Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
  • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents

Definitions

• the present invention relates, in general, to a method of suppressing an undesired immune response and to constructs suitable for use therein.
• the invention described herein was made in part in the course of work under a grant or award from the United States Army, No. DAMD 17- 86-C-6038.
• Immune responses are generally advantageous (protective) in nature, however, under certain situations, the animal body produces an immune response that is undesirable. Examples of such undesirable responses include allergic reactions, characterized by the production of IgE antibodies to extrinsic antigens, and autoimmune diseases in which the immune system reacts against self antigens.
• allergic reactions characterized by the production of IgE antibodies to extrinsic antigens
• autoimmune diseases in which the immune system reacts against self antigens.
• the immune modification methodology which forms the basis of the present series of applications is based on the premise that the immune system recognizes foreign antigens in the context of physically constrained arrays.
• arrays In order to stimulate the immune system, arrays must exceed a specific size (or geometry) and have a minimum number of physically accessible epitopes which are identical in nature (minimum valence) . Once these two parameters are met or exceeded, the immune system will respond by the production of antibodies (IgM, IgG and/or IgE) by antigen specific B-cells and by the production of T-cell factors and/or activities (T-cell 'help', cytokines, cytoxicity, etc.).
• the method to which the present invention relates is based on the finding by Applicants that this system can be manipulated by introducing synthetically derived macromolecular arrays that are "subthreshold" in geometry and/or valence and that are designed to compete with naturally occurring arrays for the suppression of autoimmune and extrinsic allergic responses.
• the technology which forms the basis of the invention is derived from the Immunon model of immune response described by Dintzis et al in Proc. Nat'l. Acad. Sci. USA, 73:3671-3675 (1976). That paper discloses the concept of there being a threshold as to the number and spacing of haptens on T-cell independent antigens in order to obtain an immunogenic response.
• the 1976 paper also discloses that the non-immunogenic polymers are suppressive of the action of immunogenic polymers towards triggering the de novo immune response in non-immunized animals. The suppressive effect of non-immunogenic polymers on the immunogenic response of immunogenic polymers is further described in Proc. Nat'l. Acad. Sci.
• the earlier applications of the present series include details of studies that were done using experimental paradigms involving T- independent antibody responses which can be assessed by the level of IgM production.
• the use of size restricted backbones of various types linear polyacrylamide, dextran, Ficoll, carboxymethyl cellulose, etc.
• IgM antibody production to small molecular weight haptens such as DNP and fluorescein is specifically described.
• DNP small molecular weight haptens
• fluorescein See Examples 1 to 7 below.
• the present application includes details of studies relating to T-cell dependent antibody production as well as T-cell responses by themselves.
• the PVA backbone structure was created by reacting low molecular weight PVA, 14 kDa, with cyanogen bromide to convert some of the hydroxyl groups on the polymer to a reactive form, and coupling those activated hydroxyl groups to amino groups on aliphatic diamine. This reaction was expected by the authors to substitute the PVA polymer molecules with a number of free aliphatic amino groups from the unreacted ends of the diamine adduct. These ends were subsequently substituted with hapten groups to form multiply substituted PVA molecules of molecular weight supposedly almost unchanged from that of the original PVA.
• This polypeptide is a commercially available randomly ordered polymer synthesized from chemically activated forms of the D-amino acids, D-lysine and D-glutamic acid, in the ratio 60:40. Katz has rationalized the findings of immune suppression as caused by the "unnatural" character of the synthetic polypeptide composed of the unusual D- a ino acids rather than the usual L- forms of the amino acids, which are found in all protein molecules. This interpretation was apparently supported by the finding that equivalent immune suppression was not observed when the carrier backbone polypeptide was synthesized from the more normal L-amino acids.
• Figure 7 Cyclodextrins as point source scaffolds.
• Figure 8. Conjugation of peptides to GMB-dextran.
• Figure 10 Amino acid analysis of a peptide-dextran conjugate.
• Figure 17 Dose-response measurements for different lots of BALB/c mice. Measurements were made on serum from individual mice. The mean of measurements on each group at each dose is shown, together with the SEM when it is larger than the symbol. Of the symbols used, the solid black dot represents ten mice per point (these points being the same as in Fig. 16) ; the open circle "o" represents five mice per point; and the symbol ⁇ represents six mice per point.
• Figure 18 Response-reduction measurements for increasing doses of nonimmunogenic polymer preparation N injected simultaneously with a constant dose of immunogenic polymer preparation S. Measurements were made on serum from individual mice. The mean of each group is shown together with the SEM when it is larger than the symbol.
• the data represent the mean of duplicate cultures with triplicate assays per culture; and SD is indicated when it is larger than the circle.
• the experimental peak response corresponds to «300 plaques per 10 6 spleen cells with a blank (without polymer) of «20 plaques per 10 ⁇ spleen cells.
• the curve gives the theoretical response expected from Eq. 1 for a peak response occurring at a polymer concentration of 0.4 ng/ml and an immunon size, q, of 10.
• FIG. 20 Dose-reduction measurements for increasing doses of nonimmunogenic polymer preparation N incubated in spleen cell culture with a constant dose (0.3 ng/ml) of immunogenic polymer preparation S. Procedures and data treatment were as in Fig. 19. The different symbols show data obtained in separate experiments. The solid curve gives the theoretical response expected from Eq. 1 for an immunon size, q, of 10 and D" x set equal to 0.4 ng/ml as derived from Fig. 19.
• Figure 21 In vitro response kinetics.
• the direct (IgM) anti-Fl response of naive spleen cells to increasing doses of F1 I10 PVA400 was measured after 3( ⁇ f),4(0), or 5(D) days of culture. All the S.D. were less than 10%.
• Figure 22 In vivo response kinetics. An optimal dose of Fl 55 PVA200 (10 ug/mouse) was injected i.p. in 0.5 ml saline, and direct (IgM) anti-Fl response was measured at times from 0 to 66 days. Each point represents 3 mice and is the mean of triplicate assays. The S.D. was less than 10%.
• FIG. 26 Inhibition of the in vitro response to Fl, 0 Fic750 by high doses of immunogenic Fl-polymers.
• the IgM PFC response to 3 ng per ml of Fl 90 Fic750 alone was assigned a value of l, and the response of cultures containing added amounts of Fl-polymer was expressed as the fractional relative response.
• -O- FL150-CMC440; -D-FL90- Fic750; ..• ⁇ ••• FL240-Fic750; -D-FL640-Fic2000; -G- FL230-PA400
• FIG. 27 Anti-fluorescein IgG antibody serum levels in 3 immunized mice as a function of time. The mice have been repeatedly injected with fluoresceinated ovalbumin on aluminum hydroxide adjuvant and allowed to rest for several weeks before the bleedings shown on the Figure. All bleedings were analyzed in the same ELISA assay at serum dilutions of 10,000 fold.
• FIG 28 Cure of anti-fluorescein IgG serum antibody level by the fluoresceinated polyacrylamide polymer FL30-Pa50, i.e., a 50 kD polyacrylamide polymer substituted with 30 fluorescein hapten groups.
• the time at which the dose of 3 mg of polymer was given has been arbitrarily designated day 0 on the time scale.
• the open circle data points are the averages of the data points shown in Figure 27 for unsuppressed mice, with standard deviations and a least square fit straight line indicated. Other data points represent 6 individual mice.
• Figure 29 Cure of anti-fluorescein IgG antibody level by the fluoresceinated dextran polymer FL25-Dex70, i.e., a 70 kD dextran polymer substituted with 25 fluorescein hapten groups.
• the time at which the dose of 1 mg of polymer was given was arbitrarily designated day 0 on the time scale.
• the open circle data points are the averages of the data points shown in Figure 27 for unsuppressed mice, with standard deviations and least square fit straight line indicated. Other data points represent 6 individual mice.
• FIG 30 Cure of anti-fluorescein IgG antibody response by the fluoresceinated dextran polymer FL30-Dex80, i.e., a 80 kD dextran polymer substituted with 30 fluorescein hapten groups.
• the time at which the dose of 3 mg of polymer was given was arbitrarily designated day 0 on the time scale.
• the open circle data points are the averages of the data points shown in Figure 27 for unsuppressed mice, with standard deviations and least square fit straight line indicated. Other data points represent 3 individual mice.
• FIG 31 Cure of anti-fluorescein IgG antibody response by the fluoresceinated dextran.
• FIG 33 Similar to Figure 31, except that mice were stimulated at the times indicated by doses of 0.1 ⁇ g of fluoresceinated ovalbumin absorbed on 1 mg of aluminum hydroxide.
• Figure 34 Reduction by cure treatment of the number of spleno ⁇ ytes producing anti-FL IgG serum antibodies.
• FIG. 37 The structures of penicillin and the penicilloyl hapten.
• R is a benzyl group for Penicillin G (benzyl penicillin) .
• the internal amide bind of the ⁇ -lactam ring is replaced by an amide bind involving a primary amine from the carrier.
• Figure 38 Administration of the suppressive polymer BPO-PA virtually abolishes the anti-BPO response ( Figure 38a) , while the anti-OA response ( Figure 38b) is unaffected. Not only does the anti-BPO titer remain undetectable for two months, but the mice are tolerized by the BPO- PA and are unresponsive to a "booster" injection given on day 110.
• Figure 40 Serum anti-BSA IgM dose- response for monomeric (•) and polymerized (•) BSA.
• CAF1/J mice were injected with 10, 100 or 1000 ⁇ g of monomeric (68 kD) or carbodiimide cross-linked 70-meric (5000 kD) preparations of BSA.
• the IgM antibody response was measured by ELISA at day 6 for serum dilutions of 200 fold. Data are the average of 3 mice per point.
• FIG 43 Serum anti-BSA dose-response for multiple injections of monomeric (•) or carbodiimide cross-linked 20-meric (•) BSA.
• the anti-BSA IgG response is shown after three injections given 30 days apart. Serum was diluted 4000 fold for ELISA assay.
• Figure 44 Effect of multiple injections on serum anti-BSA IgG response to a BSA "20-mer” given at very low dose.
• Figure 45 Serum anti-OVA IgM dose- response 5 days after injection of OVA monomer (•) or glutaraldehyde cross-linked 150-mer (•) .
• FIG 47 0 Comparison of serum IgM response generated by monomeric and various polymeric sizes of BSA ( ⁇ ) and OVA (•) .
• FIG. 49 Levels of IgG peptide (GALA)- specific antibodies in serum. Legend: Mouse #5 0—O; Mouse # 6 • •; Mouse #7
• Figure 52 Suppression of anti-histone antibody titers.
• Figure 52a is experimental group.
• Figure 52b is control group.
• Protocol Day 1 - 1, 10, 100 ⁇ g I.V. or 100 ⁇ g I.P. Day 3 - 200 ⁇ g I.P. Day 9 - 200 ⁇ g I.P. Day 16 - 200 ⁇ g I.P. Day 23 - 200 ⁇ g I.P.
• Figures 53 Specificity of suppression of anti-histone responses.
• Figure 53a Anti-N15-H2B.
• Figure 53b Anti-ssDNA.
• FIG. 54 Activation and inhibition of T-cell interleukin-2 production by soluble fluorescein polymers.
• Transfected T-cell line 1B2 was treated with phorbol ester, 3 ng per ml, and with various concentrations of soluble fluorescein polymers as indicated in the Figure. After incubation, supe natant solution was removed and assayed for IL-2 by measuring proliferation of an IL-2-dependent cell line, CTLL2. Proliferation of the CTLL2 cells is measured by the incorporation of radioactive thymidine into cellular DNA.
• the T-cell response to two fluorescein polymers of different molecular weight and valence were measured at various concentrations.
• the inhibitory polymer was added at four concentrations: none (open squares); 0.48 ⁇ g/ml (closed triangles); 4.8 ⁇ g/ml (X symbols) ; and 15 ⁇ g/ml (closed squares) .
• Figure 55 Activation and inhibition of intracellular calcium flux in T- ⁇ ells by soluble fluorescein polymers. Transfected T-cells were loaded with the calcium sensitive fluorescent dye, Indo-1 AM (Molecular Probes, Eugene, OR) . Fluorescence emission at two wavelengths, 405 and 480 n , was determined upon excitation at 355 nm for individual cells, using a Coulter MDADS flow cytometer. In the Figure, each dot represents the calcium concentration in a single cell at some instant in time, with time shown in units of
• the transfected cells were analyzed for 20 seconds and then various fluorescein polymers were added in the complete absence of phorbol ester or accessory cells.
• Substantial intracellular calcium concentration rises in at least 10% of the cells were seen when the cells were treated with the stimulatory polymer, FL50-Ficl50, at concentrations of 38 ⁇ g/ml (a), and 3.8 ⁇ g/ml (b) , but less calcium flux at 380 ⁇ g/ml (c) .
• the inhibitory polymer, FL11-Fic46 did not induce any substantial calcium flux at any measured dose, but caused substantial inhibitory effect (d and e) .
• Stimulatory polymer FL50-Ficl50, 38 ⁇ g/ml (d) and 3.8 ⁇ g/ml (e) , was added after a short incubation of the cells with inhibitory polymer. In both cases the calcium flux induced by the stimulatory polymer is almost eliminated.
• the method of the invention comprises administering to a subject suffering from an undesired immune response an effective amount of a non-immunogenic material which carries a number of antigenic domains (i.e., "epitopes" or "haptens") which correspond to the antigen, e.g. the allergen or self-antigen which causes the allergy or autoimmune disease responsible for the undesired response.
• a non-immunogenic material which carries a number of antigenic domains (i.e., "epitopes" or "haptens") which correspond to the antigen, e.g. the allergen or self-antigen which causes the allergy or autoimmune disease responsible for the undesired response.
• the haptens or epitopes bind to cell antigen receptors specific for the indicated haptens or epitopes and, provided the hapten or epitope number is sufficient and the carrier size is below an ascertainable threshold limit so as to avoid the formation of a stimulatory cluster of antigen receptor
• the disclosure of an earlier filed application of this series includes a description of constructs comprising size fractionated linear polyacrylamide chemically modified to accept DNP groups as epitopes. These conjugates can be organized into groups based on the size (molecular weight) of the backbone polymer and hapten number (number of DNP groups per average molecular weight polymer for a given group) . The combination of these two sealer quantities makes it possible to determine the role of hapten density as a separate variable. Based on the data obtained using these constructs in both in vitro and .in vivo models of immune function, certain "rules" governing B-cell activation by antigen have been elucidated and used to control the T-cell independent immune response on an antigen specific basis. These rules and their use in effecting an antigen specific alteration in immune function are included in an earlier filed application.
• Another application of this series included further exemplification in support of the application of these rules to include a variety of backbones or scaffolds and haptens, thus further documenting the "universality" of the rules elucidated in the original filing as they apply, particularly, to T-cell independent immune system activities (operationally defined as IgM production) .
• the present disclosure includes specific exemplification which makes clear the applicability of these selfsame rules to a spectrum of immune function, including T-cell dependent antibody production (operationally defined as the production of IgG and IgE) and T- cell activity as well.
• the Examples that follow include the exemplification from the parent cases and further exemplification of complex constructs involving antigens of greater diversity than simple small molecular weight haptens such as DNP and fluorescein.
• the biophysical and biochemical considerations that need to be taken into account when designing these constructs are set forth below. These include the chemistry of synthesis of the constructs and preferred methods of characterizing the final products so as to optimize fidelity to and compliance with the primary principles governing valence and size that constitute the operational underpinnings of the invention as disclosed in this series of applications.
• conjugate For a construct (conjugate) to be non- stimulatory, and hence "suppressive” or tolerogenie in nature, it must meet one or both of the following criteria: i) The "valence" of the conjugate (operationally defined as the number of "discrete antigenically recognizable moieties" per final macromolecular construct) must be less than the Immunon model threshold number (generally, less than 20) . As noted above, these moieties can be simple haptens or more complex peptides or proteins.
• each of these moieties y have multiple "antigenic facades", but for any given B-cell, capable of recognizing the moiety, it will have one and only one discrete binding region recognized by one immunoglobulin receptor of that particular B-cell even though other B-cells may recognize other regions of the moiety in question.
• Special cases such as peptides or proteins containing multiple identical peptide sequences (such as some of the sequences found in certain malaria proteins or in proteins such as hemoglobin which has repeated subunits) or carbohydrates with regularly repeating series of sugar residues (such as in bacterial polysaccharides) are considered as containing multiple "discrete antigenically recognized moieties" for purposes of defining valence; and/or ii)
• the size of the final construct must be smaller than the minimum size necessary for spanning the cluster of receptors defining the "Immunon".
• the effective size will be a function of a number of independent parameters including: geometry of the backbone or scaffold (linear, branched, globular, radial, etc.), the physical nature of the backbone (flexible, rigid, "articulated”, etc.), the hydrophilicity or hydrophobicity of the backbone, the electrostatic nature of the conjugate (the sum of charges on both the backbone and the arrayed moiety described above) , and the size, geometry and physical make-up of the moiety itself.
• Constructs suitable for use in the present invention can be produced using known means.
• the production method used is one which minimizes the possibility of polymerization as well as cross-linking between the individual molecules.
• the production method is, preferably, chosen such that only one potential reactive site per arrayed moiety is available so that the orientation of the moiety to the backbone can be controlled.
• Resulting construct preparations are, advantageously, characterized prior to use to ensure that they are substantially free from high molecular weight, potentially stimulatory molecules.
• the use of valence restricted scaffolds of defined chemistry is preferred in order to optimize reproducibility of the resulting construct.
• the fundamental concept underlying the technology upon which Applicants* invention is based is that the immune system interacts with its external milieu by the recognition of antigenic arrays of epitopes or haptens. From the biophysical or biochemical perspective, epitopes or haptens are no different from any other receptor ligand, and the soluble immunoglobulin molecules and their membrane bound relatives (such as the T-cell receptor, the B-cell receptor, etc.) are no different than any other protein receptor molecule in other biological systems. The difference lies not with the individual receptor- ligand interaction but with the mechanism of "information transfer" that occurs after the ligand is bound by the receptor.
• subthreshold and superthreshold arrays being immunologic "antagonists” and "agonists", respectively.
• antagonists For a classical pharmacologic antagonist to have acceptable potency it must bind to the receptor molecule with approximately the same degree of affinity as an agonist but in a “nonproductive" manner. That is, it must bind but not activate the secondary events caused by agonist binding.
• the corollary to an "antagonist ligand” is the "antagonist array” that can aggregate receptors in nonproductive clusters thereby preventing the formation of an immunon by an "agonist array”.
• immunologic agonists can be viewed as "superthreshold arrays” that can bind with a number of receptors that meets or exceeds the minimum necessary for immunon formation and immunologic antagonists can be viewed as "subthreshold arrays” that cannot induce immunon formation but can still occupy multiple receptors simultaneously with approximately the same degree of aggregate avidity as the superthreshold (agonist) array,
• immunon concept (mechanism) is operationally enabling, the specific chemistry of the array is unimportant as long as the biophysical rules of receptor clustering are met and the ligands being arrayed can be recognized by the intended populations of receptors.
• the targeted cell populations should not be sensitive to the exact nature of the scaffold used as long as the array is capable of interacting with the requisite number of receptors.
• the desired outcome can be achieved with a myriad different constructs as long as the principles of valence and/or size are maintained with fidelity.
• it is just as important to control the chemistry of the scaffold or backbone upon which the antigenic array is based as it is to identify and synthesize the appropriate ligand.
• One skilled in the art will appreciate that it is also important to confirm the integrity and composition of the inal construct used before it is introduced into a biological system.
• an immunon consists of eight receptors brought into a cluster
• subthreshold clusters can be achieved by presenting the immune system with rigorously defined "valence restricted" antagonist arrays wherein the number of ligands is restricted to an integral number less than eight.
• valence restricted arrays will be non-stimulatory and will prevent an antibody response to the epitope in question from developing.
• these constructs will act as competitive inhibitors to that response, i.e. they will be suppressive in nature.
• an immunon must have a finite minimum size which is determined by the maximum packing density that the requisite number of antigen receptors can achieve on the surface of the lymphocyte.
• Arrays that cannot cover this minimum area can be expected to be both non-stimulatory and suppressive no matter how many binding sites they may have.
• small ligands such as DNP, fluorescein, or small peptides
• the major determinant for array size is the scaffold, hence the size limit (preferably, less than approximately 150,000 daltons).
• the "ligands" themselves may be the controlling element with regard to the size of the final construct. In this case, even valence restricted constructs may exceed the nominal "size" criteria established for smaller epitopes.
• valence considerations will predominate over size considerations with respect to how the immune system will respond.
• An immunon cannot be expected to form if the array being introduced into the system has a subthreshold number of receptor binding sites no matter how big the array is in absolute terms.
• the first point is the preparation of the backbone scaffold so that the ligands can be covalently attached thereto in a controlled and selective manner.
• the second point is the preparation of the ligands themselves so that they can be attached to the scaffolds in a specific orientation.
• the third point is the formation and characterization of the final conjugate prior to its introduction into a biological system.
• scaffolds can be used to effect the desired outcome with respect to the formation of agonist or antagonist arrays. Some are capable of being used without selective chemical modification. The only significant restriction placed on these scaffolds is that they be freely soluble in physiologically acceptable aqueous buffers when the final constructs are prepared.
• the scaffold can be used to provide the necessary solubility for the final product even though the ligand being conjugated to the backbone is relatively insoluble. Alternatively, if the ligand in question has undesirable charge characteristics (e.g., cationicity) the scaffold can be used to counterbalance these characteristics so that the final product falls within preferred tolerances.
• the materials used are subjected to analytical and, if necessary, preparative sizing techniques (for example, size exclusion gel chromatography or ultrafiltration) to ensure homogeneity and relatively narrow mass distributions both before and after modification.
• preparative sizing techniques for example, size exclusion gel chromatography or ultrafiltration
• independent verification of mass for example, by laser light scattering and/or equilibrium ultracentrifugation
• Low molecular weight haptens The low molecular weight haptens specifically described herein were present in a form that could react directly with the available free amines on the scaffolds utilized without modification.
• the fluorescein for example, the fluorescein
• TE SHEET derivatives were formed using fluorescein isothiocyanate which rapidly reacts with available amines forming a stable thiourea linkage. Those skilled in the art will recognize that other small molecular weight haptens can also be employed using known chemical protocols.
• Peptides identified for use as a ligand can be modified so that they can be successfully arrayed and yet still be recognized by the immune system in the desired fashion.
• Naturally occurring peptides or proteins have three types of amino acid side chain moieties that can be readily used as functional groups with which to tether the peptide to the desired scaffold. These groups are: amines, as represented by the epsilon amino group of lysine and the N-terminal alpha amino group; carboxyls, as represented by the side chain carboxyl groups of aspartic or glutamic acid and the C-terminal alpha carboxyl; and the sulfhydryl group of cysteine.
• cysteine has a number of significant advantages.
• cysteine as a naturally occurring amino acid, can be incorporated into recombinantly synthesized proteins. For these and other reasons discussed in more detail below, sulfhydryl chemistry is the preferred system for conjugating peptides to various scaffolds.
• Proteins provide for significantly different considerations with respect to the immune response generated to these types of antigens. These include multiple different antigenic epitopes per protein monomer as well as different types of epitopes (sequential, linear conformational, and discontinuous conformational epitopes) .
• the first of these issues is the "mapping" of a protein's antigenic facade with smaller peptide or modified peptide based ligands.
• the second is the use of oligomeric constructs made up of the whole proteins or domains of larger proteins either crosslinked to themselves or to a scaffold.
• the third is the generation of "mimotopes" which can mimic the antigenic structure of protein epitopes but which bear little or no compositional similarity to the naturally occurring antigen.
• Reactive end groups, amino or sulfhydryl, suitable for coupling to other molecules can be produced in high yield by the following procedures which make use of the formation and selective reduction of intermediate Schiff bases:
• the saccharide material is reacted (for example, for 18 hours, at pH near 5) with ethylenediamine dihydrochloride (concentration, for example, 0.1 - 1.0 M) (or other small diamine, NH2-(CH2)n-NH2, where n is a small number 2 or greater) , in the presence of 0.01 M sodium cyanoborohydride (concentration, for example 0.01 M) .
• ethylenediamine dihydrochloride concentration, for example, 0.1 - 1.0 M
• NH2-(CH2)n-NH2 or other small diamine, NH2-(CH2)n-NH2, where n is a small number 2 or greater
• 0.01 M sodium cyanoborohydride concentration, for example 0.01 M
• the antigen is known to be a nucleic acid—double stranded DNA.
• double stranded DNA In a series of experiments designed to assess the minimum size of unmodified double stranded DNA needed for successful receptor binding it was found that approximately 40 base pairs were needed for 100% receptor binding. This requirement may be different if the double helix is covalently crosslinked instead of relying solely on the hydrogen bonding of the base pairs for stabilization.
• Naturally occurring DNA synthetic DNA or modified DNA containing phosphorothioates as opposed to naturally occurring phosphate linkages can be used to produce a successful ligand.
• Example 11 includes a description of the types of chemistries that can be employed to produce the desired epitopes possessing the necessary functional groups for covalent attachment to the appropriate scaffold. D. Conjugates
• the final steps in the preparation of a conjugate suitable for use in the method to which the invention relates is the assembly of the desired array from the appropriate scaffolds and ligands and the confirmation that the final material is, in fact, what it is intended to be. Characterization of the final constructs is an important part of the preparation and use of these materials (see Example 12) .
• the earlier filed applications of the present series relate, in large part, to the suppression of T-cell independent responses by constructs comprising size restricted backbones and small molecular weight haptens (such as DNP and fluorescein) .
• Data presented in the Examples that follow demonstrate that the same type of suppression can be obtained with more complex responses involving T-cell dependent antibody production, represented by IgG and IgE.
• the data indicate that suppression occurs at the cellular level.
• Clinically important antibody responses to extrinsic allergens (both small chemical entities and complex epitopes) represented by IgE production can be completely suppressed by constructs meeting the valence and size criteria set forth above.
• constructs of the present invention can be used to suppress autoimmune responses.
• Dextran can be considered a "prototypical" scaffold for a number of reasons: 1) it is freely soluble in aqueous buffers, 2) it can be readily modified using "off the shelf” chemistry, 3) it has been used in humans in gram quantities as a plasma expander with no significant toxicities, 4) it is available in roughly size-fractionated bulk quantities at low cost and 5) there are no known mammalian dextranases. The latter point is particularly important since one of the primary considerations of this technology is that the arrays be metabolically stable so that the desired outcome can be effected in an experimental animal or human.
• Dextran of various molecular weights (1) was first carboxymethylated with chloroacetic acid (C1CH 2 C0 2 H) at glucosyl 2'-, 3'- or 4*- hydroxyl positions to yield the corresponding carboxymethyl-dextran (2) .
• EDC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride
• dexamine (3) allows measurement of the "amine substitution density" using standard quantitative analytical chemistry techniques. Expressed as the number of ⁇ moles of amine present per milligram of dexamine (3) , the value for amine density represents the theoretical maximum substitution density for any particular peptide.
• Dexamine (3) used in the Examples described herein was found routinely to contain approximately one primary amino group (-NH 2 ) for every five glucosyl residues (or 1000 g/mole) present in the dexamine (3) sample.
• the dextran (1) used in the preparation of the peptide-dextran conjugates was a size- fractionated, average molecular weight polymer. Chloroacetic acid and ethylene diamine were purchased from Aldrich Chemical Co. l-Ethyl-3-(3- dimethylaminopropyl)carbodimide hydrochloride (EDC) and gamma-maleimido n-butyric acid N- hydroxysuccinimide ester (GMBS) were purchased from Sigma Chemical Co. Trinitrobenzensulfonic acid (TNBS) was obtained from Pierce Chemical Co.
• Phosphate-buffered saline (PBS; 150 mM NaCl, 10 mM phosphate pH 7.3 - 7.4), used in the preparation of GMB-dexamine (4) , was prepared fresh (for each day's set of reactions) from an autoclaved 10X PBS stock solution and autoclaved water with subsequent filtering through a 0.2 ⁇ m filter. (Preparation of PBS in this manner is important to the production of conjugates that are devoid of undesirable contaminants) . Dialysis tubing (6,000-8,000 mwco) was obtained from Spectrum Medical Industries, Inc.
• dextran (1) and molecular weight measurement of dextran (1) or dexamine (3) samples were carried out by size exclusion chromatography followed by equilibrium ultracentrifugation, and/or laser light scattering analysis.
• Carboxymethyl-dextran (2) was produced from dextran as follows: Sodium hydroxide (675 mmole, 135 mL of 5 M NaOH) was added to 0.3 L of water and the resulting solution chilled in an ice-water bath (0°C) with stirring. Chloroacetic
• amine substitution density was carried out as follows: Dexamine (3) was dissolved in 1 mL of 0.1 M sodium tetraborate buffer (pH 9.3) to give a solution concentration of 1-2 mg/mL. Freshly- prepared trinitrobenzenesulfonic acid (TNBS, 25 ⁇ L of a 30 mM solution in sodium tetraborate buffer) was added and the resulting (vortexed) reaction mixture stored in the dark for 2 hours at room temperature. The yellow-colored solution was then read against a reagent blank at 366 nm.
• TNBS trinitrobenzenesulfonic acid
• dexamine (3) was produced from dexamine (3) as follows: Dexamine (produced as described above) was dissolved in phosphate-buffered saline (PBS, pH 7.5) and stirred at room temperature to give a solution concentration of 5-10 mg/mL. A five ⁇ fold molar excess of GMBS (gamma-maleimido n- butyric acid J ⁇ -hydroxysuccinimide ester) relative to PBS, pH 7.5.
• GMBS gamma-maleimido n- butyric acid J ⁇ -hydroxysuccinimide ester
• ET to (dex)amine content was then dissolved in dry tetrahydrofuran (THF, stored over 4 Angstrom molecular sieves) such that a GMBS concentration of ca. 50 mg/mL was achieved.
• THF dry tetrahydrofuran
• the GMBS/THF solution was added dropwise to the stirring dexamine (3) solution and the acylation reaction allowed to proceed for 30 minutes at room temperature with the solution pH being maintained at 7.1-7.5 by the dropwise addition of 1 mM NaOH.
• GMB-dexamine (4) was then separated from excess GMBS by gel filtration of the reaction mixture on a 15 mm x 30 cm Sephadex G-25 column equilibrated in PBS (pH 7.5).
• the column effluent was monitored at 280 nm with a Pharmacia Dual Path Monitor UV-2.
• Column fractions containing GMB- dexamine (4) were combined and set aside pending the availability of a reduced cysteine (Cys)- containing peptide.
• dextran-based scaffolds can be made to have neutral, anionic or cationic characteristics using a modification of the fundamental dextran chemistry described above.
• dexamide For the preparation of anionic scaffolds, an alternative precursor dexamide is needed. These can be formed by the condensation of either L-Asn or L-Gln with dextran according to the following protocol:
• 4-Nitrophenyl chloroformate (685 mg, 3.4 mmole) was added to a solution of 1 g dextran (1) (13.87 ⁇ mole) in 60 mL of a dry DMSO-pyridine mixture (1:1, v/v) at 0°C in an ice-water bath. To this solution was added 76 mg of 4-(N,N- dimethylamino)pyridine (6.22 mmole). The reaction mixture was stirred at 0°C for 4 hours and then a fifty-fold molar excess of either L-Asn or L-Gln was added. The reaction mixture was allowed to warm slowly to room temperature and then stirring was continued for an additional 48 hours.
• the dexamide was precipitated with an excess of dry ethanol/ethyl ether (8:2, v/v) and then dried in vacuo. The dried material was dissolved in water, dialyzed for 3 days against water and then lyophilized (IX) .
• the desired dexamide can be converted into its corresponding neutral or anionic dexamine by first dissolving the dexamide in 50% aqueous acetonitrile and stirring gently at room temperature. To this solution, a several fold molar excess (relative to the calculated amide content) of iodobenzene diacetate is added and the reaction mixture is stirred overnight. (Iodobenzene diacetate will stoichiometrically convert one equivalent of a primary amide into the corresponding primary amine.) The resulting dexamine can be purified from the other reaction products by size exclusion chromatography, vacuum concentration and then lyophilization from water. Figure 3 illustrates the complete conversion of dextran to anionically charged dexamine. While not as useful as dexamine possessing an anionic character, dexamine with a net positive charge (following conjugation) might be found useful in certain limited cases.
• Preparation of this material can be carried out with ethylene diamine-containing dexamine as follows: Acylation of the (dex)amine (4) groups with J -Npys-L-Arg-OSu results in the incorporation of one equivalent of positive charge for every equivalent of acylated (dex)amine (4). Subsequent removal of the lf-Npys group (with (n-Bu) 3 P) liberates a free ⁇ -amino group which can be acylated further with GMBS or converted directly into the maleimide functional group via N- methoxycarbonyl maleimide. Longer and shorter homologs of Arg can be used in an analogous fashion (e.g. Homoarg) . (See Figure 4) B. Polyacrylamide and Poly(acrylamine- acrylic acid)
• Linear polyacrylamide was synthesized from the monomer is aqueous solution, giving polymer preparations with average molecular masses varying from 20,000 to 500,000 kDa, as determined by the methods of equilibrium ultracentrifugation, high pressure liquid chromatography (HPLC) , size- exclusion gel chromatography (SEC) and laser light scattering. Preparations were size fractionated on appropriate gel filtration chromatography columns, Sepharose CL-2B, CL-4B or CL-6B, Pharmacia, to yield center cuts with relatively narrow molecular mass distributions, as measured by HPLC. Usually, such polyacrylamide preparations were chemically substituted with amino groups to prepare them for later coupling with hapten reagents.
• poly(acrylamine-acrylie acid) was synthesized using a modification of the iodobenzene diacetate reaction described above with commercially available size-fractionated random copolymers of acrylamide acrylic acid. Specifically, _E,J- bis(trifluoroacetoxy)iodobenzene is dissolved in DMF to which an equal volume of water is slowly added with continuous stirring. The size fractionated polymer is then added to the reaction mixture and stirred overnight at room temperature. It is then transferred to a separatory funnel, washed with water equivalent to three times the volume of the reaction mixture, and extracted with diethyl ether (4 extractions with 4X volume) .
• Ficolls like dextrans, are polysaccharide polymers that have free hydroxyls that can be modified using the same chemistry as described for the conversion of dextran to the various dexamines (neutral, anionic, and cationic) . In addition, bulk quantities of these materials are available from commercial sources in various molecular weight ranges.
• dextran based scaffolds and Fieoll based scaffolds are more globular or "three dimensional" in nature while dextrans are more linear and branched. As can be seen from the data presented in the Examples that follow, this difference does not seem to carry with it significant functional consequences with respect to the production or use of agonist or antagonist arrays.
• carboxymethyl cellulose is very similar to both Fieoll and dextran from the biochemical perspective.
• This polymer has a net anionic character from the beginning and, as a result, can be used to produce anionic scaffolds.
• the chemistry needed to modify this polymer is essentially the same as that for dextran once it has been carboxymethylated.
• polyvinyl alcohol While polyvinyl alcohol is not carbohydrate based, it does possess free hydroxyl moieties that can be modified by the same chemistry as described above for dextran.
• Higher molecular weight scaffolds can be produced by the introduction of various water soluble crosslinking agents such as water soluble carbodiimides at various concentrations. The resulting crosslinked material can then be subjected to the same type of size exclusion chromatography and molecular mass analysis as that for the other polymers described above.
• Proteins or other polypeptides behave in many respects like the poly(D-Glu/D-Lys) eopolymer with respect to the availability of both carboxyl and amino groups for chemical modification.
• proteins can be crosslinked and fractionated with respect to size in a manner similar to the crosslinking and separation of the poly (D-Glu/D-Lys) described above.
• the fractions can be effectively segregated into what would be the equivalent to valence restricted oligomers (dimers, trimers, etc.).
• these constructs can be used as agonist and/or antagonist arrays without further modification.
• recombinant DNA/protein engineering technologies have evolved to the point that fusion proteins made up of a core "scaffold" with recombinantly produced oligomeric representations of other proteins or protein domains can be constructed.
• the final product can be formulated to represent valence restricted arrays of the desired "epitope or ligand" just as if they had been chemically crosslinked or conjugated to a valence restricted carrier.
• streptavidin has a relatively unique structure that can be used to form tetrameric arrays by the introduction of a biotin moiety onto the desired ligand. Streptavidin has four binding sites for biotin that have such high affinity for this moiety that once bound are essentially the same as a covalent linkage. In addition, streptavidin is freely soluble and has an isoelectric point near neutrality so that undesirable charge characteristics can be avoided.
• valence-restricted scaffolds for producing agonist or antagonist arrays.
• Illustrated below are a number of valence- restricted or "point source” scaffolds that can be utilized for these purposes.
• point source scaffolds One skilled in the art will realize that these scaffolds are only a few of the types of potential valence-restricted scaffolds that can be constructed to meet this need.
• maleimide/succinimide moiety as a representative reactive group for this series of scaffolds, several potential compounds can be synthesized from commercially available starting materials. Illustrated in Figure 6 is a sampling of these types of "point source scaffolds”.
• An alternative type of scaffold can also be made that has the capability of being varied with respect tc both the effective size and valence of the final construct.
• An example of this type of scaffold using beta-cyclodextrin as a template is illustrated below wherein the valence can be controlled with precisely defined chemistry and the arm length using various types of flexible spacers such as polyethylene glycol.
• the cyciodextrins (CD) are oligosaccharides made up of glucose units that are linked through ⁇ 1—>4 glycosidic bonds. In the resulting torus shaped molecule, the primary and secondary hydroxyls are positioned on opposing faces.
• Perderivitization of ⁇ , ⁇ or ⁇ CD provides the corresponding 6, 7 and 8 valenced products.
• Each of these compounds can also be mono-functionalized.
• Treatment of ⁇ -CD, the most readily available substrate, with a bifunctionalized protecting group will lead to the bis protected product. This in turn can provide the 2 or 5 substituted products. Accordingly, reaction with two linker groups leads to products with valences of 3 and 4. Thus it is possible to attach between 1 and 8 epitopes to CD by judicious use of protecting groups.
• valence and "arm length" can be varied to produce what can be considered as a radially disbursed array or "octopus-like" scaffold for ligand presentation.
• This type of array is optimal for receptor/ligand interactions when the receptor population is relatively free to move in the cell surface membrane.
• the chemistry of the "arms” can be varied to produce scaffolds with relatively free range of motion to arms with progressively less flexibility. Disclosed below are some of the chemistries that can be employed to make these types of constructs for use in suppressing an undesirable antigen specific immune response.
• ⁇ -Cyclodextrin 1 was transformed into its heptaamino derivative 2, using literature procedures (Boger et al, Helvetica Chimica Acta 1978, 61:2910) (Scheme 1).
• the extended arm product 3 was produced as follows heptaamino ⁇ -cyclodextrin (2) (3.0 g, 2.15 mmol) and triethylamine (2.4 mL, 17.2 mmol) were dissolved in 50 mL DMF. EDC (3.78 g, 19.3 mmol) was added followed by Boc- ⁇ - aminocaproic acid (5.53 g, 19.3 mmol). The reaction mixture was stirred overnight, at which time 200 mL water was added and a precipitate formed. The solid product was filtered, washed with water and dried under vacuum to yield 4.78 g (85%) of the Boc protected 3. Deprotection was effected as follows the product was dissolved in 50 mL of HC1 saturated dioxane (4N) and stirred for 3 h. Evaporation followed to yield 3, 2.99 g (75%) .
• CS-0001 was produced from 3 as follows.
• the extended arm ⁇ -cyclodextrin (3) (500 mg, 0.23 mmol) was dissolved in 60 mL of 0.1M NH 4 C0 3 .
• GMBS (2.25 g, 8.05 mmol) was dissolved in 40 mL THF and added to the reaction mixture, which was stirred overnight. The mixture was evaporated and then purified by RPHPLC to yield 251 mg (36%) of CS-0001.
• the fluorescein specific construct Cl- 374 was produced from 3 as follows.
• the extended arm ⁇ -cyclodextrin (3) (40 mg, 0.019 mmol) was dissolved in 10 mL of 0.1M NaHC0 3 .
• Fluorescein isothiocyanate (200 mg, 0.52 mmol) was added and the resulting mixture was stirred overnight.
• the orange solution was then ultrafiltered through a YM3 membrane until the filtrate remained uncolored.
• the remaining orange retentate was purified by RPHPLC to yield Cl-374, 20mg (16%).
• the fourteen armed scaffold (6) was produced as follows. Heptaamino ⁇ -cyclodextrin (2) (500 mg, 0.36 mmol) was dissolved in 4 mL DMF. J ⁇ -t-Boc-N ⁇ -t-Boc-L-lysine-iV-hydroxysuccinimide ester (4.56 mg, 10.1 mmol) was added followed by 2V-methylmorpholine (320 ⁇ L, 2.88 mmol). The reaction mixture was stirred overnight at which time 25 mL of water was added and a precipitate formed. The solid product was filtered, washed with water and dried. The resulting solid was dissolved in HCl saturated dioxane and stirred 3 h. Evaporation produced 575 mg (63%) of 5.
• This fourteen armed product-5 (400 mg, 0.16 mmol) was dissolved in 4 mL DMF. Boc- ⁇ - aminoacaproic-W-hydroxysuccinimide ester (2.89 g, 8.85 mmol) was added followed by N- methylmorpholine (485 ⁇ L, 4.42 mmol). The resulting mixture was stirred overnight, at which time 25 L of water was added to effect precipitation of the product.
• the fourteen armed scaffold was isolated upon filtration, washed with water, and dried. The resulting solid was immediately dissolved in HCl saturated dioxane and stired 3 h. Evaporation yielded 96 mg (60%) of 6. These compounds have all exhibited satisfactory 'H NMR, mass spectral analysis and amino acid analysis.
• Other arms can consist of polyethylene glycol units or some other hydrophillic polymeric subunit. Spacers of this sort would permit exploration of distances between receptors.
• a heterobifunctional linker with amine and hydroxyl termini can be functionalized such that an activating group can be fashioned at the hydroxyl terminus. This can in turn be displaced by the amines of compounds 2 or 3.
• a scaffold such as the ones previously described, containing longer spacer arms, will result.
• the peptides destined for incorporation into peptide-dextran conjugates were generated by solid phase peptide synthesis using a standard stepwise elongation of the peptide chain.
• solid phase peptide synthesis begins with ⁇ -deprotection of the amino acid residue attached to the synthesis resin. This step is followed by neutralization and washing of the deprotected amino acid-containing resin which prepares it to receive (i.e. react with) the next amino acid, itself activated to facilitate the formation of the first peptide bond (-NH-CO-) . A subsequent washing of the now (di)peptide-containing resin is then followed by the same series of events which are continued until the desired peptide has been produced.
• the finished peptide is then cleaved off of the resin under conditions which simultaneously remove some or all of the individual amino acid side-chain protecting groups.
• Specific protecting groups designed to be removed under different conditions than that used for resin cleavage are frequently employed so as to render subsequent conjugation to backbone more controllable. All reagents used in the studies described herein were obtained from standard commercial sources.
• Solid phase peptide synthesis was carried out on either an Applied Biosystems (ABI) 430A or Biosearch 9600 automated peptide synthesizer using ⁇ -tert-butyloxycarbonyl (N-t-BOC) protection. Trifunctional amino acids other than Cys were protected with (protecting) groups compatible with standard N-t-BOC solid phase peptide synthesis.
• N-t-BOC-L-Cys was S_-protected with the p_- methylbenzyl (Meb, HF labile) , acetamidomethyl (Acm, HF stable) or nitropyridinesulfenyl (Npys, HF stable) group depending on the need for HF labile or HF stable sulfhydryl protection.
• a Cys residue to either the N- or C- terminus of a peptide destined for incorporation into a conjugate provided the peptide with a nucleophilic moiety in the form of the Cys sulhydryl (-SH) group.
• -SH Cys sulhydryl
• other -SH containing residues can be substituted for cysteine in order to provide an alternative conjugation moiety.
• Finished peptidyl-resins were dried in vacuo and then placed in the reactors of a Biosearch HF cleavage apparatus or a Peninsula Laboratories Type I HF apparatus. Peptides were cleaved from the resin using standard HF procedures. After HF removal in vacuo, the resin was washed well with diethyl ether and the peptide then extracted from the resin with trifluoracetic acid (with subsequent precipitation of the peptide via the addition of diethyl ether) or with 10-30% aqueous acetic acid (with subsequent lyophilization) .
• Synthetic peptides purified by reverse phase high performance liquid chromatography were processed on a Waters Delta-Prep 3000 preparative chromatography system (47 mm x 30 cm Delta-Pak radial compression cartidge containing 300 Angstrom, 15 ⁇ m C Cincinnati) equipped with a variable wavelength detector.
• HPLC reverse phase high performance liquid chromatography
• peptides were eluted over a 40 minute period with a linear acetonitrile gradient (0% - 100%) containing a constant concentration of trifluoroacetic acid (0.1% v/v).
• the purification was monitored at 215 nm and the homogeneity of purified material was established by analytical HPLC on a Waters Delta- Pak C Cincinnati column (300 Angstrom, 15 ⁇ m C Compute; column dimensions: 3.9 mm x 30 cm) using the same gradient.
• Epitope mapping relates to the characterization of specific regions of a protein that are being recognized by the immune system. It is unlikely that peptide residues in the "core" of a globular protein are being recognized by the immune system at least as far as the development of a humoral response is concerned. As a result, the surface map of a protein with respect to the different epitopes can be used to design and synthesize peptides that can be incorporated into the desired array. An example of this type of epitope mapping is illustrated by the identification of the histone antigen recognized by the NZB/NZW mouse.
• N-Acetyl-Cys-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Lys-Ala- Val-Thr-Lys-Ala-Gln-Lys-CONH 2 Lupus 3 •
• N-Acety 1-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Lys-Ala-Val- Thr-Lys-Ala-Gln-Lys-Cys-CONH 2 Lupus 4 •
• N-Acetyl-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser- Lys-CONH 2 N-Ac- [ Lupus 2 ' ( 5-15) ] -CONH 2
• N-Acetyl-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys- Gly-Ser-Lys-CONH 2 N-Ac- [Lupus 2 ' ( 3-15) ] -CONH 2
• N-Acetyl-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys- Lys-Gly-Ser-Lys-CONH 2 N-Ac- [Lupus 2 ' (2-15) ] - C0NH 2
• N-Acetyl-Glu-Pro-Ala-Lys-Ser-Ala-Pro-CONH 2 N-Ac- [ Lupus 2 ' ( 2 -8 ) ] -CONHj
• the peptide chosen to be incorporated into a suppressive conjugate obviously had to include enough immunological "information" to be recognized by the murine immune system but also had to address the net positive (charge) character of residues 3-12.
• N-Ac-Glu 2 which is not required immunologically, was included as were two non-histone C-terminal glutamic acid (Glu) residues.
• Glu non-histone C-terminal glutamic acid
• the final target peptide designated N-Ac-[Lupus 2' (2-13)]- Glu-Glu-Cys-CONH 2 (see Table 2) , was then used for conjugation.
• highly cationic epitopes may need to be compensated for, particularly when they are arrayed in a multivalent way. In this case, such compensation was effected by adding additional anionic amino acids to the defined epitope.
• an anionic scaffold could have been used. In either case, the desired outcome is to have an overall charge neutral or slightly anionic construct so as to avoid non-specific adherence of these compounds to anionic surfaces such as cell membranes.
• the antigenic facade of the H2B histone protein consists of a single continuous peptide sequence that was capable of accommodating the entire population of antibodies generated by a population of mice. And, while each individual mouse recognized a discrete region within the entire epitope, the entire population of mice could be dealt with using a single peptide ligand. This is unlikely to be the rule for other proteins such as Ragweed antigen E where multiple discrete epitopes are more likely to be encountered. Again, a certain amount of microheterogeneity within a population with respect to a given epitope is likely; no single epitope can be expected to predominate over all the others for the entire population.
• one of at least two alternatives can be employed. Either multiple ligands can be synthesized and presented either as a mixture of arrays each with a specific ligand or an array of a mixture of ligands (an artificial protein from an antigenic perspective) wherein each array contains a valence-restricted representation of the relevant ligands. Another alternative is to produce valence-restricted arrays of the protein in question. Where these types of constructs are determined to be the most appropriate means for manipulating the immune response for a specific antigenic protein, the following synthetic approaches can be used.
• mapping the antigenic facade of a protein is to produce oligomeric
• BSA was polymerized to itself through the use of a water soluble carbodiimide, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a reagent which links a free carboxyl group on one molecule to a free amino group on a neighboring molecule through an amide bond.
• OVA was polymerized to itself by the use of glutaraldehyde, a reagent which links a free amino group on one molecule to a free group on a neighboring molecule.
• the molecular weight for each fraction was then determined by the use of the Model E analytical ultracentrifuge under equilibrium conditions. During this prolonged series of slow fractionation steps, molecules which were unstable to any of the many steps involved in processing, handling, or storage were fractionated away from the samples, yielding a series of preparations, each of which contained a relatively narrow range of molecular sizes of substantial time stability.
• mice were then injected into mice intra-peritoneally, without the use of any adjuvant, in order to determine their relative immunogenicity.
• the level of immune response was determined by measuring serum IgM or IgG antibody levels against BSA or OVA by standard solid state ELISA technique.
• the desired protein can be biotinylated in such a manner that only one biotin moiety is incorporated per protein monomer. This can be accomplished by reacting a significant molar excess of the protein monomer in dilute solution with a modified biotin molecule capable of reacting with either free amines or carboxyl groups on the protein. These conditions yield a predominance of "mono-functionalized" protein molecules with a minimum of multiply derivatized protein monomers. These biotinylated proteins can then be arrayed in a rigorously tetravalent fashion with streptavidin with any polymeric constructs removed by size exclusion chromatography.
• valence restricted scaffold Similar "mono-functionalization" of a protein ligand can be achieved using many different chemistries with the functionalized protein then being arrayed on a valence restricted scaffold to achieve the same endpoint. These valence restricted arrays can then be used to manipulate the immune system in the desired fashion. Finally, as previously mentioned, epitopes represented by well defined protein domains or whole proteins can be incorporated into genetically engineered constructs having the desired valence for use as either a portion of or as a completely independent valence restricted array.
• Mimotopes Immunoglobulins and their related surface bound receptors are predominantly concerned with the physical structure (shape, hydrophobicity or hydrophilicity, hydrogen bond donor or acceptor groups, etc) and charge of the antigen in question.
• the specific "content" of the antigen with respect to peptide sequence, carbohydrate content, etc. is only significant as it contributes to the "fit” of the ligand to the receptor.
• mimotope The relationship of a ligand identified in this manner to the "natural" ligand to which the immune system's response is directed is limited solely to their structural similarity. Such a ligand has been given the term "mimotope" to represent the ability of this type of ligand to mimic a naturally occurring epitope.
• mimotopes can be modified to enhance their binding to the targeted receptor population using standard chemical modification techniques and substitutions. Mimotopes generated by a random process may require modification prior to their being conjugated to a scaffold to yield an agonist or antagonist array.
• Salmon testes DNA (Sigma) was digested with Aspergilus orvzae SI nuclease (Pharmacia) in order to eliminate "nicked" DNA. The product of this reaction was then subjected to partial digestion with bovine pancreatic Deoxyribonuclease I (BRL Gibco) in the presence of manganese ions. In the presence of manganese ions, bovine pancreatic DNase I cleaves both strands of a DNA duplex at approximately the same site to yield fragments of DNA that are blunt-ended or have protruding termini only one or two nucleotides in length (Melgar and Goldthwaite, 1968) .
• the DNA (0.64 ⁇ mole) was converted into the bis-5'-(1- methyl)phosphorimidazolide with EDC (0.15 M) in 1- methylimidazole buffer (0.1 M pH 6). Subsequent coupling to (S-3-nitro-2-pyridinesulfenyl)- cysteamine ( (S-Npys)-Cmn, 0.2 M) then afforded the bis-phosphoramidate; i.e. the conjugatable
• EE- Aminolink 2 (Applied Biosystems) .
• the Aminolink 2 reagent was used according to the manufacturer's recommended protocols. After removal from the solid support and deprotection according to the 5. manufacturer's protocols, the DNA was purified by gel filtration on a 1.6 cm X 16 cm column of Sephadex G-50 (fine mesh, Pharmacia) . The column was eluted with 0.5 M NH 4 0H. Fractions were collected and those fractions containing the DNA were pooled and lyophilized. The DNA was resuspended in water and the concentration determined by measuring the OD 260 . For single- stranded DNA, 1 OD 260 equals 40 ⁇ g/ml DNA.
• Derivatization is carried out with one of two activated S_-containing amino acids: N-acetyl-S.- Npys-L-cysteine-N-hydroxysuccinimide ester or N_- (succinyl-N-hydroxysuccinimide ester)-S-Npys- cysteamine.
• a reactive primary amino group can also be incorporated at the 5'-end of the synthetic DNA 40-mer via coupling of a modified nucleotide available from Glen Research.
• This nucleotide a modified thymidine, contains a trifluoroacetylated primary amine attached to the base moiety by a 10 atom spacer group.
• this method (of amine incorporation) has the advantage of verification of incorporation of the nucleotide bearing the protected amino group (via standard DNA colorimetric coupling assays).
• Chemical 5'- phosphorylation of synthetic DNA is also possible and yields DNA that is functionally identical to the size-fractionated DNA described above except that the resulting DNA is mono-functionalized.
• the DNA intended for conjugation is derivatized with a protected thiol- containing moiety that when deprotected and reacted with maleimide containing scaffolds will conjugate to the scaffold in a manner analogous to the thiol-containing peptides described above.
• these modified DNA analogues now contain a residue that can be used to unambiguously confirm and quantitate covalent attachment of the DNA to the desired scaffold.
• Small molecular weight haptens As mentioned below, the small molecular weight haptens chosen for investigation all included reactive groups that allowed for easy covalent attachment to the desired scaffolds. All of the conjugations were done in aqueous solution and the unconjugated small molecular weight haptens removed by dialysis, ultrafiltration or size exclusion chromatography.
• the reaction between a cysteine (Cys)- containing peptide and GMB-dexamine (4) is but one example of the well-known tendency of thiol (-SH) nucleophiles to react with , ⁇ -unsaturated carbonyl systems.
• This reaction is referred to as conjugate- or 1,4-addition and is used for the covalent attachment of peptides to dextran ( Figure 8) .
• conjugation chemistries can be employed to accommodate any particular set of combinations of backbone or hapten.
• Alternative chemistries that take advantage of a reactive thiol include reactions with haloalkanes or haloacetamides.
• the maleimide functional group has been observed to be extremely stable over the pH range of the conjugation reaction (pH 5-7) . Specifically, the succinimido-form of dexamine produced by hydrolysis of the maleimide double bond could not be detected after 2 days of exposure of GMB-dexamine (4) to typical conjugation reaction conditions. Therefore, the hydrolysis of GMB-dexamine (4) does not appear to be a factor (i.e. a side reaction) influencing conjugation yield.
• Cysteine (Cys)-containing peptides and GMB-dexamine (4) were prepared as described above.
• DTT dithiothreitol
• PBS Phosphate-buffered saline
• the peptide solution was then added to a 50-fold molar excess of reductacryl resin (DTT equivalents) in a glass reaction vessel equipped with a cinder glass bottom and a nitrogen (gas) inlet. Reduction of the peptide was carried out for 30-45 minutes at room temperature with gentle mixing of the reaction mixture promoted by nitrogen bubbling. The fully-reduced Cys-containing peptide was then added slowly to a freshly-prepared solution of GMB-dexamine (4) in PBS and the resulting conjugation reaction allowed to proceed for 2 hours at room temperature (reaction pH 5-7) .
• DTT equivalents reductacryl resin
• mercaptoethanol can be replaced with either ercaptoacetic acid (MAA) or mercaptosuccinic acid (MSA) which introduce one or two equivalents of negative charge, respectively, for each maleimide group.
• MAA ercaptoacetic acid
• MSA mercaptosuccinic acid
• Peptide-dextran conjugates present in the quenched reaction mixtures were purified/isolated by either extensive dialysis or ultrafiltration against PBS. Such treatment effectively removes mercaptoethanol or other quenching reagent and unconjugated peptide from the finished conjugate.
• reaction "contaminant” the hypothesis that noncovalently- associated peptide should not contribute significantly to either the immunogenicity or to the immunosuppressive nature of a conjugate is reasonable when one considers what is expected of a conjugate in the context of the Immunon model of immune response.
• Quenched conjugation reaction mixtures were transferred jLn toto to 12,000-14,000 mwco dialysis tubing and then dialyzed against PBS according to the following schedule: 24 hours against PBS containing ca. 0.02% (w/v) NaN 3 (2 PBS changes) , 24 hours against PBS (3 PBS changes) and then 24 hours against one-tenth strength (i.e. 15 mM NaCl and 1 mM phosphate, pH 7.3-7.4) PBS (3 PBS changes) .
• Ultrafiltration was carried out in an A icon 8200 ultrafiltration vessel equipped with either a 5,000 or 10,000 mwco filter as follows: The conjugation reaction mixture was diluted to 200 mL total volume with PBS that contained ca.
• nucleic Acids As discussed above, the DNA used for conjugation is modified in such a manner that it will behave similarly to peptides when conjugated. Discussed below are some of the methods by which either the size fractionated DNA or the synthetic DNA is attached to the desired scaffold.
• SH-containing DNA, deprotection and conjugation is carried out as described above for size- fractionated DNA.
• Single stranded DNA-containing (dextran) conjugate is then exposed to the complimentary DNA strand (also 40 nucleotides in length) to afford the double stranded DNA- containing (dextran) conjugate. It is at this point that efforts to stabilize the resulting DNA duplex can be undertaken.
• Both the highly specific reagents: mitomycin C and the less specific psoralen can be used to cross-link the individual strands of the dextran-bound DNA duplex in an attempt to decrease the rate of exonuclease and (perhaps) endonuclease digestion.
• DNA-Dex conjugate Alternatively, chemically modified DNA analogues such as phosphorothioates can be utilized. These nucleic acid analogues are known to be resistent to endonuclease and exonuclease digestion.
• conjugates are subjected to rigorous analysis with respect to both content and overall structure so as to assure the final product meets the criteria established for agonist or antagonist arrays as desired. Described below are representative analytical procedures for conjugates in general (haptens, peptides and nucleic acids) and the specific peptide containing conjugates used in the Examples set forth herein.
• the primary analytical reference standard for each type of polymeric material was a dry weight analysis for the actual amount of polymer mass present in a given type of polymer preparation. Dry weight was determined after the thorough vacuum drying of polymer samples and appropriate dialysate samples. ii) Spectral analysis
• the haptens used in these studies were chosen, in part, so that they had identifiable spectral absorption bands at wavelengths in the visible or near ultraviolet regions.
• the amount of hapten chemically coupled to polymer molecules in a preparation was usually determined from the comparison of the optical absorption due to the hapten groups and the total mass of the polymer preparation, as determined by dry weight analysis or refractive index increment analysis, iii) Chemical analysis
• SEC methods permit the convenient determination of relative molecular mass by comparison of the chromatographic column retention times of unknown samples and homogeneous standard samples using standard HPLC techniques.
• the standardization polymer materials have to be relatively homogeneous and independently calibrated for molecular mass by some absolute experimental procedure, such as equilibrium ultracentrifugation or low angle laser light scattering. Because the SEC method is very sensitive to any physical interactions between the column support and the polymer molecule, the column retention times must be calibrated for each and every type of haptenated polymer molecule. Such calibration is sensitive to the physical and chemical nature of the polymer molecule, the chemical nature and number of haptens, the net electrical charge on the molecule, etc.
• the weight average molecular weight of the haptenated polymer was approximately 68 kDa.
• Low angle laser light scattering When appropriately combined with an experimentally determined refractive index increment, low angle light scattering methods yield a value for the absolute weight average molecular weight of a polymer preparation. For large molecules, the method requires measurements at a number of concentrations and angles, followed by extrapolation both to zero polymer concentration and to zero angle of measurement. If combined with the separation of molecules of different molecular sizes using SEC, this light scattering method yields a dependable determination both of the molecular mass and physical size distributions in a very small quantity of polymer preparation. Measurements of this type were routinely carried out using a high pressure liquid chromatography apparatus, Hewlett- Packard HP1090M, and a low angle laser light
• SHEET scattering device Wyatt Technology Dawn. Size exclusion columns were Toyo Soda TSK GMPW gel columns or Pharmacia Superose 6 or 12 columns or combinations thereof, appropriately chosen to separate the molecular sizes present in the particular sample. When low molecular weight samples were inadequately separated from salt a column of Sephadex G15 was added to increase resolution. When polymers were substituted with high amounts of haptens having appreciable hydrophobic character, such as dinitrophenyl or fluorescein, there was significant interaction between the hapten and the column material, causing interference with the size exclusion based separation. When this effect occurred, it was minimized by using 20% acetonitrile in the column buffer.
• conjugate conjugate peptide and carbohydrate content.
• acid hydrolysis does not permit recovery of the peptide or carbohydrate portions as intact entities, such recovery is not necessary for the evaluation of conjugate peptide substitution density. That is, simply by recovering and quantitating the amino acids derived from the conjugated peptide and GMB-dexamine it is possible to assess the moles of bound peptide and the moles of recovered dexamine, respectively.
• GABA gamma-aminobutyric acid
• S_-2-(2R,2S_-succinyl)- L-Cys which distinguishes covalently-bound from noncovalently-bound conjugate peptide (because only covalently-bound peptide is S_-succinylated) and the amino acids derived from the conjugated peptide.
• the phenylthiocarbamyl (PTC)-derivative of S-2-(2R,2S_-succinyl)-L-Cys having a retention time of 1.33 minutes on the PICO-TAG HPLC column, is well separated from any other PTC-derivative.
• the PTC-derivative of gamma-aminobutyric acid (PTC-GABA) coelutes with that of arginine (Arg) . While this is inconvenient when Arg-containing peptide-dextran conjugates are being analyzed, difference analysis (i.e.
• S -containing "marker" amino acids can be of value when different peptides are conjugated to the same sample of GMB-dexamine (4) .
• non-sulfur containing amino acids may function in a similar context (i.e. as marker amino acids) .
• amino acids not normally found in the biologically relevant portion of peptides to be conjugated such as 5-aminovaleric acid (5-AVA) , e-aminocaproic acid (e-ACA) , ⁇ -alanine ( ⁇ -Ala) , norleucine (Nle) , norvaline (Nva) and ⁇ - aminobutyric acid ( ⁇ -ABA)
• markers can be regarded as "markers” that specify the amount of a particular covalently-attached peptide.
• markers that specify the amount of a particular covalently-attached peptide.
• these amino acids may also be thought of as "spacer" elements that provide distance between the chemically reactive (i.e. S-containing) and biologically relevant portions of a peptide destined for incorporation into a peptide-dextran conjugate.
• CI-0060 Lupus 2' : Pro-Glu-Pro-Ala-Lys- S er-Ala- Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Cys-CO H 2
• CI-0060 Lupus 2': Pro-Glu-Pro-Ala-Lys-Ser-Ala- Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Cys-CONH 2
• the pmole ratio of an amino acid near the N-terminus (Glu) to an amino acid near the C-terminus (Gly) should be equal to one for the lupus peptide (CI-0060)/1420K dextran conjugate prepared for this experiment.
• the fact that this ratio is significantly less than one in the case where the impure C-terminal Cys-containing peptide was conjugated (Table 5) is consistent with a mixture of peptides actually participating in the conjugation process.
• CI-0134 Ac-Cys- ( e -ACA) -Ala-Asp-Ser-Gly- Glu-Gly-Asp-Phe-Leu-Ala-Glu- Gly-Gly-Gly-Val-Arg-Gly-Pro- Arg-Val-Val-Val- (d) Tyr-C0 2 H
• CI-0134 Ac-Cys-(e-ACA)-Ala-Asp-Ser-Gly Glu-Gly-Asp-Phe-Leu-Ala-Glu- Gly-Gly-Gly-Val-Arg-Gly-Pro- Arg-Val-Val-Val-(d)Tyr-C0 2 H
• Purified peptide-dextran conjugates were routinely dissolved in HPLC-grade water at a concentration of ca. 1 mg/mL. An appropriate aliquot was removed, dried in vacuo and then subjected to the Waters PICO-TAG chemistry (see above) for amino acid analysis.
• Is -Acetyl-S-3-(3R.,3S-succinimido)-L-Cys was prepared as follows: To a stirred solution of N-Ac-L-Cys (0.082 g, 0.50 mmole) in 10 mL of H 2 0 was added NMM (0.101 g, 1 mmole) and maleimide (0.0485 g, 0.50 mmole). The reaction mixture was stirred overnight at room temperature and then transferred in toto to a 25 nu. x 22 cm Dowex AG50W-X 4 column. The column was eluted with H 2 0 and fractions (25 x 8 mL) were collected and analyzed by TLC.
• a dialytic or ultrafiltrative purification has proven very satisfactory in the initial stages of the preparation of peptide- dextran conjugates. Certain applications may, however, require conjugate preparations that are completely devoid of high or low molecular weight impurities.
• Molecular exclusion chromatography of dextran samples on Superose 12 or Superose 6 can be very effective as a means of sample purification.
• a commercially available analytical Superose 12 column (Pharmacia) attached to an in- house fast protein liquid chromatography (FPLC) system will separate fluoresceinated dextran (Fl- Dex) samples reasonably well.
• a preparative Superose 12 column (separation range: 1,000 - 3 x 10 s g/mole) could be used to purify large-scale reaction mixtures of peptide-dextran conjugates. Separation results obtained from the preparative Superose 12 column using Fl-Dex standards suggest that this type of chromatography may be useful both as a means of conjugate purification and (expensive) peptide recovery.
• the conjugates used in the anti-histone, anti-OVA and anti-EALA studies described herein were characterized as follows:
• CI-0159 N-Ac-Cys-(e-ACA)-Glu-Ala-His"'- Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg 33 '-CONH 2 .
• CC-0010 Cys-Gly-Ala-Gly-(Glu-Ala-Leu- Ala) 6 -Gly-Ala-Gly-Arg-Gly-Asp-Ser-Pro-Ala-CONH 2 .
• Linear polyacrylamide substituted with Dnp hapten groups was prepared as described above.
• linear polyacrylamide (Gelamide 250-American Cyanamid) with average molecular weight 5 x 10 6 was substituted with ethylene diamine in a manner analogous to that previously used for polyacrylamide beads (Inman et al. Biochemistry 8, 4074-4082 (1969)).
• Dnp derivatives were obtained by shaking the ethylene diamine substituted derivatives with excess fluorodinitrobenzene followed by extensive dialysis. The degree of substitution was determined from measurement of dry weight and optical absorbance at 360 nm. Preparations were labeled with '"I substitution levels of approximately one per 2500 monomer units were obtained, corresponding to less than one t2S I per molecule labeled.
• Dnp-substituted polymers were fractionated by gel filtration through 1 m long columns of Bio-Gel A-0.5 M agarose beads. These original fractions were further fractionated three more times to obtain relatively homogeneous preparations, as determined by sedimentation equilibrium measurement in the analytical ultracentrifuge.
• Polymer B was not immunogenic while Polymer D was (see Table 1, 1976 paper noted above) .
• Polymers B and D were subjected to further column fractionation on Sepharose C1-4B. Two preparations (N and S) were separated for further testing. Preparation N was a central subfraction of polymer B and preparation S was a central subfraction of polymer D. Measurement of partial specific volume (0.690 ml/g) and extrapolation of sedimentation equilibrium molecular weight to zero concentration gave values of 60,000 for N and 130,000 for S. These values together with dry weight and absorbance at 360 nm show N to contain 19 Dnp groups per molecule [7-9 "effective” or appropriately spaced] whereas S contains 43 Dnp groups per molecule (14-21 “effective”) . Polymers N and B had almost identical "epitope densities" or degrees of substitution by hapten per molecular size unit.
• Polymer preparations were injected intraperitoneally in BALB/c mice in 0.5 ml of isotonic saline. After 6 days, blood was collected by bleeding from the tail, and the serum was stored at -30°C until analysis. The concentration in serum of IgM antibody against Dnp was determined by a solid-phase binding assay. Surfaces covalently coated with Dnp-substituted gelatin served to bind the anti-Dnp mouse antibody, whose presence was then measured by a second incubation with I'"-labeled rabbit antibody against mouse IgM antibody.
• mice were killed by cervical dislocation, and their spleens were minced in RPMI-1640 medium and pressed through a stainless steel mesh (60 x 60 mesh; 0.019-cm diameter). Cellular debris was allowed to settle, and the supernatant containing a dispersed-cell suspension was decanted, freed of erythrocytes by osmotic shock, and washed. Suspensions of nucleated spleen cells were then incubated with or without appropriate polymer in 60 x 15 mm tissue culture dishes containing 5 x 10 7 viable cells in a final volume of 7.5 ml.
• the incubation was carried out in 5% C0 2 /95% water-saturated air at 37.0°C.
• the incubation medium consisted of RPMI 1640 medium enriched with 5% (vol/vol) heat-inactivated fetal calf serum, 2% (vol/vol) heat-inactivated horse serum, 4 mM glutamine, 100 units of penicillin and 100 ⁇ g of streptomycin per ml, and 50 ⁇ M 2-mercaptoethanol.
• Fig. 16 The immunological response in BALB/c mice 6 days after injection of various doses of immunogenic polymer preparation S, as measured by the concentration of serum IgM molecules reactive toward Dnp groups, is shown in Fig. 16.
• the mice in this experiment came in a single shipment of uniform age from the supplier and were divided into groups of 10. Members of each group were injected with the same dose, and all groups were handled as uniformly as possible.
• the solid curve in Fig. 16 is the theoretical response curve expected from Eq 1
• Fig. 17 compares the dose-response curves of three separate shipments of BALB/c mice and illustrates both group-dependent variability of response of individual mice at each dose and some change of shape of the dose-response curve from group to group.
• the variable immunological response given by different groups of mice is a well-known phenomenon, having been observed both in studies using whole animals and in those using cell cultures. It probably is dependent on factors in the previous history and handling of the animals, such as exposure to bacteria, viruses, and parasites, which might influence the "antigenic naivete" of the animals, as well as exposure to environmental shocks such as heat and cold during shipment.
• the wider experimental curve may be explained in the following way:
• the theoretical curve in Fig 16 is based on the assumption that all cells responding to the immunogen have receptor molecules with the same binding constant for Dnp groups. This assumption of complete homogeneity is unlikely to be true. If cells that bind immunogen and respond to it have protein receptors with differing binding constants for Dnp, then the predicted response should be the sum of a number of individual cellular response curves. Each curve would be like that in Fig. 16, but those with lower binding constants would be displaced to the right by an amount proportional to the ratios between their binding constants for Dnp. Inspection of Figs.
• Fig. 19 shows the results of such an in vitro experiment as compared with a visually fitted theoretical curve calculated from Eq. 1. This agreement between experiment and theory for the in vitro experiment with cultured spleen cells (Fig. 19) is approximately as good as it was for the in vivo experiment with whole mice (Fig. 16) . In both cases, the measured response curve is somewhat broader than that predicted from a model based on a homogeneous hapten binding constant in the responding cells.
• Fig. 20 Of particular significance to the present invention are measurements of the inhibition of immune response in vitro with increasing amounts of nonimmunogenic polymer which are shown in Fig. 20.
• the solid line is not fitted to the data but is calculated directly from Eq. 1 by using the value of the maximum-response dose, r f x of 0.4 ng/ml from Fig. 19. There is s substantial agreement between the experimental points and the calculated theoretical curve.
• the blood volume and extracellular fluid volume of a mouse are each « 1 ml, so the optimal immunogenic polymer does in vivo is « 1 ⁇ g/ml.
• the optimal immunogenic polymer does in vivo is « 1 ⁇ g/ml.
• the almost 1000-fold sensitivity difference is largely explained by rapid removal in vivo of polymer molecules by phagocytes located throughout the body.
• Studies with I25 I-labeled preparations of the polymers, as described in the above-noted 1976 paper showed that the bulk of the injected polymer is quickly removed from the circulation by Kupffer cells in the liver and phagocytic cells in other tissues.
• polymer N fails to stimulate at any dose, it inhibits polymer S at the same dose where polymer S is maximally stimulatory. This indicates a competition for surface receptors. Because both polymer preparations have almost identical "epitope densities" with a common carrier chemistry, this finding is in disagreement with theories that explain immunogenicity by invoking epitope density or polyclonal (i.e., nonspecific) activation by the "carrier.”
• a specific immunogenic signal is generated by the formation of immunons on the surface of a responsive cell
• an immunon will form only after a sufficient number of surface receptors are clustered
• specific clustering of surface receptors occurs as a consequence of their being bound to linked haptens. This binding is specific for the hapten-receptor interaction and does not primarily depend on the "scaffolding" to which the haptens are attached.
• the underlying physical scaffold that links the haptens may be molecular in nature or may consist of a surface on which small hapten-containing structures are aggregated, as on the surface of an "antigen-presenting cell.”
• Nonspecific stimuli such as mitogens, lectins, antibodies against cell surface proteins, and activating or inhibiting factors from other cells, may well influence the level of "irritability" of the responding cell, making it more or less likely to respond to a given amount of immunogenic signal or even to respond in the absence of specific signals.
• Factors from T cells and macrophages have previously been shown to enhance antibody responses nonspecifically. Mitogens are known to stimulate cells nonspecifically to secrete antibodies.
• the suppressive effect of the nonimmunogenic polymer, on the immunogenic polymer, as illustrated above, can be used to control undesired immune response.
• the amount of nonimmunogenic polymer so used will necessarily vary depending on the specific immune response which is involved, the polymer carrier, the effective number of epitopes involved, body weight and other factors. It is believed, however, that the administration of from 0.5 to 50 mg/kg body weight would be effective in controlling undesired immune response.
• the administration may be effected by, for example, injection using a sterile solution of the non-immunogenic polymer.
• Example 7 Extension of Immunon Model to Alternative Haptens and Carriers
• the invention is not dependent on the nature of the hapten or carrier but on the molecular mass of the carrier and the hapten density, these physical characteristics (molecular mass, hapten density) determining whether or not the matter is immunogenic or non-immunogenic or suppressive.
• This is further illustrated by the following additional disclosure and exemplification of tests done using fluoresceinated carriers.
• the molecular characteristics of five chemically different fluoresceinated (Fl)-polymers were systematically varied, and their ability to stimulate an anti-hapten immune response was measured.
• the polymers used as carriers were carefully size-fractionated and consisted of one natural polymer (dextran) , one modified natural polymer (carboxymethyl cellulose) , and three synthetic polymers (Fieoll, polyvinyl alcohol, and polyacrylamide) .
• the carriers varied in physical structure from the highly cross-linked Fieoll, to the somewhat branched dextran to the linear polyacrylamide, carboxymethyl cellulose and polyvinyl alcohol.
• Polymers were haptenated with Fl and size-fractionated so as to yield a panel of molecules with varying molecular mass, hapten valence and hapten density. Anti-Fl response to these haptenated polymers was measured in vivo after i.p.
• the immunogenicity of soluble haptenated polymers depends on predictable physical molecular characteristics, and is relatively independent of the chemical composition and conformation of the carrier polymer.
• Polymer carriers selected to be haptenated were dextran (T2000, T500 and T70 - Pharmacia) ; Fieoll (400 and 70 - Pharmacia) ; carboxymethyl cellulose (medium viscosity - Sigma) ; polyvinyl alcohol (average molecular weight 115,000 - Aldrich); and linear polyacrylamide (synthesized in aqueous solution from crystalline acrylamide) .
• the polymer carriers were conjugated with fluorescein by the following procedures: Reactive carboxyl groups were generated in polyacrylamide by partial hydrolysis in 0.05M Na 2 CO 3 -0.05M NaHC0 3 , pH 10.1, at 20°C (3).
• Amino groups were introduced into such deamidated polyacrylamide and also into dextran, Fieoll, polyvinyl alcohol and carboxymethyl cellulose according to the procedures disclosed by Inman, J. Immunol. 114:7044. Subsequently, the amino groups on the polymers were conjugated to excess fluorescein isothiocyanate at pH 9.2 in 0.1M Na 2 B 4 0 7 . The polymers were then dialyzed exhaustively against the buffer used for subsequent gel filtration (0.1M NaCl, 0.001M EDTA, 0.02% NaN 3 , 0.01M KP0 4 , pH 7.4).
• Fl-polymers were then repeatedly fractionated over 95 cm columns of Sepharose CL- 2B, CL-4B and/or CL-6B; center cuts were taken repeatedly to give preparations of relatively narrow molecular weight distributions.
• Fl content was determined by measuring optical density at 496 nm in 0.01 M Na 2 B 4 0 7 using a molar extinction coefficient of 72,000 for Fl. This measurement together with polymer dry weight measurement permitted calculation of epitope density.
• Molecular mass was determined by sedimentation equilibrium analysis in the analytical ultracentrifuge as known in the art (Proc. Natl. Acad. Sci 73:3671 1976). Measurements were performed at several polymer concentrations by using the short column method, and molecular mass was obtained by extrapolation to zero polymer concentration. Polymers used in experiments were dialyzed against PBS and were sterilized by filtration with the use of 0.22- ⁇ m Nucleopore filters.
• Indicator cells in the plaque assay were hapten substituted at low density in order to minimize assay response to low affinity (i.e., non-specific) antibody.
• Substituted indicator cells were prepared by mixing 1 ml of packed burro red blood cells (BRBC) with a solution of 1 mg of fluorescein isothiocyanate dissolved in 9 ml of borate buffered saline (BBS; 0.9% NaCl containing lOmM sodium borate, pH 9.2). The mixture was then stirred for 1 hour at room temperature in the dark. The cells were centrifugally washed first in BBS and then 3 or 4 times in PBS. They were stored in PBS containing 0.11% glycylglycine for no longer than one week.
• BRBC packed burro red blood cells
• BBS borate buffered saline
• composition and characteristics of the haptenated polymers used herein are listed in Table 1 (see page 31) .
• FIG. 21 shows dose-response curves of the primary in vitro anti-hapten response of naive spleen cells to Fl-PVA after various times of incubation. The peak in vitro response occurred after three days of incubation. The kinetics of the primary in vivo anti-hapten response to the optimal dose of the same polymer are pictured in Figure 22. Spleen PFC peaked at about 4 days.
• In vivo anti-hapten dose-response curves generated by four different fluoresceinated polymers, Fl-Dex, Fl-Fic, Fl-CMC and Fl-PVA, are shown in Figure 23.
• In vivo dose response curves, shown in Figure 2 include the curve generated by an additional polymer Fl-PA. These curves are representative of the responses generated by all the immunogenic polymers used in this study. Each dose-response curve is bell-shaped, initially increasing with the dose of antigen until a maximum is attained and then decreasing at higher doses of antigen.
• N.D. not determined It is to be noted that the subscript number after the hapten abbreviation refers to the number of haptens per molecule (hapten valence) , while the number after the carrier abbreviations refers to the molecular mass in kD.
• FlistDex400 refers to a molecule with 65 fluorescein groups on a dextran carrier, with a total molecular mass of 400,000 daltons.
• the group of polymers listed above the dotted line were immunogenic and the group below the dotted line were nonimmunogenic. Both groups included molecules with each of the five kinds of polymer carriers studied: Fl-Fic, Fl-Dex, Fl-PA, Fl-CMC and Fl-PVA. Thus all five Fl-polymers have the potential to be either immunogenic or nonimmunogenic, irrespective of the chemical composition of the polymeric carrier. Examination of the molecular characteristics of the polymers in Table 8 indicates that immunogenicity is directly related to the molecular mass and the hapten valence. All polymers above the dotted line, had a hapten valence greater than 20 and a molecular mass larger than 100,000 daltons and were immunogenic.
• Polymers below the dotted line had a molecular mass less than 100,000 daltons and were not immunogenic at any dose tested.
• the hapten densities in both groups had approximately the same range: between 0.12 and 0.59 millimoles of fluorescein per gram of polymer. Thus, hapten density by itself was not a predictor of the presence or absence of immunogenicity.
• Naive spleen cells were cultured with a series of solutions formulated to contain increasing concentrations of the nonimmunogenic polymers together with a constant concentration of the immunogenic polymer Fl, 0 Fic750.
• the inhibitory ability of the nonimmunogenic polymers increases with increasing concentration until complete inhibition of the anti-Fl response to the immunogenic polymer is reached at inhibitor concentrations between approximately 1 and 10 ng per ml.
• Figure 25 demonstrates “cross- inhibition", whereby Fl on the backbone carriers
• EET PVA, Dex, or CMC can inhibit the anti-Fl response stimulated by Fl-Fic.
• the data indicate that the inhibitory potentials of these nonimmunogenic Fl- polymers are largely independent of specific carrier chemistry.
• Figure 25 shows that the irrelevant hapten, Dnp, on a PA carrier could not inhibit the anti-Fl response.
• Carrier- independent inhibition is further evidenced in Table 9, where the ability of four nonimmunogenic Fl-polymers to inhibit the immune response to four immunogenic polymers with different carrier backbones is shown.
• Figure 26 shows inhibition curves generated by five chemically different Fl-polymers when mixed in increasing amounts with a constant amount of Fl, 0 Fic750.
• One of the curves illustrates the self-inhibition caused by adding increasing amounts of Fl, 0 Fic750 to an optimally immunogenic concentration of the same polymer.
• inhibition increases with dose. Although this may be termed "high-dose" inhibition, the actual in vitro molar concentration of inhibitor necessary for 50% inhibition of the response to Fl 90 Fic750 did not exceed 30 pM for any of the Fl-polymers, and for F1 105 CMC440, it was as low as 2 pM. The influence of hapten density and molecular mass individually on inhibitory ability was also measured.
• Table 10 compares the inhibitory abilities of pairs of Fl-polymers with similar molecular mass, but differing hapten densities. In each pair of molecules where the molecular mass was kept constant, the polymer with the higher hapten density was the better inhibitor, i.e., lower concentrations were required to cause a 50% inhibition of the response to Fl TO Fic750.
• Table 11 compares the inhibitory abilities of two sets of polymers, one set with CMC as the carrier, and the other set with Fieoll as the carrier.
• the hapten densities in each set are similar, but the molecular weights differ.
• Included in the CMC carrier set are two nonimmunogenic polymers (F1 6 CMC27 and F1 4 CMC15) ; one nonimmunogenic polymer (Fl 1 Fic40) is included in the Fie carrier set. In each set, regardless of immunogenic potential, the polymer with the higher molecular weight is the better inhibitor.
• Table 11 Effect of Molecular Mass on Inhibitory Ability
• haptenated proteins such as hen egg ovalbumin (OVA) or bovine serum albumin (BSA)
• OVA hen egg ovalbumin
• BSA bovine serum albumin
• the response that results may contain high levels of both IgG and IgE antibodies directed against the hapten which is coupled to the injected protein.
• IgG serum anti-fluorescein IgG response levels of three individual mice, which had been immunized by this protocol with fluorescein substituted OVA over a time period of several months and then were followed for a number of weeks without further exposure to antigenic material, is shown in Figure 27.
• mice were part of a large cohort which had all been immunized simultaneously according to the same protocol. Some of these mice were then injected intraperitoneally with polymers which we had previously determined were inhibitory. Such polymers were soluble fluoresceinated polymers of high hapten substitution density, but with molecular weights under 100,000. These polymers were injected to test their ability to suppress an ongoing high level anti-fluorescein IgG antibody response (cure) . The results from the injection of three different such polymers on the serum levels of individual mice are shown in Figures 28, 29 and 30, where the time scale of bleedings is the same as in Figure 27.
• the sixth mouse showed a sharp drop to a low level, followed by a slow and steady decline thereafter.
• the data in Figure 30 show that a dose of 1 mg of FL30Dex80 caused a very substantial, but not total, suppression of the serum level of anti-FL IgG antibody. However, a subsequent dose of 3 mg brought about total suppression.
• the particular polymer used in this experiment was fluoresceinated dextran containing 30 fluorescein groups substituted on dextran with an average molecular mass of 80 kDa for resulting polymer (FL30-Dex80) as determined by high pressure liquid chromatography analysis. Since the mice had high circulating levels of antibody against fluorescein at the time they were "cured", it was important to administer the curative dose in steps over an extended time interval in order to avoid undesirable side reactions in the recipient animals. Accordingly, doses were increased a maximum of 10 fold every 2 hours beginning with an initial dose of 1 ⁇ g. This protocol produced no visual evidence of distress in the animal during or following the administration of the FL-Dextran. Cure doses were administered on day 35 when the immune response was very substantial, and also on day 95, following the attempt to restimulate the animals on day 75.
• Figures 31, 32 and 33 show the dramatic decrease in the anti-FL antibody levels following the cure dose, as well as the lack of response to a subsequent restimulation on day 75.
• Figures 31, 32 and 33 include data from mice which had been previously stimulated with high (10 ⁇ g) , medium (l ⁇ g) and low (0.1 ⁇ g) doses of the stimulatory antigen, FL-OVA on aluminum hydroxide.
• Figures 31, 32 and 33 clearly show that the serum anti-FLU IgG antibody level is very substantially reduced by a single cure treatment in each case, and is not restimulated by repeating the original process of stimulation.
• the measured suppression increases as the dose of immunogen which produced the immune response decreases, and 2.
• the measured suppression increases as the amount of epitope (fluorescein) coupled to the gelatin used for the spot-ELISA assay decreases, (i.e. the percent inhibition measured with 0.08 FL per gelatin is larger than when measured with 0.2 FL per gelatin) .
• More measurements have indicated that this trend holds true across a wide range of hapten substitution density in the assays for cells producing anti-Fl antibodies in both the spot-ELISA assay for antibody-producing cells and the ELISA assay for serum levels of IgG antibody.
• a highly specific biological assay for the presence of such antibodies of the IgE class is the passive cutaneous anaphylaxis (PCA) test, wherein a few microliters of diluted serum from the animal under analysis is injected into the skin of a test animal. An hour or two later the test animal is injected intravenously with the relevant antigen in a saline solution containing soluble dye. In the skin regions where injected serum IgE antibodies against the antigen are present, visible dye color appears as a result of the activation of mast cells with subsequent release of mediator causing vascular leak. The greatest dilution of injected serum which will provoke an observable skin response is a measure of the IgE antibody titer in that serum.
• PCA passive cutaneous anaphylaxis
• Figure 36 demonstrates that, after substantial levels of IgE anti-fluorescein antibody had been developed, a single injection of suppressive Fl-Dex brought the serum level of such IgE antibody to a very low level for several weeks (actually, the level was so low that it was experimentally indistinguishable from the background level of the measurement) . Furthermore, the mice so treated were completely resistant to boosting with a repeated dose of antigen, whereas the control mice showed a very substantial increase in their IgE serum titer when boosted. It appears that the suppressive polymer injection caused a long lasting "cure" of an established allergic type response in the mice.
• penicillin allergy is among the most clinically distressing drug allergy since administration of penicillin (or its related compounds) is still the treatment of choice for many diseases.
• many people are allergic (i.e. show immediate-type hypersensitivity reactions) to penicillin or become so while undergoing loncj-term penicillin therapy.
• mice Female mice were obtained from Cumberland Farms, Clinton, TN and were approximately 10 weeks old when first immunized. Male Sprague-Dawley rats weighing 320- 380 g were obtained from Holtzman Co. , Madison, I.
• Bovine serum albumin Fraction V was obtained from Miles Laboratories, Inc. Penicillin G (sodium salt) and crystallized chicken ovalbumin (OVA) were obtained from Sigma Chemical Corporation, St. Louis, MO, p_- Chloromercuribenzoate (PCMB) was obtained from Calbiochem, Los Angeles, CA, Evans Blue was purchased from Eastman Kodak, Rochester, N.Y. and ethylene diamine (EDA) was ordered from MCB Manufacturing Chemicals, Cincinnati, OH.
• BSA Bovine serum albumin
• Penicillin G sodium salt
• OVA crystallized chicken ovalbumin
• PCMB p_- Chloromercuribenzoate
• PCMB p_- Chloromercuribenzoate
• EDA ethylene diamine
• Hapten-Carrier Conjugates Benzylpenicilloyl-bovine serum albumin (BPO-BSA) and benzylpenicilloyl-ovalbumin (BPO-OVA) were prepared by incubating BSA or OVA with Penicillin G (benzylpenicillin) in 0.5 M K 2 C0 3 , pH 10.0 at room temperature (Nakawaga et al, Int. Archs. Allergy Appl. Immunol. 63:212 (1980)). Various incubation times yielded different epitope densities. The number of haptens per carrier is denoted by subscript, i.e. OVA substituted with four BPO groups is BPO.-OVA.
• the degree of substitution was determined by a modification of the penamaldate assay (Parker, C.W. Methods in Immunology and Immunochemistry, Williams and Chase eds. Vol. I, p. 133, Academic Press, NY (1967)).
• 0.1 ml of 2 x 10 "3 M PCMB in 0.05 M carbonate, pH 9.2 is added to 1.0 ml of the penicilloyl-carrier conjugate in 0.05 M carbonate, pH 9.2.
• BPO-PA Benzylpenicilloyl-polyacrylamide
• IgE content was determined by a modification of the passive cutaneous anaphylaxis (PCA) assay (Ovary, Int. Archs. Allergy Appl. Immun. 3:293 (1953)). Equal volumes of serum from mice in each group were pooled and 0.1 ml volumes of diluted serum were injected into the skin of rats. After two hours, 4 mg BPO.-BSA plus 10 mg Evans Blue Dye in 0.5 ml PBS was injected intravenously (i.v.) and twenty minutes later the rats were sacrificed, skinned, and the titer (the reciprocal of the highest dilution yielding a lesion at least 5 mm in diameter) was determined.
• PCA passive cutaneous anaphylaxis
• mice injected with BPO.-OVA on Al(OH) 3 gel developed an anti-BPO IgE response (Figure 38) as measured by PCA assay.
• This anti-BPO response is the murine correlate of human penicillin allergy.
• Four mice were injected with a suppressive dose of 1 mg of the polyacrylamide haptenated with BPO (BPO-PA) .
• BPO-PA polyacrylamide haptenated with BPO
• serum levels of anti- BPO IgE in the experimental group declined by greater than 98% (Figure 38a) while the levels of anti-OVA IgE remained constant ( Figure 38b) . That is, the response of the experimental group to the BPO hapten, after suppression, was less than 1/80 of the control group response.
• mice were immunized and subsequently boosted twice vith Fl-BSA adsorbed on to aluminum hydroxide to raise high titre IgG anti-Fl antibodies. These mice were divided into groups and treated either with the valence-restricted scaffold bearing seven FITC groups (CI-0374) as described in Example 1H above at three different doses or with dextran of 70,000 dalton substituted with 60 FITC groups (CI-0323) at a dose shown in previous experiments to be optimally suppressive. Another group was immunized with the buffer alone as a control. Mice were bled at intervals following these treatments and sera were assayed by ELISA for IgG anti-Fl antibodies as described in previous Examples. It is apparent (see Figure 39) that the valence-restricted scaffold can induce dose-dependent, long-lasting suppression of the anti-FL response similar to that induced by the Fl-dextran construct.
• BSA of differing molecular weight is compared, in Figure 41, the immunogenicity, as measured by IgM levels at day 6, is found to increase most rapidly at the higher molecular weights, but is strongly dose dependent at all molecular weights.
• Figure 42 illustrates experiments in which mice were given three injections 30 days apart of 1, 10, or 100 ⁇ g of BSA polymers in saline. As was true for single injections, the anti-BSA IgG serum levels were strongly dose and polymer size dependent. Figure 43 indicates that monomeric BSA is not very effective in producing an anti-BSA IgG response even after repeated injections in saline at doses up to 100 ⁇ g.
• fluorescein was coupled to BSA polymer at a number of different levels of substitution, and the immune response was determined after several injections, as shown in Figure 48.
• Example 11 Suppression of antibody responses to peptides from extrinsic antigens and autoimmune antibody responses against epitopes on endogenous proteins.
• mice - Balb/c femal mice were obtained from either the Jackson Laboratory, Bar Harbor, ME or Harlan/Sprague Dawley, Indianapolis, IN. They were used at 8-10 weeks of age.
• mice were given a single intraperitoneal injection of 10-50 ⁇ g peptide-BSA conjugate adsorbed on aluminum hydroxide. Test bleeds were taken at various times thereafter and anti-peptide IgG Ab titers measured. To test the immunogenicity of peptide-dextran conjugates, mice were injected intraperitoneally with 100 ⁇ g doses of dextran backbone to which peptides were conjugated at various substitutions ratios. Bleeds were taken at weekly intervals and levels of IgM and IgG peptide-specific Abs in the sera were measured (Figure 49) .
• the suppressive peptide-dextran conjugates were administered in the following way unless otherwise indicated: 1, 10 and 100 ⁇ g doses were injected at 2-hourly intervals, with the 1 and 10 ⁇ g doses being given intravenously whereas the high dose was given intraperitoneally ("cure" treatment) . Subsequent doses to maintain suppression were given at weekly intervals.
• ELISA assay - Antibody titers were measured by standard solid-phase ELISA assay. Microtiter plates (Immunolon II, Dynatech Labs,
• peptide-gelatin conjugates were coated overnight at 4°C with peptide-gelatin conjugates at 0.1 ⁇ g/well. After blocking plates with PBS/gelatin, various dilutions of antisera were added and incubated at room temperature for two hours. Plates were washed and antibody binding was detected with horseradish peroxidase-conjugated isotype-specific antibodies (Kirkegaard and Perry Labs, Gaithersburg, MD) followed by the ABTS substrate. Data are expressed as OD_ 05-I of the ABTS product. Antibodies directed against linker regions were detected using an irrelevant peptide-gelatin preparation and these readings were subtracted from those for specific binding.
• the first peptide chosen for study was a sixfold repeat of a glutamic acid-alanine- leucine-alanine sequence (EALA using single letter amino acid code) followed by the peptide sequence glycine-alanine-glycine-arginine-glycine-aspartic acid-serine-proline-alanine-amide. This peptide will be referred to herein as (EALA) .
• EALA-dextran conjugates were synthesized on 84,000 MW Dextran (EALA-Dex,) that were verified to be non-immunogenic. Using these conjugates, the ongoing anti-EALA IgG response elicited by EALA-BSA was suppressed. Forty-six days after injection of EALA-BSA, these mice were split into two groups one of which received EALA- dex ⁇ in increasing doses of 1, 10 and 100 ⁇ g dextran backbone and the other of which received three injections of buffer alone. The mice were bled three days later and at three day intervals thereafter and the antisera tested for any reduction in their anti-EALA IgG titers.
• FIG. 50 clearly indicates that EALA-dex 8 prevented the rise in Ab levels that continued to progress in the untreated mice. The low levels persisted for at least 21 days. At this point another cure was performed using the same regimen to see if the low response of suppressed mice could be effectively abolished altogether. Although the response was not reduced further by EALA-dex 8 . administration, it was maintained at low levels for at least another 14 days while that of the control mice still appeared to increase.
• This peptide referred to as 159, represents residues 331-339 of chicken ovalbumin (OVA) .
• IgG antibodies were raised to 104 by injection of Balb/c mice with 104-BSA adsorbed on to alum. High titers were raised both to 104 and to 159 ( Figure 51) . Forty days after the immunizing injection (day 0 in the Figure) , groups of eight mice were either injected with saline or with 1, 10 and 100 ⁇ g dextran of 40,000 molecular weight conjugated with 159 at a ratio of 10 moles peptide/mole dextran (ie 159 10 -dex 40 ) . Mice were subsequently bled at day 3 and then at 7 day intervals and assayed for responses to both 159 and 104.
• Figure 51 shows the responses after the cure and indicates that the response to 159 was dramatically reduced immediately after treatment but that the antibody titer rebounded virtually to control levels within 14 days.
• the response to 104 in treated mice followed the same pattern of reduction and recovery as the 159 response but was only reduced at day 3 to about 60% of the precure response whereas the 159 response is reduced to about 9% of precure levels.
• approximately 40% of the response to 104 is removed by administration of 159 10 -dex 40 indicating that this portion of the 104 response is directed against epitopes in the 159 sequence.
• up to 40% of the 104 response can indeed be completed successfully with soluble 159 in solution phase competition assays (data not shown) .
• mice were again cured (day 77) with 1, 10 and 100 ⁇ g 159-dex and subsequently boosted intraperitoneally with 104- BSA, as were the control mice. It is clear that cured mice can withstand this challenge since although the antibody responses of control mice were substantially boosted (as would be expected) , those of the cure mice were not significantly changed from pre-challenge levels. It appears therefore that the effect of
• 159 10 -dex 40 is not only to suppress the ongoing anti-159 antibody response but also to inactivate the specific memory B cells such that they can no longer respond to the challenge with 104-BSA. This indicates that the suppression occurs at the level of the specific B cells and is not just an apparent suppression caused by anti-159 antibodies being absorbed to the circulating conjugates and thus being effectively removed from the sera.
• Spleens from individual mice were enumerated for cells secreting anti-159 antibodies. Two x 10 5 cells were added to each well.
• mice In many cases, in order for the symptoms to be maintained, continued, regular administration of antigen is required otherwise the disease process wanes. The relevance and applicability of these models to spontaneously occurring autoimmune processes in man is unclear. There is, however, a model of autoimmune disease in mice (the NZB/NZW mouse model of human systemic lupus erythematosus— urine lupus) that parallels the human with great fidelity.
• mice Animals suffering from murine lupus exhibit the production of both anti-histone as well as anti-DNA antibodies.
• the distribution of antibodies directed against histone proteins in these mice was shown to be predominantly limited to the amino terminal region of H2B. In fact, in greater than 90% of the mice tested the antigenic region of this protein was found to reside between residues 3 and 12 inclusively 3 12
• T-cell line Measurements were made of biologically relevant responses of a T-cell line, after exposure to defined, soluble, polymeric molecules containing haptens capable of binding specifically to the T-cell surface antigen receptors.
• the responses obtained in these experiments with T- cell lines were found to be in close agreement with the expectations based on the Immunon model. This was true for both the dose-response behavior of the T-cells to individual haptenated polymer preparations and the dose-inhibition behavior observed when stimulatory polymers and non- stimulatory (suppressive) polymers were administrated together.
• the Jurkat T-cell was transfected with genes encoding both the alpha and the beta polypeptide chains of a fluorescein- specific human T-cell antigen receptor. This transfected Jurkat line was shown to be functional, since it could produce the lymphokine, interleukin-2, upon treatment with conventional T-.
• soluble fluoresceinated polymers i.e., those of high molecular mass and containing a large number of fluorescein epitopes, caused functional activation of the T-cell transfectants. Activation of the T-cells by these soluble polymers was demonstrated by either of two different assays:
• Figure 54 demonstrates, for a particular pair of fluoresceinated polymers, a representative example of the experimental data described above.
• Figure 54 (a) shows that a heavily fluorescein- substituted Fieoll preparation of molecular mass over 100 kDa, FL50-Ficl50, activated the transfected Jurkat T-cells to produce interleukin- 2, as measured by tritiated thymidine incorporation by an IL-2 sensitive cell line.
• the dose-response stimulation curve is bell shaped, as was observed in the similar mouse B-cell studies previously described.
• the same Figure shows that a fluorescein-substituted dextran, FL8-Dex21, of a similar epitope density but molecular mass well below 100 kDa, was not capable of stimulating the same transfected T- cells at any comparable dose.
• Figure 54 (b) shows that when the two polymers were simultaneously added to the transfected T-cells, increasing amounts of the non-stimulatory smaller polymer can be clearly seen to inhibit increasingly the activating ability of the larger, stimulatory, polymer in a dose-dependent manner. Similar activation and inhibitory effects were observed when intracellular calcium flux was measured for the transfected T-cells using soluble fluoresceinated polymers, Figure 55.
• a large highly substituted polymer FL50-Ficl50 stimulated the rapid activation of intracellular calcium flux when added at low or moderate dose (a and b) , but not at high dose, (c) .
• a non-stimulatory polymer of smaller size but similar epitope density, FL11- Fic46 caused a lack of response by the cells to stimulatory polymer, again demonstrating competitive inhibition (d and e) .

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