL. Plasma was
• SUBSTITUTE SHEET collected 8-10 min. after challenge to determine the histamine response.
• mice treated with MHC-l produced high levels of IgGl antibody (11-fold higher than untreated controls) . This result was statistically significant (greater than 95% confidence) . 100% of the MHC-l treated mice responded to the antigen, in contrast to only 67% of untreated controls. The average ratio of histamine related in response to antigen to the level of antigen-specific IgGl was decreased by a factor of greater than 7 in the treated mice.
• histamine ng/ml mean 2773 107 5728 (SEM) (2239) (31) (1858) histamine/IgGl ratio arbitrary units 3.8 0.55 (SD) (4.9) (0.38)
• Bleeds from all groups post-treatment and before sensitization were all negative for antigen-specific antibody, i.e., no detectable response to MHC-l during treatment.
• Comparisons were by Students t, on logarithmic data, P ⁇ 0.05 is significant.

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