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WO1994014976A1 - Complexe modulateur de la reponse immunitaire, et ses utilisations - Google Patents

Complexe modulateur de la reponse immunitaire, et ses utilisations Download PDF

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Publication number
WO1994014976A1
WO1994014976A1 PCT/US1993/012479 US9312479W WO9414976A1 WO 1994014976 A1 WO1994014976 A1 WO 1994014976A1 US 9312479 W US9312479 W US 9312479W WO 9414976 A1 WO9414976 A1 WO 9414976A1
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WIPO (PCT)
Prior art keywords
antigen
fragment
receptor
hel
binding
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PCT/US1993/012479
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English (en)
Inventor
Salvatore V. Pizzo
Charleen T. Chu
Tim D. Oury
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Duke University
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Priority to AU58739/94A priority Critical patent/AU5873994A/en
Publication of WO1994014976A1 publication Critical patent/WO1994014976A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Definitions

  • the present invention is a Continuation-In-Part of co-pending application Serial No. 07/992,899, filed December 18, 1992, incorporated herein by reference in its entirety, to which the above-identified a; ; cation claims the benefit of priority pursuant to 35 U.S.C. ⁇ 120.
  • the present invention relates generally to the field of immunology and, more particularly, to the modulation of the immune response to a variety of antigens, including the enhancement of host immune-competence and the preparation and administration of vaccines for prevention and treatment of disease states.
  • antigens are "presented” to. the immune system by antigen presenting cells (APCs), including, for instance, macrophages, dendritic cells and B-cells in the context of major histocompatibility complex molecules (MHCs) which are present on the APC surface.
  • APCs antigen presenting cells
  • MHCs major histocompatibility complex molecules
  • natural antigens and molecules supplied as immunogens are thought to be taken up and partially digested by the APCs, so that smaller pieces of the original antigen are then expressed on the cell surface in the context of MHC molecules.
  • T-lymphocytes in contrast to B-lymphocytes, are relatively unable to interact with soluble antigen.
  • T-lymphocytes require antigen to be processed and then expressed on the cell surface of APCs in the context of MHC molecules as noted above.
  • T-cells and more particularly, the so called "T-cell receptors,” are able to recognize the antigen in the form of a bimolecular ligand composed of the processed antigen and one or more MHC molecules.
  • the APC In addition to presenting antigens on MHC molecules, the APC must be activated to express costimulatory molecules, such as B7/BB1, before effective stimulation of T-cells can occur.
  • epitopes on proteins are generally unrecognized or only weakly recognized by the immune system. These epitopes therefore elicit little or no antibody or other immune response, or at most, only a weak response. It has therefore been difficult and in some instances, impossible to raise antibodies against such epitopes.
  • other epitopes elicit extraordinarily strong immune responses, in some instances, to the exclusion (or partial exclusion) of other epitopes within the same antigen molecule.
  • Such epitopes can be termed "immunodominant. "
  • HIV human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • the predominant vaccine strategy has focused on the use of the envelope protein antigens gpl20 and gpl60 of HIV-1 produced by recombinant DNA technology.
  • the full promise of their use in vaccines cannot presently be realized unless they are administered along with an effective adjuvant.
  • An adjuvant should usually be a non-toxic agent that provokes specific responses to antigens.
  • an adjuvant can function by creating a depot at the site of injection that prolongs the release of antigens with antigen-presenting cells. It may also function by activating macrophages to synthesize and/or release costimulatory molecules, cytokines, and other mediators which in turn activate effector T cells or antibody-forming B cells. The net result is that an adjuvant augments specific humoral and cell-mediated immunities with a lower dose of antigen required.
  • the agents that have been commonly used as adjuvants can be broadly categorized into four groups of which the following two are most significant.
  • the first, and the only clinically acceptable group comprises the gels of aluminum (e.g. alum) and calcium salts.
  • alum is a weak adjuvant and its formulation in laboratory tests of HIV and SIV antigens has been found to be inadequate.
  • the second, and perhaps the most potent group includes pure compounds and undefined mixtures derived from mycobacterial cell walls.
  • Mixtures such as Freund's complete adjuvant (FCA) and lipopolysaccharides (LPS) are the best known examples.
  • FCA and LPS produce side effects. They are pyrogenic and induce arthritis in rats and anterior uveitis in rabbits.
  • B-cells possess specific receptors, surface Ig, for capturing the Ag they present efficiently (86,87), macrophages and other non-B APCs must utilize other mechanisms. These may include phagocytosis of paniculate or cellular Ag and enhanced endocytosis of opsonized Ag or immune complexes. Yet, the efficient uptake and presentation of soluble Ag by these non-B cell APCs in naive animals is not fully understood. A receptor-mediated process might be involved.
  • the macrophages are of particular interest by virtue of the central role that they play in the regulation of the activities of other cells of the immune system. Macrophage act as effector cells in microbial and tumor cell killing as well, and are believed to secrete numerous cytokines that orchestrate many of the diverse aspects of the immune response. The ability of macrophage to regulate a range of immunologic events is in part a function of their expression of I vision surface antigens. The expression of membrane I a antigens is essential for the induction of specific T cell responses to antigens [Unanue (1981), ADV. IMMUNOL. 31: 1-136].
  • the ⁇ -macroglobulins and the complement components C3, C4, and C5 comprise a superfamily of structurally related proteins.
  • the ⁇ -macroglobulin family includes proteinase-binding globulins of both a ⁇ and ⁇ 2 mobilities.
  • the most extensively studied ⁇ -macroglobulin is human ⁇ 2 -macroglobulin ( ⁇ 2 M), a large tetrameric protein capable of covalently binding other proteins (6-7, 20, 63-68) and targeting them to cells bearing the ⁇ 2 M receptor (14, 15, 41 , 68).
  • ⁇ 2 M can incorporate proteins and peptides bearing nucleophilic amino acid side chains in the relatively nonselective manner.
  • ⁇ 2 M, C3 and C4 are evolutionarily related thioester-containing proteins that undergo conformational and functional changes upon limited proteolysis (57, 88), resulting in possible formation of thioester-mediated covalent bonds with targets such as proteinases, cell-surface carbohydrates or immune complexes, respectively.
  • ⁇ 2 M 3 Human ⁇ 2 -macroglobulin ( ⁇ 2 M) 3 is an abundant protein. It consists of four identical subunits arranged to form a double-sided molecular "trap” (4). This trap is sprung when proteolytic cleavage within a highly susceptible stretch of amino acids, the "bait region", initiates an electrophoretically detectable conformational change that entraps the proteinase (5).
  • the resulting receptor-recognized f- ⁇ 2 M is efficiently internalized by macrophages, dendritic cells, and other cells that express ⁇ 2 M receptors [reviewed in (9); see also (69)], which has recently been cloned and sequenced (10, 11).
  • Receptor-recognized ⁇ -macroglobulins from different animal species cross-react with similar affinities for the ⁇ 2 M receptor regardless of the proteinase used [See (9, 12, 13) for review].
  • the additional binding of nonproteolytic proteins does not appear to affect the rate of internalization even when artificial crosslinking is employed (14-16). Therefore, regardless of the mechanism of binding, proteins complexed with f- ⁇ 2 M can be effectively internalized.
  • a proteinase-activated antigen capture and delivery system that binds antigens in a nonselective but irreversible manner might be expected to play a particularly important role during the primary antigen exposure in vivo. In vitro studies, however, do not fully reflect this situation. Unlike T-hybridoma clones, which often do not require costimulatory signals, the responses of naive T-cells encountered during a primary exposure are dependent upon costimulation (70, 71). Furthermore, the relative roles of macrophages, dendritic cells, B cells, and Langerhans cells in mediating in vivo immune responses are still unclear.
  • soluble antigens may be presented primarily by dendritic cells (72, 73), with macrophages mediating the presentation of paniculate antigens or inducing tolerance (74). Thus, in vivo studies are necessary to establish whether antigen delivery by ⁇ 2 M is sufficient for the induction of fully competent helper T- cells.
  • the foregoing articles had urged that the receptor for ⁇ 2 M had played a role in the delivery of various proteins.
  • Two references by Osada et al. merit specific mention for their purported teachings.
  • the invention described herein relates to the modulation of the immunogenicity of an antigen. More particularly, the invention relates to enhancing the immunogenicity of an antigen.
  • the present invention relates to modifying the antigenicity or immunogenicity of an antigen by administering a receptor binding form of a ⁇ -macroglobulin, in particular ⁇ 2 M, or a fragment thereof with the antigen.
  • a complex between the antigen and ⁇ 2 -macroglobulin ( ⁇ 2 M) or an active fragment thereof is formed.
  • Such complex may be introduced to a cell culture or host, or to a target tissue or organ where it is believed that ⁇ 2 M augments presentation of the desired antigen and the development of the corresponding immune response will occur.
  • the complex of the present invention comprises a covalent binding between the antigen of interest and ⁇ 2 -macroglobulin or an active fragment thereof.
  • suitable antigens include nucleophiles, and extend to and include peptides, proteins, carbohydrates, cytokines, growth factors, hormones, enzymes, toxins, nucleic acids such as anti-sense RNA, as well as other drugs or oligonucleotides.
  • ⁇ 2 -macroglobulin of a thiolester bond which is susceptible to nucleophilic attack when ⁇ 2 -macroglobulin is activated by proteinases, ammonia or small amines, is believed to account for the efficient formation of the present complex.
  • Complexes with ⁇ 2 M or fragments thereof can be formed with chemical crosslinking agents as well.
  • the high affinity that activated ⁇ 2 - macroglobulin and C-terminal fragments thereof demonstrate for its cellular receptor is believed to account for the efficient presentation of the antigen and significant increase in the speed and magnitude of the immune response that is achieved.
  • the present invention relates to a method for modifying immune recognition of epitopes by the immune system.
  • This may involve endogenous or foreign molecules (immunogens), and may entail altering the antigenicity thereof or altering the immune response thereto, including without limitation modulating the immune response to (non-modified) naturally occurring endogenous or foreign molecules represented by the immunogen.
  • the epitopes of a particular immunogen may be essentially unrecognized, or may be inactive epitopes on otherwise antigenic molecules. Similarly, such epitopes may otherwise be only weakly recognized or responded to by the immune system under normal conditions.
  • such epitopes may be dominantly recognized by the immune system, such that other epitopes on the same immunogen molecule do not elicit immune responses.
  • the immunogenicity of a T cell epitope containing a strongly nucleophilic residue such as lysine is modified, i.e. , decreased, by covalent binding of lysine to the thioester group on the ⁇ 2 -macroglobulin.
  • One of the advantages of the present invention and a particular feature thereof resides in the fact that the complex prepared by the covalent binding of ⁇ 2 M or its fragments to a given antigen, or the development of the constructs disclosed herein comprising the C-terminal receptor binding regions of ⁇ 2 M disposed either singly or in tandem with respective antigens covalently bound thereto, can be administered as a vaccine without need for an adjuvant.
  • the immune response achieved by administration of antigen in accordance with the present invention equals or exceeds both in vitro and in vivo, those levels that would be achieved with conventional formulations including adjuvant.
  • the preparation of vaccines in accordance with the present invention represents a significant improvement and offers the promise of a far more efficient vehicle for antigen presentation, and one which will avoid many of the drawbacks such as toxicity and the like that are experienced with current adjuvant-containing formulations.
  • the complex and/or constructs prepared in accordance with the present invention have particular utility in their direction against macrophage, and other cells that bind or internalize ⁇ 2 M.
  • antigens, immunogens or immune modulating molecules that may be associated in the complex and/or constructs of the present invention is equally diverse, as it extends from oligonucleotides, proteins, peptides, cytokines, toxins, enzymes, growth factors, antisense RNA and drugs, to other carbohydrates that may exhibit some desired modulatory effect on the target cells.
  • the invention is therefore contemplated to extend to these variations within its spirit and scope.
  • a further advantage of the invention is that it provides for independently targeting a receptor binding ⁇ 2 M or fragment thereof, as well as complexes of the invention comprising these components, for endocytosis or for cell signalling and activation. Proper activation of the APC is necessary for effective antigen presentation and effective stimulation of the immune response in general.
  • fusion protein might be prepared between the C- terminal fragment of ⁇ 2 M and the protein to be delivered, in which the C-terminal portion of the fusion protein contains the receptor recognition site for the ⁇ 2 M receptor.
  • the carboxyl terminal fragment of ⁇ 2 M or other ⁇ macroglobulins may be prepared to include a chemically cross-linkable group, such as sulphydryl groups, so that covalent binding can then be made to the antigen, immunogen, or the like, of interest for eventual presentation and activation.
  • the effective concentration of the material being delivered may be potentiated while desirably limiting the total protein concentration.
  • the C-terminal construct containing engineered cysteine may be prepared in a tandem arrangement, or with a polyvalent crosslinker attached to it, wherein a plurality of antigens may be bound by like cross-linking groups. In such instance, one may associate a plurality of diverse, complementary antigens to bind to a corresponding set of receptors. This strategy may be useful in the preparation and presentation of polyvalent vaccines.
  • constructs of the present invention may be prepared recombinantly, by the initial combination of the antigen with a C-terminal fragment, and the subsequent introduction of the resulting construct within a vector for expression in a suitable host.
  • the exact parameters and protocols followed in this preparation are within the skill of a molecular biologist, and will most likely vary depending on the particular antigen, fragment and host.
  • both positive and negative regulation of the antigenicity of epitopes can be achieved.
  • by rendering epitopes recognized, or recognizable, antibodies can be raised to recognize and bind to the antigen.
  • Enhanced antigenicity and the ability to raise antibodies to otherwise weak, scarce or ineffective epitopes finds great utility not only, for example, in vaccine applications in animals, including humans, but also in producing antibodies which can be used as reagents for, among other uses, binding, identifying, characterizing and precipitating epitopes and antigens, such as the production of antibodies against scarce antigens for research purposes.
  • the immunogenicity of a given antigen is enhanced according to the methods of the invention.
  • this invention contemplates the downregulation or suppression of immune responses to immunodominant epitopes, by the preferential stimulation of immune responses to otherwise "subordinate" epitopes, or by the introduction of agents or factors that on presentation, would selectively suppress the immunogenicity of the target epitope.
  • This additional ability to modulate antigenicity may be useful, for example, in immunizing animals, including humans, and also in producing antibodies which are reactive towards otherwise silent or weakly antigenic epitopes.
  • Such antibodies are also useful for, among other things, binding, identifying, characterizing and precipitating epitopes and antigens in vivo and in vitro.
  • Another preferred embodiment of the invention utilizes antigen presenting cells (APCs) and the major histocompatibility complex (MHC) present on the surface of such cells.
  • the antigen is complexed initially with ⁇ 2 -macroglobulin or an active fragment thereof, as described above.
  • This complex is then combined with APCs having MHC present on the cell surface as well as receptors for ⁇ 2 -macroglobulin, the antigen or the ⁇ 2 M-antigen complex, until processing of the antigen is effective for rendering the epitope recognizable.
  • the processed and displayed antigen is then available to react with other components of the immune system which recognize the epitope or the complex-modified epitope in the context of the ABC.
  • the invention described herein also preferably includes the antibodies produced by the methods described herein or in response to the immunogens, modified as described herein, said antibodies including monoclonal, polyclonal and chimeric antibodies, as well as immortal strains of cells which produce such antibodies, for example hybridomas which produce monoclonal antibodies which recognize the molecules and other antigens of interest.
  • such antibodies can be prepared against epitopes on the antigen that are normally secondary or even suppressed.
  • the invention also encompasses cellular immune system components, e.g. , T- lymphocytes raised in response to such antigens or immunogens, pharmaceutical compositions containing the antigens, antibodies or cellular immune system components and various methods of use.
  • cellular immune system components e.g. , T- lymphocytes raised in response to such antigens or immunogens
  • pharmaceutical compositions containing the antigens, antibodies or cellular immune system components and various methods of use.
  • the invention provides for enhancing the efficiency o immunizations. This can have useful application not only for potential therapeutic interventions, in particular vaccinations, but also for production of antibodies or primed lymphocytes (T or B) against scarce antigens for research purposes.
  • T or B primed lymphocytes
  • ⁇ 2 M complexed hen egg white lysozyme undergoes enhanced macrophage uptake, processing, and presentation to T- hybridoma clones in vitro compared to free antigen; antibody production in rabbits using two antigens complexed with either human ⁇ 2 M (H ⁇ 2 M) or a homologous protein purified from rabbit plasma, ⁇ macroglobulin (R ⁇ ,M) was evaluated, and was found that complexing the Ag to ⁇ 2 M resulted in 10-500-fold higher IgG titers compared to uncomplexed controls; proteinase-treated ⁇ 2 M complexed with insulin protects the antigen from degradation; and a C-terminal fragment of ⁇ 2 M that lacks the cis-DPP/oxidation reactive site binds an ⁇ 2 M-receptor and induces cell stimulation.
  • HEL human ⁇ 2 M
  • R ⁇ ,M ⁇ macroglobulin
  • FIGURE 1 depicts the binding of 125 I-HEL to different conformational forms of ⁇ 2 M.
  • a two-fold molar excess of l25 I-HEL was incubated for 30 min at room temperature with 1.4 ⁇ M amounts of native ⁇ 2 M, ⁇ 2 M undergoing PPE induced conformational change, or pre-formed f- ⁇ 2 M.
  • the PPE was inhibited by addition of 100 mM dichloroisocoumarin after 10 min. Samples were analyzed by SDS-PAGE under reducing (R) and nonreducing (NR) conditions (A & B) and by native pore limit gel electrophoresis (C & D).
  • the arrow represents the position of migration for HEL that is covalently complexed to the C-terminal half of the "bait-region "-cleaved ⁇ 2 M, when analyzed after reduction.
  • Molecular weight standards are indicated on the left: denatured but nonreduced ⁇ 2 M (360 kDa), reduced ⁇ 2 M (180 kDa), phosphorylase b (91 A kDa), BSA (66.2 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa).
  • Positions of migration for ⁇ 2 M conformational forms were determined using native ⁇ 2 M (s- ⁇ 2 M) and methylamine-treated ⁇ 2 M (f- ⁇ 2 M).
  • FIGURE 2 comprises two graphs: (A) Sensitivity of I23 I-HEL- ⁇ 2 M complex formation to ⁇ -aminopropionitrile. 123 I-HEL, ⁇ 2 M, and PPE (molar ratios of 0.3:1:2) were coincubated for 10 min at room temperature in HEPES buffer containing 0-200 mM ⁇ -aminopropionitrile. Reactions were terminated and samples analyzed by native pore limit gel electrophoresis (•) or nonreducing SDS gel electrophoresis ( ⁇ ) as described in METHODS. Values are expressed as percent of control incubations in the absence of ⁇ -aminopropionitrile, and reflect mean ⁇ SD from duplicate determinations.
  • FIGURE 3 is a graph of specific uptake of 12 T-HEL derivatives by macrophages.
  • Log dilutions of either 123 I-HEL (•), or ,25 I-HEL- ⁇ 2 M-PPE complexes ( ⁇ ) were incubated with macrophage monolayers at 37 °C for increasing periods of time as described in METHODS.
  • Total cell associated radioactivity was determined, and specific association was calculated by subtracting the nonspecific binding determined from incubations with 100-fold molar excesses of unlabelled HEL or of ⁇ 2 M-methylamine.
  • a representative time course for uptake of 100 nM concentrations of the HEL derivatives is shown. Error bars represent one SD from quadruplet samples.
  • FIGURE 5 is a graph depicting the results of antigen presentation by macrophages pulsed with different forms of HEL.
  • Macrophages were pulsed with free HEL (•), HEL- ⁇ 2 M-PPE complexes ( ⁇ ), or free HEL in the presence of equimolar amounts of ⁇ 2 M-methylamine (A), for two h before extensive washing to remove excess Ag.
  • Pulsed macrophages were assayed for their ability to stimulate IL-2 secretion by HEL-specific T-hybridomas as described in METHODS. Values are expressed as mean + SD from triplicate samples, and are representative of four independent assays.
  • FIGURE 6 is a graph depicting the time and concentration dependence of presentation to HEL-specific T-cells.
  • Macrophages were pulsed with log dilutions of either free HEL (•) or of HEL- ⁇ 2 M-PPE complexes ( ⁇ ) for different time periods ranging from 15 min to 3 h.
  • Pulsed macrophages were assessed for their ability to stimulate IL-2 secretion by 3A9 T-hybridomas.
  • the 24 h time points were obtained from coincubating macrophages and T-hybridomas with the Ag.
  • Supernatants from these all experiments were analyzed for IL-2 activity as described in METHODS.
  • a detectable response was defined as > 1 SD above the baseline, which was calculated from six incubations with no added Ag.
  • the minimum concentrations of the two Ag forms that were required to achieve a measurable T-hybridoma response are plotted against time. Error bars represent one SD derived from triplicate samples.
  • FIGURE 7 is a graph illustrating the stimulation of T-hybridomas by macrophages in the continued presence of Ag.
  • Peritoneal macrophages and 3A9 T-hybridoma cells were co-incubated for 24 h with varying concentrations of HEL (•), HEL- ⁇ 2 M-PPE complexes ( ⁇ ), or equivalent concentrations of control ⁇ 2 M-PPE complexes (A).
  • Control curves from similar incubations that lacked 3A9 cells ( ⁇ ) or macrophages (D) are also shown.
  • IL-2 secretion during this 24 h period was quantified as described in METHODS. Error bars represent one SD derived from triplicate samples.
  • FIGURE 8 denotes a primary IgG response to injections of HEL.
  • Pathogen-free NZW rabbits were injected s.c. with the equivalent of 124 ⁇ g HEL. Shown here are the rates of substrate hydrolysis (alkaline-phosphatase coupled 2° Ab) plotted against reciprocal dilutions of sera obtained two weeks after the injection.
  • Four rabbits were injected with H ⁇ 2 M-HEL-PPE complexes in HEPES buffer ( ⁇ ), three with HEL emulsified in CFA ( ⁇ ), three with free HEL in HEPES (O), two with HEL mixed with s- ⁇ 2 M ( ⁇ ), and two with HEL mixed with preformed f- ⁇ 2 M (methylamine-treated) (A).
  • the symbol represents the mean + SD.
  • a representative curve for preimmune sera is shown (•).
  • FIGURE 9 depicts anti-HEL IgG titers elicited by rabbit (R) ⁇ 2 M complexes compared with H ⁇ 2 M complexes.
  • Sera were collected from each rabbit weekly after a single injection at Week 0 with the indicated equivalent doses of HEL.
  • the maximum dilution factor which yielded substrate hydrolysis rates of at least 1 mOD/min were defined as the end titer.
  • a zero titer indicates that substrate hydrolysis was not detectable in a 100-fold dilution.
  • FIGURE 10 depicts anti-PPE antibody responses.
  • Each rabbit was injected s.c. with equivalent to 200 ⁇ g PPE.
  • Substrate hydrolysis rates in Week 3 sera are shown in comparison with preimmune sera (D).
  • the rabbits received either H ⁇ 2 M-HEL-PPE complexes ( ⁇ ), inhibited PPE alone (O), or inhibited PPE + 6 mg of BSA ( ⁇ ).
  • the symbol represents the mean from two rabbits, and the bars show the high-low range of the values.
  • FIGURE 11 presents a schematic illustrating a potential role for ⁇ 2 M (and other ⁇ - macroglobulins) in Ag processing by macrophages.
  • the ⁇ 2 M conformational forms are derived from a previously published model, which was based on electron micrographs (4). (1) Diffusion of proteins in and out of the s- ⁇ 2 M "trap.” (2) Proteolytic cleavage of the ⁇ 2 M "bait” region [See (5)], results in capture of the proteinase (stippled "pacman") and potential Ag (striped polygon).
  • FIGURE 12 presents an autoradiogram of a non-reduced SDS-PAGE with labelled insulin. Lanes A-G are described in Example 3, infra.
  • FIGURE 13 presents graphs reporting changes in the level of intracellular calcium, [Ca 2+ ];, observed in TG-elicited macrophages.
  • A A representative response of a single cell on addition of methylamine-treated ⁇ 2 M (40 nM); in this study, 45 individual cells were evaluated.
  • B A representative response of a single cell on addition of the 20 kDa RBF of rat ⁇ 2 M (•) (40 nM), or buffer (O); in this study, 250 cells and 15 cells were evaluated, respectively, after addition of RBF or buffer.
  • FIGURE 14 presents a graph showing the percent change in the concentration of intracellular calcium upon exposure to varying concentrations of either ⁇ 2 M- methylamine ( ⁇ ) or RBF (O). Calcium mobilization was evaluated as described in the legend to FIGURE 13.
  • FIGURE 15 presents graphs showing increased histone-III phosphorylation resulting from exposure of macrophages to ⁇ 2 M-methylamine, ⁇ ,-inhibitor 3 and RBF. Histone phosphorylation is a consequence of PKC activity.
  • A 33 P incorporation after treatment with various combinations of buffer, the 20 kDa RBF from rat ⁇ ,M, and the PKC inhibitor staurosporin. The presence or absence of one or more of these reagents is shown in the Figure.
  • FIGURE 16 presents graphs showing movement of tritiated [ 3 H]-phorbol dibutyrate ([ 3 H]-PDBu) to cell membranes in response to treatment with ⁇ 2 M, rat ⁇ ,-inhibitor 3 , and rat ⁇ -M RBF. Movement of the diacylglycerol analog [ 3 H]- PDBu demonstrates activation of PKC. TG-elicited macrophages were cultured as described. The macrophages were exposed to the ligands for 20 min, after which they were assayed for PKC activation by binding of 3 H-phorbol dibutyrate as previously described (Misra, U.K. and Sahyonn, N.E.
  • immunogen refers to any substance, such as a molecule, cell, virus or fragment of such molecule, cell or virus which can be administered to an individual in an effort to elicit an immune response.
  • immunogen thus simply refers to such substances which are or can be administered or otherwise used to raise antibodies or cellular immune system components, such as by "priming".
  • molecule refers to a molecule or molecular fragment of the antigen unless otherwise specified.
  • the immunogen can be the cell, virus or component thereof, which can be disposed in a complex or construct in accordance with the present invention to enhance the immune response thereto.
  • the term "immunogen” therefore encompasses antigenic compounds, such as foreign proteins as well as species which are essentially non- antigenic in the absence of the treatment described herein, cells, viruses, and cellular and viral components.
  • antigen which may be abbreviated "Ag,” refers to substances, e.g., molecules which induce an immune response. It thus can refer to any molecule contacted by the immune system, and may include without limitation, proteins, nucleic acids and the like, and may even extend to carbohydrates capable of presentation in accordance herewith. Generally, each antigen typically comprises one or more epitopes.
  • the antigens described herein or epitopes thereon do not substantially induce an immune response or other immunological reaction upon injection or other exposure to a normal, substantially immunocompetent host. They may also include scarce antigens that are difficult to obtain or purify, or antigens that require adjuvant or administration in large amounts ( ⁇ M) for efficient immune responses. Based on the foregoing, “antigenicity” and “immunogenicity” are used interchangeably.
  • protein refers to synthetically produced and naturally occurring polypeptides, fragments of polypeptides and derivatives thereof which may provoke an immune response, either in vitro or in vivo.
  • protein refers to synthetically produced and naturally occurring polypeptides, fragments of polypeptides and derivatives thereof which may provoke an immune response, either in vitro or in vivo.
  • the description below utilizes the term “protein” but these teachings also apply to other compounds which either contain protein residues or that are otherwise structurally similar. Oligonucleotides, carbohydrates, and amine-containing lipids, as well as other reactive biomolecules may be mentioned as non-limiting examples. The teachings contained herein are therefore not to be limited to proteins or fragments thereof.
  • immunocompetent refers to the immune response which can be elicited in a normal mammalian host with the antigen of interest, when the antigen in question is administered without the modifications and preparation described herein.
  • the immunogen can simply be administered to the host in unmodified form, and the normal immune response evaluated. Thus, using art recognized methods, this control is readily ascertained without resort to undue experimentation.
  • antibody refers to immunoglobulins, including whole antibodies as well as fragments thereof, such as Fab, F(ab' 2 or dAb, that recognize or bind to specific epitopes.
  • the term thus encompasses, inter alia, polyclonal, monoclonal and chimeric antibodies, the last mentioned being described in detail in U.S. Pat. Nos. 4,816,397 and 4,816,567, which are incorporated herein by reference.
  • An antibody “preparation” thus contains such antibodies or fragments thereof, which are reactive with an antigen when at least a portion of the individual immunoglobulin molecules in the preparation recognize (i.e., bind to) the antigen.
  • An antibody preparation is therefore termed "non-reactive" with the antigen when the binding of the individual immunoglobulin molecules to the antigen is not detectable by commonly used methods.
  • an antibody is said to "recognize” an epitope if it binds to the epitope.
  • “recognition” involves the antibody binding reaction with an epitope, which may include the typical binding mechanisms and methods.
  • Binding is thus used in the conventional sense, and does not require the formation of chemical bonds.
  • epitope is used to identify one or more portions of an antigen or an immunogen which is recognized or recognizable by antibodies or other immune system components.
  • epitope region refers to the epitope and the surrounding area in the vicinity of the epitope, taking into account three dimensional space. Hence, this may take into account the tertiary and quaternary structure of the antigen.
  • Processing and “presentation” refer to the mechanisms by which the antigen is taken up, altered and made available to the immune system. Presentation also includes, when appropriate, complexation or binding with MHC and other molecular events associated with generating an effective T-cell response. In certain instances, processing entails the uptake and partial proteolytic degradation of the antigen by APCs, as well as display on the APC surface in the context of MHC.
  • reaction and “complex” as well as derivatives thereof, when used in this general sense, and are not to be construed as requiring any particular reaction mechanism or sequence.
  • MHC refers to major histocompatibility complex, a series of compounds which is normally present to a greater or lesser degree on the surface of, among others, antigen presenting cells.
  • MHC functions to "signal" cellular immune system components, e.g., T-lymphocytes, to recognize and react with the antigen presenting cell and/or the antigen bound to said cell and/or the MHCs thereof.
  • signal is used in the general sense to refer to the initiation of the reaction between T-cells and APCs bearing processed antigen in the context of MHC. As such the “signal” may involve any reaction between these components which causes the antigen to become recognized by antibodies, an antibody preparation or by the cellular immune system components.
  • ⁇ 2 -macroglobulin and its abbreviation " ⁇ 2 M” are to be used interchangeably.
  • ⁇ 2 M the abbreviation of ⁇ 2 M
  • the use of ⁇ 2 - macroglobulin in accordance with the present invention is believed to be more generally applicable to ⁇ -macroglobulins and to the macroglobulin family, and the scope of the invention is to be interpreted in this broader fashion.
  • ⁇ 2 M refers to human ⁇ 2 M, or a receptor-binding fragment thereof.
  • this term includes, but is by no means limited to, rat ⁇ 2 M (a homotetramer); rat ⁇ jM (a homotetramer); rabbit ⁇ ,M (a homotetramer); human pregnancy zone protein (a homodimer); cow ⁇ 2 M (a homotetramer); dog ⁇ 2 M (a homotetramer); duck ovostatin, or ovomacroglobulin (a homotetramer); hen ovostatin, or ovomacroglobulin (a homotetramer); rat ⁇ ,-inhibitor-3 (a monomer); frog ⁇ 2 M (a homotetramer); as well as receptor-binding fragments thereof.
  • receptor-binding refers to the ability to bind to a specific receptor on an APC.
  • the receptor may mediate endocytosis, signalling and cell activation, or both. It is presently believed that there are two receptors for ⁇ 2 M. One receptor mediates signalling, and thus cellular activation and growth. The other receptor mediates endocytosis.
  • a C-terminal fragment of ⁇ 2 M induces macrophage activation. When this fragment lacks a cis-dichlorodiamine platinum (cis- DDP)/oxidation sensitive reaction site, it appears to bind to the signalling receptor but not as well as the endocytic receptor. When the C-terminal fragment includes the cis-DDP/oxidation sensitive reaction site, it appears to bind to both receptors.
  • slow (s)- ⁇ 2 M the native conformation of ⁇ 2 M that is not receptor-recognized
  • fast (f)- ⁇ 2 M the receptor-recognized forms of ⁇ 2 M derived from treatment with proteinase or with methylamine
  • H ⁇ 2 M human ⁇ 2 M
  • R ⁇ ,M rabbit ⁇ r macroglobulin, the ⁇ 2 M-equivalent purified from rabbit plasma
  • HEL hen egg lysozyme
  • PPE porcine pancreatic elastase
  • APC antigen presenting cell.
  • a method for enhancing the presentation, recognition and uptake of antigens comprises administering said antigen with a material selected from the group consisting of ⁇ 2 - macroglobulin and an active fragment thereof and plural such active fragments thereof, and directing said ⁇ 2 M to the target cellular mass to which presentation of said antigen is intended.
  • the antigen is in a complex with the ⁇ 2 M or fragment thereof. More preferably, the antigen is in covalently associated with the ⁇ 2 M or fragment thereof.
  • the present invention advantageously provides for forming complexes comprising substantially a single antigen or a few antigens, rather than a diverse population of proteins.
  • the term substantially indicates that greater than 30% of the antigen in the complex is a specific antigen; preferably greater than 50%; more preferably greater than 75%; and most preferably greater than about 90%.
  • the active fragments of ⁇ 2 -macroglobulin may comprise the carboxyl terminal region thereof and said region including the receptor recognition site, and said region having associated therewith chemical cross-linking moieties.
  • exemplary such moieties would comprise a cysteine residue or other moiety providing a disulfide bond for covalent attachment to the antigens in object.
  • native ⁇ 2 M can be used to entrap and covalently or non-covalently complex with an antigen of interest.
  • the trapping and complex formation occur by activation, e.g. , with proteolysis.
  • fast ⁇ 2 M which is ⁇ 2 M treated with an activating agent such as ammonia or methylamine, or with proteinase
  • This activated form of ⁇ 2 M can bind to the endocytic receptor and the signalling receptor.
  • Antigen can be covalently conjugated to the f- ⁇ 2 M using a bifunctional crosslinking agent, as is well known in the art.
  • the antigen is crosslinked to the thioester on ⁇ 2 M, or to a site proximal to the thioester, so that the antigen can benefit from the protection provided by the ⁇ 2 M trap.
  • trapping of an antigen in a complex of intact ⁇ 2 M can protect the antigen from degradation by sterically hindering access of hydrolytic enzymes specific for the antigen. Protection by ⁇ 2 M is most efficient when the antigen in the complex is covalently associated with ⁇ 2 M via the thioester, or by conjugation to immediately adjacent or proximal residues.
  • the invention provides for targeting conjugation of antigen to the thioester or an immediately proximal residue.
  • the invention provides for crosslinking an antigen via the free thiol group (one per ⁇ 2 M subunit) that results from treatment of ⁇ 2 M with methylamine. Any bifunctional crosslinking agent known in the art can be used to form the conjugates of the invention.
  • the ⁇ 2 M can be a C-terminal fragment of ⁇ 2 M containing an oxidation sensitive/cis-dichlorodiamine platinum (cis-DDP) reactive site, but lacking the "bait" region and the thioester group. This fragment may be referred to as the receptor binding domain (RED) of ⁇ 2 M.
  • the C-terminal fragment can have a molecular weight of approximately 40 kDa.
  • a 40 kDa fragment of rat or human ⁇ 2 M can be prepared (e.g. , Enghild et al. (1989), BIOCHEMISTRY 28: 1406-1412; Gordon (1976), BIOCHEM. J.
  • the 40 kDa fragment appears to bind to both the endocytic receptor and the signalling receptor.
  • the fragment contains oxidation/cis-DDP-sensitive residues that appear to enhance binding to the endocytic ⁇ 2 M receptor.
  • the RBD is a good candidate for targeted antigen delivery, since antigen attached to this fragment may undergo endocytic processing more readily.
  • a smaller C-terminal fragment that lacks the oxidation sensitive/cis-DDP reactive site but that contains the receptor recognition site of ⁇ 2 M can be used.
  • the fragment may be referred to as the receptor binding fragment (RBF).
  • the fragment has an apparent molecular weight of approximately 20 kDa.
  • the fragment can be obtained from the limited proteolytic digest of ⁇ 2 M under acidic conditions (Enghild et al. (1989), BIOCHEMISTRY 28: 1406-1412; Van Leuven et al. (1986), J. BIOL. CHEM. 261:6933-6937; Sottrup-Jensen et al. (1986), FEBS LETT.
  • This fragment has been designated as the 20 kDa fragment, although the apparent molecular weight of the fragment from a natural protein can be closer to 30 kDa in reduced SDS-PAGE (Enghild et al., supra; see, e.g. , Salvesen et al. (1992), FEBS 313: 198-202).
  • this fragment can be produced recombinantly, as described in Salvesen et al., 1992, supra.
  • this fragment shows enhanced signalling activity, but apparently is not taken up by the endocytic receptor.
  • a recombinant 20 kDa fragment from rat ⁇ ,M (Salvesen et al. , 1992, supra) is as effective or more effective than f- ⁇ 2 M on a molar basis in activating cells.
  • This activation efficiency occurs despite the observation that the fragment binds to macrophages with about 100-fold lower affinity than f- ⁇ 2 M (Salvesen et al., supra).
  • the RBF demonstrates chemoattractant activity, as treatment of macrophages with this molecule induces polarization, which is the first step in chemoattraction.
  • a C-terminal fragment of ⁇ 2 M such as the 40 kDa fragment or the 20 kDa fragment
  • the recombinant fragment can be expressed in a glycosylated form, e.g. , by expression in a yeast, baculovirus, or mammalian expression system; or in a non-glycosylated form, e.g. , by expression in a bacterial expression system.
  • the fragment is expressed in a baculovirus expression system, which can provide for high yield of a glycosylated fragment, while avoiding the problem of endotoxin contamination that accompanies expression in bacterial systems, such as E. coli.
  • the invention contemplates identifying peptides that can direct binding to ⁇ M receptors and activation of APCs.
  • the invention further contemplates conjugating antigens to such peptides for modulating, and preferably increasing, the immune response to such antigens.
  • Such peptides can be produced recombinantly, or by chemical synthesis.
  • a 39 kDa receptor associated protein (RAP) (Williams et al. (1992), J. BIOL. CHEM. 267:9035-40) is capable of competing with all of these ligands, including ⁇ 2 M, although none of the ligands themselves compete with ⁇ 2 M. It is this receptor that is believed to mediate endocytosis.
  • the other receptor is believed to mediate signalling.
  • ⁇ 2 M binding of ⁇ 2 M to its receptor elicits intracellular signalling cascades, including an increase in intracellular Ca 2+ concentration; an increase in cyclic AMP; generation of inositol triphosphates and tetraphosphates (Uhing et al. (1991), BIOCHIM. BIOPHYS. ACTA 1093:115-120; Misra et al. (1993), BIOCHEM. J. 290:885-891); and activation of protein kinase C (data contained herein). These events are characteristic of G-protein coupled receptor signalling cascades.
  • cis-DDP treatment of f- ⁇ 2 M or the 40 kDa fragment of ⁇ 2 M reduces the binding affinity to macrophages.
  • the Kj for binding of f- ⁇ 2 M to murine macrophages increases from 0.5 nM to 11.0 nM upon treatment with cis- DDP; the K d for binding of the 40 kDa RBD increases from 5 nM to 50 nM.
  • the K d of the 20 kDa RBD is not affected by treatment with cis-DDP.
  • treatment of f- ⁇ 2 M and the 40 kDa RBD with cis-DDP has no effect on signalling ability.
  • the inhibitor of ligand binding to the endocytic receptor, receptor associated protein (RAP), abolishes f- ⁇ 2 M binding to the endocytic receptor, but failed either to elicit a calcium signal or to antagonize intracellular signals elicited by either f- ⁇ 2 M or RBF.
  • binding to the signalling receptor can occur independent of binding to the endocytic receptor.
  • the invention provides for specifically targeting the signalling receptor and activating macrophages or other APCs.
  • ⁇ 2 M-mediated signalling can include one or more of the following: activation of protein kinase C; phosphorylation of histones; and transport of diacylglycerol (DAG) analogs to the cell membrane.
  • DAG diacylglycerol
  • ⁇ 2 M, or a fragment thereof, that is capable of binding to a receptor is useful for immunomodulation whether or not the ⁇ 2 M forms a complex with an antigen.
  • the 20 kDa RBF targets the signalling receptor.
  • cis-DDP or oxidized f- ⁇ 2 M or the 40 kDa RBD specifically targets the signalling receptor, as treatment with cis-DDP or oxidation appears to significantly diminish binding to the endocytic receptor.
  • Oxidation can be effected by treatment with an oxidant, such as but not limited to peroxide, in particular hydrogen peroxide, hypochlorous acid, or chloramines, or with a free radical such as an oxygen radical.
  • an oxidant such as but not limited to peroxide, in particular hydrogen peroxide, hypochlorous acid, or chloramines
  • a free radical such as an oxygen radical.
  • specific targeting of the endocytic or signalling receptor can be accomplished by various strategies.
  • the relative cellular expression of one or the other receptor can be altered, e.g. , using anti-sense technology or specific cytokines, such as 7-interferon or other hormones that downregulate the endocytic receptor.
  • competitors for the endocytic receptor such as but not limited to RAP, can be provided.
  • Specifically targeting the signalling receptor without binding to the endocytic receptor, can promote signalling for a longer time period, and provide for a longer acting complex, as the RBF and cis-DDP treated or oxidized f- ⁇ 2 M or RBD are cleared more slowly.
  • a further advantage of the RBF is that its small size facilitates production, purification and administration.
  • the RBF is as potent or more potent than f- ⁇ 2 M for inducing signalling.
  • 40 nM of the 20 kDa RBF was found to be equivalent to 200 nM ⁇ 2 M in a signal cascade assay.
  • 2 ⁇ m doses of RBF stimulated such massive calcium fluxes that it became cytotoxic; an effect not seen with ⁇ m doses of ⁇ 2 M.
  • the invention further provides for immunostimulation without specifically targeting an antigen by providing ⁇ 2 M or fragments thereof that are targeted specifically to the signalling receptor and not to the endocytic receptor.
  • ⁇ 2 M or fragments thereof that are targeted specifically to the signalling receptor and not to the endocytic receptor.
  • f- ⁇ 2 M that has not been cis-DDP treated or oxidized can nevertheless enhance an immune response.
  • ⁇ 2 M or a fragment thereof targeted specifically to the signalling receptor can be administered to a subject in whom immunomodulation, in particular, immunostimulation, is desired.
  • such administration is concomitant with or in an admixture with an antigen or vaccine.
  • the signalling receptor targeted ⁇ 2 M or fragment thereof of the invention can be used in conjunction or synergy with additional immunomodulatory agents, such as but not limited to lymphokines (e.g. , interleukin (IL)-1, IL-2, IL-3, IL-4, IL-6, etc.), cytokines (e.g. , macrophage inflammatory proteins, interferons, tumor necrosis factor, colony stimulating factors, etc.), growth factors, and the like, to achieve greater immunomodulation, and particularly immunostimulation.
  • lymphokines e.g. , interleukin (IL)-1, IL-2, IL-3, IL-4, IL-6, etc.
  • cytokines e.g. , macrophage inflammatory proteins, interferons, tumor necrosis factor, colony stimulating factors, etc.
  • growth factors e.g. , and the like.
  • very high doses of the RBF can be used to selectively injure immune cells bearing the signalling ⁇ 2
  • Suitable antigens intended for the practice of the present invention may possess nucleophilic groups, as ⁇ 2 M exhibits a particular facility for the covalent attachment of nucleophilic moieties. They may also include other molecules for which a chemical crosslinker is available that allows attachment to engineered carboxyl terminus ⁇ -macroglobulin derivatives. Exemplary antigens may be selected from peptides, proteins, cytokines, growth factors, hormones, enzymes, toxins, nucleic acids, in particular, anti-sense RNA, as well as other drugs, oligonucleotides and carbohydrates.
  • the invention includes multiple active fragments of ⁇ 2 M associated in tandem relationship, such as carboxyl terminal moieties with cross-linking and/or receptor recognition sites associated therewith, disposed in direct connection with each other.
  • multiple antigens both identical and diverse may be associated with such corresponding multiple fragments to facilitate a polyvalent vaccine delivery construct.
  • polyvalent construct may be achieved with a single fragment to which is attached an appropriate polyvalent moiety. Both concepts and constructs are included within the scope of the present invention.
  • the present invention is predicated on the discovery that the formation of the complex or construct between the antigen and ⁇ 2 M or its active fragments results in significant improvement in antigen presentation in vitro and more importantly, a dramatic increase in immune activity as measured by the development of antibodies to the antigen stimulus in vivo.
  • This significant increase in activity is one aspect of the invention, the other being the ability of the complex or constructs of the present invention to be presented without use or inclusion of an adjuvant.
  • improvement in immune response is achieved over like formulations with adjuvant.
  • the ability to delete adjuvant from the formulations prepared in the present invention represents a further efficiency and likewise eliminates the potential for deleterious reactions and delays in uptake that have been experienced with adjuvant-containing formulations.
  • the present invention further extends to methods for the preparation of antibodies to such antigens, including where desired, the preparation of monoclonal and chimeric antibodies based upon those raised against the complexes and/or constructs of the present invention, as well as "primed" lymphocytes specific for the antigens.
  • the present invention can be used as a means for stimulating antigenicity and immune-competence in instances where the particular antigen has previously failed to elicit immunologically or therapeutically significant arousal and activity in the host.
  • the present invention is primarily directed to the administration of antigens recognized by the macrophage in view of the existence on the macrophage of receptors for ⁇ 2 -macroglobulin.
  • other APCs may possess receptors for ⁇ 2 M and the present invention is accordingly intended to extend to the presentation of antigen to these other APCs.
  • a "modified immune response” By combining the antigen with ⁇ 2 -macroglobulin or an active fragment thereof to form the complex or construct of the invention and using the complex or construct as the immunogen, a "modified immune response" can be achieved.
  • the immunogen can be used to raise antibodies which are specific to epitopes either weakly or not previously recognized.
  • the modified immune response may involve non-antibody immune system components, e.g. , T- lymphocytes, which may recognize an epitope not previously presented or recognized.
  • the "modified immune response” is largely directed to the previously weakly or unrecognized epitope on the antigen treated, or epitopes requiring adjuvant or use of large amounts of antigen, all as described herein.
  • the preferred embodiments of the invention utilize the complex or construct as the immunogen, and seek to raise or react said complex or construct with antibodies which also recognize the same or a different epitope which is present on the molecule.
  • the so-called modified immune response therefore involves the generation of antibodies which are not otherwise efficiently formed or observed in vitro or in vivo. It may also involve generation of antibodies or stimulation of lymphocytes that would not otherwise occur in the absence of noxious adjuvants not approved for human usage.
  • such antibodies can be generated by immunization in the absence of adjuvant.
  • the immunogen can be used to inoculate a mammal to raise antibodies to the newly recognizable epitope, and to produce antiserum or vaccine preparations, and the like.
  • antibody molecules can be cleaved to form antibody fragments, which can be recombined in vitro to form chimeric antibodies which recognize or bind to newly recognizable epitopes on the antigen.
  • modified immune response is not limited to a conventional immune response, or to increases or decreases in the extent or severity thereof.
  • both positive and negative regulation of the antigenicity of epitopes can be achieved.
  • by rendering epitopes recognized, or recognizable antibodies can be raised to recognize and bind to the antigen.
  • Enhanced antigenicity and the ability to raise antibodies to otherwise weak, scarce or ineffective epitopes finds great utility not only, for example, in vaccine applications in animals, including humans, but also in producing antibodies which can be used as reagents for, among other uses, binding, identifying, characterizing and precipitating epitopes and antigens, such as the production of antibodies against scarce antigens for research purposes.
  • imunodominancy of particular epitopes on a molecule may be modified.
  • Certain antigens containing more than one epitope have characteristic immune responses based upon the dominance of one epitope over the other(s).
  • This aspect of the invention enhances the recognition of the subordinate epitope(s) by either preparing and administering a complex or construct of the invention to potentiate the recognition and activation of the subordinate epitope(s), or by preparing and administering a complex or construct bearing an agent that will be recognized by the dominant epitope and suppress the recognition of the same by antigen.
  • a multivalent construct could be prepared that bears both the antigen for the subordinate epitope and the inhibitor or down regulator for the immunodominant epitope. This allows other silent or recessive epitope(s) to be expressed.
  • the present constructs may be prepared recombinantly. This, again, can be undertaken by the incorporation of the antigen within the carboxyl terminal fragment, inserting the resulting construct into an expression vector, transfecting the vector into a host and allowing the host to express the construct with the antigen added thereto.
  • An expression vector may be prepared that codes for expression of a mutein of the molecule of interest, which vector may be transfected into a host cell, such that the cell is caused to express the mutein.
  • the mutein may comprise the original amino acid sequence of the construct, conserved variants thereof or portions thereof, substituted with or having inserted therein codons for expression of the antigen in the primary sequence of the mutein, in the instance where the antigen is capable of such replication.
  • a further embodiment may for example, take advantage of APC receptor proteins which recognize and bind to polypeptide molecules present on the antigen or in the complex or construct of the invention.
  • Antigen uptake by the APCs can occur via nonspecific mechanisms, and may be followed by display of the antigen in association with MHC on the cell surface.
  • K G G G C G G E G G G G Y G G G (SEQ ID NO: l) 5 10 15 can be prepared synthetically.
  • Lys ! can provide a side for crosslinking to Glx of the ⁇ 2 M thioester; Cys 5 can provide a site for labelling with
  • Glu g provides a cleavage site for V8 protease [MW 28 kilo-
  • Tyr 13 can provide a site for 25 I-labelling.
  • a complex of this peptide and ⁇ 2 M can be formed by PPE activation of ⁇ M in the presence of peptide. This complex can then be treated with V8 protease, and the amount of ,25 I released from the complex measured to determine whether the peptide is protected from V8 protease.
  • the 14 C provides a control for covalent crosslinking to ⁇ 2 M. If this model peptide is protected once bound to ⁇ 2 M, it follows that ⁇ 2 M can protect peptides in general.
  • the biological processes within the APCs can be controlled to enable one to qualitate or quantitate the binding of the complex or construct.
  • the incubation time and temperature can be adjusted to achieve complete internalization by APCs, complete binding of the complex or construct, and like parameters.
  • an appropriate temperature e.g., about 4°C
  • an appropriate time period e.g., about one hour
  • binding of the complex or construct to APC cell surfaces can be quantitated, since internalization can be effectively decreased or shut down.
  • APC/complex or construct incubation temperature and/or time period e.g. , up to about 37°C for about one hour, internalization can be evaluated.
  • the antibodies described herein are typically those which recognize the epitopes on the antigens which are made recognizable, enhanced or suppressed as described above. By injecting this type of antigen into a mammal, such as through a hyperimmunization protocol, modulated antibody responses or CTL responses to the epitopes can be achieved.
  • the antibodies which are disclosed herein may be polyclonal, monoclonal or chimeric antibodies, and may be raised to recognize the desired epitope and used in a variety of diagnostic, therapeutic and research applications.
  • the antibodies can be used to screen expression libraries to ultimately obtain the gene that encodes proteins bearing the epitope evaluated.
  • antibodies that recognize the antigen presented can be employed or measured in intact animals to better elucidate the biological role that the protein plays, or to assess the state of immune response or immunologic memory more effectively.
  • the present invention also includes the immunogens which are produced and used as described herein in form.
  • the preferred immunogen is an antigen prepared in a complex or construct of the invention, which has at least one epitope.
  • the immunogen has modified antigenicity due to the presence of, reaction with or linkage to the ⁇ 2 M molecule or construct.
  • the immunogen induces the formation or proliferation of T-cells of antibodies which recognize the protein in its modified form or in its non-modified form.
  • the present invention provides for formation of covalent complexes of antigen and ⁇ 2 M or C-terminal fragments thereof.
  • covalent complexes are advantageously formed by reaction of a nucleophile on the antigen, such as an amine functional group (e.g., the e-amino group of lysine, or a ⁇ -amino group of a peptide) or hydroxyl, with the thioester of ⁇ 2 M during activation of ⁇ 2 M with protease.
  • a nucleophile on the antigen such as an amine functional group (e.g., the e-amino group of lysine, or a ⁇ -amino group of a peptide) or hydroxyl
  • the invention contemplates covalently coupling an antigen to ⁇ 2 M or fragments thereof using any bifunctional crosslinking agent known in the art.
  • the antigen used in an immunogenic complex of the invention is a synthetic HIV peptide, e.g. , as described in Hart et al. ((1991), PROC. NATL. ACAD. SCI. U.S.A. 88:9448-52).
  • Such synthetic peptides combine neutralizing B-cell sites from the third variable region (V3) of the HIV envelope peptide gpl20, with the gpl20 T-helper epitope T-l.
  • T1-SP10 synthetic peptides
  • the peptide Tl-SPIOMN(A) (MW 4771), which has the following amino acid sequence: KQIINMWQEVGKAMYACTRPNYNKRKRIHIGPGRAFYTTK (SEQ ID NO:2), can be complexed with ⁇ 2 M, or a C-terminal fragment thereof, either via the endogenous ⁇ 2 M thioester or using a bifunctional crosslinker, such as the homo-bifunctional sulfhydryl reactive crosslinker bis-moleimidohexane (BMH) to methamine (ma)-treated ⁇ 2 M, e.g.
  • BMH homo-bifunctional sulfhydryl reactive crosslinker bis-moleimidohexane
  • the present invention also contemplates diagnostic and therapeutic applications for these agents. Accordingly, the antigens or antibodies thereto may be prepared for use in a variety of these methods.
  • any of these agents may be labeled or unlabeled as appropriate.
  • the labelled component is the antibody, but it is possible to label the antigen or the ⁇ 2 M component, MHC or APCs as well.
  • a radioimmunoassay may be conducted, using for example, an antibody or ligand, that may either be labeled or unlabeled. Labelling may be accomplished, e.g., by radioactive addition, reduction with sodium borohydride or radioiodination.
  • Labels most commonly employed are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light and others.
  • Suitable radioactive elements may be selected for the group consisting of 3 H, 14 C, 32 P, 33 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, «°Y, 125 I, 131 I, and 186 Re.
  • known currently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, thermometric, amperometric or gasometric techniques known in the art.
  • the enzyme may be conjugated to the antigens or antibodies, their binding partners or carrier molecules, by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Also, and in a preferred embodiment of the present invention, the enzymes themselves may be modified into advanced glycosylation endproducts by reaction with sugars as set forth herein.
  • a control quantity of a binding partner to a antigen complex/construct may be prepared and optionally labeled, such as with an enzyme, a compound that fluoresces and/or a radioactive element, and may then be introduced into a tissue or fluid sample taken from a mammal in order to assess, e.g., the amount of antigen present therein.
  • the labeled material or its binding partner(s) After the labeled material or its binding partner(s) has had an opportunity to react with the sample, the resulting complex may be examined by known techniques, which vary according to the nature of the label attached. In this manner the antigen receptor, the activity and effect of MHC, or the epitope recognized by the antibody can be evaluated.
  • the antigen complex/construct forms complexes with one or more binding partners and one member may be labeled with a detectable label.
  • a complex has formed and, if desired, the amount thereof, can be determined by the detection methods described herein.
  • One preferred diagnostic method included herein involves the determination of T- lymphocyte levels, function or activity in a sample taken from a mammal.
  • the immunogen comprising the present complex/construct is incubated with APCs, after which T-lymphocytes taken from the mammal are added.
  • the level, function or activity of the T-lymphocytes taken from the mammal can then be compared to a standard.
  • the APCs can be associated with a solid support.
  • the immunogen is combined with APCs at a temperature which is effective to cause binding between the APCs and the immunogen. This can be accomplished without allowing substantial internalization by the APCs. In this manner, antigen binding to said APCs can be evaluated. Also, by increasing the temperature, APC internalization of antigen and subsequent cell metabolic processes can be evaluated.
  • Therapeutic treatments and diagnostic methods can be performed using any or all of the various components and processes described herein.
  • an isolated protein can be derived from the tumor, abnormal cells or infectious organism, and this protein can be used as an antigen and prepared in a complex or construct.
  • Antibodies to this protein can be elicited using the methods for enhanced antigen presentation disclosed herein and used to identify, characterize, bind, inhibit or inactivate, as desired, previously unknown or ineffective epitopes on the tumor, abnormal cell, bacterial or viral protein. This information, in turn, is useful for developing drugs which combat such afflictions, such as agonists, antagonists and the like.
  • the antibodies described above can be raised to have direct diagnostic or therapeutic utility, particularly in oncologic, autoimmune and infectious disease treatments.
  • a preferred use for the antigen complex/constructs described herein is in the form of a vaccine which can be used to immunize mammalian patients in need of such treatment.
  • a vaccine which can be used to immunize mammalian patients in need of such treatment.
  • antibodies can be raised to the particular immunogen and immunogen-specific lymphocytes can be primed and activated, which are effective for treating disease or preventing the development or spread thereof.
  • the invention provides a vaccine against HIV.
  • the preferred non-cellular components which recognize antigen and which are used to characterize epitopes presented in accordance with the invention include the antibodies raised to an antigen which are not normally elicited in the absence of the methods described herein. Also, as noted above, the most preferred antibodies are raised to antigen in the complex/construct, but recognize the non- modified molecule.
  • Hen egg white lysozyme (HEL) was purchased from Boehringer Mannheim (Indianapolis, IN). ⁇ 2 M was purified as previously described (22, 23), except that buffers made from pyrogen-free sterile water (Abbott Laboratories, Chicago, IL) were used for extensive washing and elution. Fractions were analyzed by pore limit gel electrophoresis in a tris/boric acid/EDTA (TBE) buffered system [(24), modified from (25)], and those fractions containing any trace amounts of "fast” form were discarded, resulting in material that was >98% native, as determined by DTNB titration (20, 24).
  • TBE tris/boric acid/EDTA
  • Porcine pancreatic elastase (PPE) of the higher purity grade was purchased from Sigma (St. Louis, MO). Human neutrophil elastase was the gift of Drs. James Travis and Wieslaw Watorek, University of Georgia, Athens, GA. Carrier-free Na ,2 T and
  • HEL incorporation Characterization of HEL incorporation.
  • the binding of radiolabelled HEL to ⁇ 2 M was analyzed by systematically varying conditions as previously described for insulin (20). Incubation products were analyzed by gel electrophoresis and autoradiography using native 4-20% TBE gels (24) and denaturing 5-15% polyacrylamide gradient gels in an ammediol-buffered SDS system (29). Radioactive bands were excised and counted after autoradiography.
  • HEL- ⁇ 2 M-PPE complexes were prepared in 20 mM HEPES, 150 mM NaCl, pH 7.4 (HEPES buffer) by incubating 1.4 mM ⁇ 2 M with a two-fold molar excess of PPE for 15 min at room temperature in the presence of a 100-fold molar excess of HEL.
  • ⁇ 2 M-PPE complexes were formed in similar incubations omitting the HEL.
  • Ag preparations were concentrated in sterilized CentriprepTM-30 concentrators (Amicon, Beverly, MA), and filtered through 0.22 mm filters (Millipore, Bedford, MA). Ag dilutions were prepared immediately before use in 96- well polypropylene plates (Costar, Cambridge, MA).
  • Peptone-induced peritoneal macrophages were harvested from female CBA/J mice (Jackson Laboratories, Bar Harbor, ME) and allowed to adhere to 96-well tissue culture plates (Costar, Cambridge, MA) for 2 h before use (32).
  • the 3A9 T-cell hybridoma line specific for residues 52-61 of HEL (33, 34), and the IL-2-dependent CTL line (CTLL-2) (35) were kindly provided by Peter Cresswell, Yale University, New Haven, CT.
  • Both cell lines were grown in RPMI 1640 (GIBCO, Gaithersburg, MD), supplemented with 10% heat-inactivated newborn calf serum (Hyclone Laboratories, Inc., Logan, UT), 1 % (v/v) 1-glutamine (200 mM, GIBCO), and 0.5% (v/v) gentamicin solution (10 mg/ml, GIBCO).
  • the CTLL cell media was further supplemented with 20% rat spleen cell Con A supernatant, prepared as previously described (36).
  • the Coomassie-stained SDS gel (Panel A) reveals a typical fragmentation pattern for ⁇ -macroglobulins (38).
  • the 720 kDa protein dissociates into disulfide-linked dimers (360 kDa).
  • ⁇ M migrates as its constituent 180 kDa subunits. Denaturation and boiling promotes autolytic cleavage at the thiolesters, resulting in the characteristic 120 kDa and 60 kDa bands (39).
  • Proteinase-treated ⁇ 2 M migrates as a doublet (-90 kDa) (40) when examined by reducing SDS-PAGE (Panel A, lane c); the binding of HEL to the thiolester-containing fragment resulted in the appearance of a new band whose position is marked by the arrow (Panel A, lane b). All of the bound 125 I-HEL migrated at this position.
  • HEL- ⁇ 2 M-PPE complexes for use in the cellular assays, dose response studies were conducted to determine the stoichiometry of the bound components. Under saturating conditions, about one mole HEL (Figure 2B) and one mole of PPE (not shown) were bound to each mole of ⁇ 2 M. About 85% of HEL binding was covalent and resistant to reduction, again supporting the existence of a 7-glutamyl linkage. The use of human neutrophil elastase as the activating proteinase also resulted in a maximum of one mole HEL bound per mole ⁇ 2 M, but in this case, binding was 100% covalent.
  • Small primary amines such as methylamine
  • Methylamine-treated ⁇ 2 M interacts with the receptor in a manner indistinguishable from ⁇ 2 M-proteinase complexes (23).
  • it can be used either as a specific competitor for ⁇ 2 M-proteinase complexes or as a method to investigate the effects of ⁇ 2 M receptor-binding in a context free of proteinases.
  • macrophages were pulsed with log-dilutions of Ag for varying amounts of time ranging from 15 min to 3 h.
  • the resulting dose response curves for different macrophage-Ag incubation times were analyzed to determine the minimal concentration of Ag required to enable activation of T-hybridomas by the macrophages.
  • DISCUSSION ⁇ 2 M displays an unique capacity, upon proteolytic activation, for rapidly forming essentially irreversible complexes with proteins possessing dissimilar structures.
  • the inter-related mechanisms of trapping and of covalent crosslinking allow it to complex with a wide variety of proteinases and other proteins, including large proteinases up to 90-110 kDa (5, 12, 44).
  • the capture of nonproteolytic proteins such as insulin is very efficient, occurring readily at physiologic concentrations of reactants (20).
  • the resulting complexes bind the ⁇ 2 M receptor and are effectively internalized.
  • ⁇ 2 M has been shown to covalently bind proteins as diverse as lysozyme, aprotinin, inactive forms of trypsin (7), insulin (8, 20), luteinizing hormone and possibly streptokinase (unpublished observations). All these appear to interact with ⁇ 2 M by nucleophilic attack at the thiolester Glx, as was originally described for the covalent attachment of ly sine-containing proteinases (7). This is an efficient process since crosslinking occurs during a reactive intermediate state that decays in seconds (8, 20).
  • Growth factors that appear to bind ⁇ 2 M include platelet-derived growth factor, transforming growth factor-b, IL-lb, IL-6, and basic fibroblast growth factor (Reviewed in (21)). The association of these different proteins attests to the versatility of the ⁇ 2 M "trap.”
  • proteins may be captured by additional mechanisms.
  • Basic proteins can adhere to ⁇ 2 M in a manner distinct from trapping (18).
  • Additional forms of covalent bonds are also possible.
  • the free thiols released by thiolester decay have been suggested as potential sites for thiol-disulfide interchange (19).
  • ⁇ 2 M may act to target potential Ag to macrophages within areas of inflammation. That is, by forming covalent complexes with differing proteins, ⁇ 2 M may be acting as a carrier or adaptor molecule that mediates rapid internalization of these proteins. The receptor recognition site at the C-terminus of each ⁇ M subunit is masked until after proteolytic activation (13), during which complex formation with a variety of proteins can occur. Because complex formation depends upon proteolytic activity, which is usually tightly controlled in vivo, the proteins carried by ⁇ 2 M into the macrophages would be limited to those that are present in areas of enhanced proteolytic activity, as might be expected in areas of inflammation.
  • ⁇ 2 M can be activated by completely unrelated proteinases, both pathogen- and host-derived proteinases could serve this purpose.
  • This proteolytic activity may also serve to cleave large Ag into smaller fragments that can enter the trap, while the covalent binding mechanism ensures capture of even the smaller peptides.
  • Human ⁇ 2 M is actively secreted by fibroblasts (62) and macrophages (63), which ' also secrete increased amounts of proteinases under inflammatory conditions (64).
  • the native form is present in plasma at levels greater than 3 ⁇ M (18, 48), as well as in extravascular fluids (48). Changes in vascular permeability may result in leakage of serum proteins into sites of inflammation.
  • Bovine ⁇ 2 M is present in serum typically used to supplement culture media. Thus, native ⁇ 2 M would likely be present in areas of inflammation, as well as in many in vitro presentation systems. There are no known examples of complete ⁇ 2 M deficiency in mammals (57), lending further support to the importance of this conserved family of thiolester-containing proteins.
  • Example 1 demonstrates the effects of complexing ⁇ 2 M with hen egg lysozyme (HEL) upon Ag uptake and presentation to HEL-specific murine T-cell hybridomas (see also 20).
  • HEL hen egg lysozyme
  • HEL was purchased from Boehringer Mannheim (Indianapolis, IN). H ⁇ 2 M was purified as previously described, employing extensive dialysis against dH 2 O to precipitate out the receptor-recognized f- ⁇ 2 M conformational forms [Example 1, (22,23,68)]. The purified H ⁇ 2 M was >98% in the native s- ⁇ 2 M conformation, as determined by 5,5'-dithiobis-(2-nitrobenzoic acid) titration of thiols released following proteolysis (20). R ⁇ ,M was purified by similar methods from titrated rabbit plasma (Pel-Freeze, Rogers, AR).
  • ⁇ iM was not stable to freeze-thaw cycles, and was stored at 0°C.
  • PPE of the highest available purity grade, low-endotoxin BSA, affinity-purified alkaline phosphatase-conjugated goat anti-[rabbit IgG], and p-nitrophenyl phosphate tablets were purchased from Sigma (St. Louis, MO). Endotoxin standards and Pyrotell Limulus amebocyte lysate reagents were obtained from Associates of Cape Cod (Woods Hole, MA).
  • ⁇ 2 M-HEL-PPE complexes were prepared as described in Example 1 for ⁇ 2 M in 20 mM HEPES, 150 mM NaCl, pH 7.4 (HEPES buffer) (see 68). Incorporation ratios of 0.8 moles HEL/mole H ⁇ 2 M and 0.14-0.32 moles HEL/mole R ⁇ ,M were achieved. H ⁇ 2 M-methylamine was formed as previously described (31). both methylamine treatment and PPE treatment resulted in the complete transformation of s- ⁇ 2 M to f- ⁇ 2 M. All sterile Ag preparations contained less than 100 pg endotoxin/ml as determined by Limulus amebocyte lysate clotting times.
  • NZW rabbits Injection of Ag into NZW rabbits.
  • Pathogen-free NZW rabbits were purchased from Robinson's Services (Winston-Salem, NC) and housed in specific pathogen- free facilities at the Duke Vivarium.
  • Twenty-two rabbits were injected with either HEL alone, H ⁇ 2 M-HEL-PPE complexes, HEL mixed with ⁇ 2 M-methylamine (f- ⁇ 2 M), HEL mixed with s- ⁇ 2 M, or R ⁇ ,M-HEL-PPE complexes.
  • six rabbits were injected with HEL emulsified in CFA.
  • PPE that was previously inhibited with 3,4-dichloroisocoumarin (100 ⁇ M) was injected alone or mixed with BSA into four more rabbits to deliver the same amount of PPE as received by rabbits injected with H ⁇ 2 M-HEL-PPE complexes. All rabbits were bled before injection at Week 0 to obtain preimmune sera, which were used to verify the absence of preexisting antibodies directed against HEL, H ⁇ 2 M, R ⁇ 2 M, or PPE. Blood was allowed to clot at 37°C, then spun down, and the sera were stored at -20°C until shortly before use.
  • Antibody capture ELISAs Costar 96-well RIA/EIA plates were incubated overnight at 4°C with 8 ⁇ g/nl of HEL, PPE, BSA, R ⁇ -M, or H ⁇ 2 M-methylamine, in PBS pH 7.3. Coated plates were washed and blocked with PBS containing 5 % Carnation non-fat dry milk and 0.05% Tween 80 (blocking buffer) for 2 h at room temperature. Plates were then incubated with 100 ⁇ l of sera diluted in blocking buffer for 1 h at room temperature, followed by 100 ⁇ l (1:2000 dilution) of alkaline phosphatase-coupled anti-[rabbit IgG].
  • the substrate p-nitrophenyl phosphate (1 mg/ml in 0.1 M glycine, 1 mM MgCl 2 , 1 mM ZnCl 2 , pH 10.4) was added. Alkaline phosphatase activity was followed kinetically at 37 °C using a THERMOmaxTM microplate reader (Molecular Devices, Menlo Park, CA).
  • HEL elicits higher IgG titers after multiple injections. End titers for each of the protein components that was injected — HEL, H ⁇ 2 M, PPE, or BSA -- were calculated for each rabbit at Week 3, after injections with the different Ag preparations (125 ⁇ g HEL equiv.) at Week 0 and 2 (Table I). Titers for the four rabbits that received either free HEL or HEL mixed with s- ⁇ 2 M were indistinguishable. The rabbits that received H ⁇ 2 M-complexed HEL displayed consistently higher anti-HEL titers, as did the rabbits that received CFA (Table I). Titers were also calculated for an arbitrarily defined endpoint, 5mOD/min, with equivalent results (not shown).
  • Ra j M-HEL complexes also elicit efficient primary IgG responses.
  • the control experiments described above establish that Ag complexed directly with ⁇ 2 M demonstrates the greatest enhancement in immunogenicity. However, it is possible that the Ag are carried into cells through another uptake mechanism after forming immune complexes with anti-H ⁇ 2 M antibodies. Although there were no detectable anti-H ⁇ 2 M titers in the preimmune sera (Table I, legend), a particularly rapid immune response directed against H ⁇ 2 M may have resulted in some immune complex formation during the second injection at Week 2. Thus, we developed a purification procedure for a rabbit homologue of H ⁇ 2 M (R ⁇ jM) and studied the development of a primary IgG response following a single injection equivalent to 40 ⁇ g of HEL.
  • ⁇ 2 M Although its unique capacity for inhibiting a wide range of unrelated proteinases has been a major focus of study, there are indications that the reaction of ⁇ 2 M with proteinases may subserve broader roles. In addition to binding potential Ag and delivering them to macrophages, ⁇ 2 M has been reported to bind many growth regulating substances [Reviewed in 90-92]. Although growth factor binding may simply reflect its general ability to interact with many different proteins, ⁇ 2 M could potentially alter their biodistributions and activities. In addition, we have recently shown that the binding of f- ⁇ 2 M to its receptors on macrophages results in several intracellular signalling events (93). These observations suggest that ⁇ 2 M may function as a sensor for situations involving increased proteolysis.
  • Receptor ligation of f- ⁇ 2 M initiates macrophage second messenger responses, including effects on inositol trisphosphates, intracellular Ca 2+ , cAMP, prostaglandins and protein kinase C (93, 95, Example 4).
  • F- ⁇ 2 M has been described as chemotactic for macrophages (96), eliciting a "spread out" macrophage morphology (52), and an intracellular signalling pattern reminiscent of chemoattractants (93, 97).
  • the addition of higher doses of f- ⁇ 2 M in vivo may have affected leukocyte mobility.
  • Other effects of ⁇ 2 M-elicited second messengers may include APC activation and regulation of costimulatory signals and molecules.
  • ⁇ 2 M a potential role for ⁇ 2 M in Ag processing is shown in Figure 11.
  • Areas of inflammation contain high levels of s- ⁇ 2 M both from increased plasma extravasation and from increased local synthesis (99).
  • fibroblasts and macrophages 62, 63). These sites would also possess enriched levels of inflammatory proteinases and foreign proteins.
  • S- ⁇ 2 M which is not receptor-recognized, would react with the proteinases in the presence of these antigenic proteins, resulting in receptor-recognized f- ⁇ 2 M forms carrying a mixture of proteins.
  • the bound proteins Upon endocytosis into macrophages or other APCs that express the receptor, the bound proteins would be processed and presented for surveillance by T-cells.
  • the cells that express the ⁇ 2 M receptor are monocytes/macrophages (41) fibroblasts (100, 101), and dermal dendritic cells (102).
  • monocytes/macrophages 41) fibroblasts (100, 101), and dermal dendritic cells (102).
  • monocytes/macrophages 41) fibroblasts (100, 101)
  • dermal dendritic cells (102).
  • these nonphagocytic APCs may also express the ⁇ 2 M receptor.
  • ⁇ M complexes formed during proteolytic activation of ⁇ M undergo receptor-mediated endocytosis into macrophages, dendritic cells, and other cells bearing the ⁇ 2 M receptor, resulting in enhanced cellular humoral immune responses in vitro and in vivo.
  • ligation of the ⁇ 2 M receptor itself may also affect the immune response in vivo.
  • Species specific or autologous ⁇ 2 M may prove to be an effective, nonirritating alternative to traditional adjuvants.
  • the ⁇ 2 M trap has been considered to prevent diffusion of fairly large molecules, e.g. , proteinases. This conclusion results from the observation that anything larger than about 10,000 to 15,000 molecular weight cannot access the active site of a trapped proteinase which is believed to be deeply buried within the ⁇ 2 M molecule. As discussed in the Background of the Invention, this steric inhibition that results from trapping is the mechanism by which ⁇ 2 M inhibits proteinases.
  • Nonproteolytic proteins up to about 22,000 to 23,000 molecular weight can fall out of the closed trap if they are not covalently bound.
  • the present Example demonstrates that ⁇ 2 M can protect bound antigens. Insulin was labelled with 125 I. The labelled insulin (1.4 ⁇ m) was complexed with ⁇ 2 M (1.4 ⁇ m) by treatment with elastase (2.5 ⁇ m) for 10 min. Then the remaining elastase was irreversibly inhibited by treatment with dichloroisocoumarin (DO), an active site directed inhibitor. These reaction solutions were termed ⁇ 2 M-insulin complexes.
  • DO dichloroisocoumarin
  • reaction solutions were prepared: (A) 125 I insulin alone; (B) a preformed ⁇ 2 M-insulin complex, incubated for 10 min with elastase; (C) a preformed ⁇ 2 M-insulin complex treated for 2 min with elastase; (D) ⁇ 2 M and insulin treated with elastase for 10 min (i.e.
  • FIG 12 is an autoradiogram of a non-reduced SDS-PAGE gel in which the lanes correspond to the reaction solutions described above.
  • ⁇ 2 M 25 I-insulin-labelled ⁇ 2 M is found in lanes B, C, D, E and F.
  • Lane A contains non-degraded insulin
  • lane G contains both non- degraded and degraded insulin.
  • the insulin complexed with ⁇ 2 M was slightly degraded with 10 min exposure to elastase (lane B), but not with 2 min exposure (lane C).
  • insulin reacted with elastase prior to complex formation was significantly degraded (lanes E and F).
  • a small amount of degradation was found when insulin was reacted with elastase and ⁇ 2 M at the same time (lane D).
  • This Example demonstrates the ability of the RBF of ⁇ 2 M to activate the ⁇ 2 M signalling receptor.
  • Evidence for signalling receptor activation included increase in intracellular calcium concentration; cholera toxin-induced ADP-ribosylation of the 43 kDa G-protein; and protein kinase C (PKC) activation, as measured by histone phosphorylation and movement of tritium labelled phorbol dibutyrate [ 3 H]- PDBu, a DAG analog, to the cell membrane.
  • PKC Measurements Thioglycollate-elicited murine peritoneal macrophages were plated at a density of 6x10° cells in 35 mm Petri dishes containing RPMI 1640 medium. After 16 to 18 h, the medium was aspirated and the cells washed three times in HHBSS. Native ⁇ jl 3 , ⁇ ,I 3 -methylamine or buffer each containing 75 ⁇ M Ca 2+ was then added. The cells were incubated for 20 min at 37°C in a humidified incubator under 5% CO 2 .
  • the reaction was terminated by aspirating the buffer and addition of a volume of buffer containing 20 mM HEPES, 10 mM EGTA, 2 mM EDTA, 5 mM DTT, 20 ⁇ g/ml leupeptin, 1 mM PMSF, 0.25M sucrose, 1 % nonidet P40, pH 7.4.
  • the cells were scraped, transferred to tubes and sonicated on ice (five 10 s bursts with 30 s intervals). The sonicate was left on ice for 20 min and then centrifuged at 100,000 x g for 60 min at 4°C.
  • the supernatant was then applied to a DE 52 column pre-equilibrated with 20 mM HEPES, 10 mM EGTA, 2 mM EDTA, 5 mM DTT, 1 mM PMSF, 20 ⁇ g/ml leupeptin, pH 7.4.
  • the column was eluted with the same buffer containing 300 mM NaCl at a flow rate of 8 ml/h.
  • the PKC activity was determined by histone Ills phosphorylation using [ 32 P]- ⁇ -ATP (Sahyoun, N.E. et al. , 1989, J. BIOL. CHEM. 264: 1062-1067) and by [ 3 H]PDBu binding (Misra and Sahyoun, 1987, BIOCHEM. BIOPHYS. RES. COMM. 145:760-767).
  • the 20 kDa RBF was obtained as described in Salvesen et al. ((1992), FEBS 313: 198-202). This is the C-terminal RBF of rat ⁇ ,M expressed in E. coli.
  • the purified protein was passed serially through three 2 ml Detoxi-Gel columns (Pierce Immunochemicals) with regeneration of the columns between runs, until endotoxin levels were ⁇ 10 pg/ml in the 1.3 ⁇ m stock solution as determined by Pyrotell Limulus amebocyte clotting times (Association of Cape Cod, Woods Hole, MA) using a THERMOmax microplate reader (Molecular Devices, Menlo Park, CA) (see Example 1, supra).
  • the PKC inhibitor staurosporin inhibits this increase. Increased histone phosphorylation, that could be inhibited with staurosporin, was observed upon activation with ⁇ 2 M-methylamine, rat ⁇ j-inhibitbr 3 (a monomeric ⁇ 2 M homolog), and RBF. Notably, RBF present at a 5-fold lower concentration than either the tetrameric or monomeric proteins (40 nM for RBF versus 200 nM for the proteins) was nevertheless comparably effective at enhancing PKC activation.
  • Movement of the diacylglycerol (DAG) [ 3 H]-PDBu to cell membranes also demonstrates PKC activation.
  • Increased binding of the labelled DAG analog in TG-elicited murine macrophages was evaluated after treatment with buffer, slow and fast ⁇ 2 M, slow and fast ⁇ ,-inhibitor 3 , and the 20 kDa RBF C-terminal fragment from rat ⁇ jM.
  • the slow form macroglobulins do not bind receptor, so these samples serve as negative controls.
  • the fast form of both intact ligands was formed by treatment with methylamine.
  • the amount of label moved to the membrane was about the same with 40 nM of the RBF and 200 nM of either intact ligand.
  • the RBF also stimulated increased cholera toxin-induced ADP-ribosylation of the 43 kDa G-protein found in macrophages when compared to buffer (data not shown).
  • the 20 kDa receptor binding fragment binds to a signalling receptor for ⁇ 2 M on macrophages that is novel and distinct from the previously described endocytic ⁇ 2 M receptor, although the two receptor proteins may interact. Binding to this second receptor stimulates cellular activation. From the range of assays performed, it appears that all of the signal events resulting from ⁇ 2 M binding to APCs occur upon binding the 20 kDa fragment as well.
  • the 20 kDa fragment was found to be as potent on a molar basis as intact f- ⁇ 2 M at stimulating increased concentration of [Ca + ]*, and about 5-fold more potent on a molar basis at PKC activation than either f- ⁇ 2 M and f- ⁇ r inhibitor ⁇
  • T cell growth factor parameters of production and a quantitative microassay for activity.
  • Murine T cell proliferation can be specifically augmented by macrophages fed with specific antigen: ⁇ 2 -macroglobulin conjugate. Biochem. Biophys. Res. Commun. 146:23. 54. Osada, T., N. Noro, Y. Kuroda, and A. Ikai. 1988. Antibodies against viral proteins can be produced effectively in response to the increased uptake of alpha 2 -macroglobulin: viral protein conjugate by macrophages. Biochem. Biophys. Res. Commun. 150:883.
  • LRP low-density-lipoprotein receptor-related protein
  • MOLECULE TYPE peptide
  • HYPOTHETICAL YES
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • HIV HIV CD8+ cytotoxic T cells in vivo by carrier-free HIV synthetic peptides
  • Lys Gin lie lie Asn Met Trp Gin Glu Val Gly Lys Ala Met Tyr Ala 1 5 10 15

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Abstract

Est décrit un procédé pour stimuler la présentation de l'antigène, dans lequel un antigène est modifié par incorporation ou couplage avec une α2-macroglobuline (α2M), son produit de recombinaison, ou bien son fragment réactif. L'antigène ainsi préparé est capable de déclencher une réponse immunitaire améliorée de la part d'épitopes silencieux, rares ou faibles. Ceci peut consister en un processus d'activation réelle, un glissement de dominance en faveur d'un épitope différent par diminution de la reconnaissance d'un épitope immunodominant, ou bien un autre mécanisme. Sont également décrits les anticorps qui reconnaissent ces épitopes, des procédés de traitement et d'utilisation, notamment la préparation de vaccins monovalents et polyvalents, de produits de recombinaison de α2M, ainsi que des techniques et des trousses de dosage pour mettre en ÷uvre de tels procédés.
PCT/US1993/012479 1992-12-18 1993-12-20 Complexe modulateur de la reponse immunitaire, et ses utilisations WO1994014976A1 (fr)

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US6403092B1 (en) 1998-04-01 2002-06-11 Duke University Immune response modulator alpha-2 macroglobulin complex
AU754988B2 (en) * 1998-04-01 2002-11-28 Duke University Immune response modulator alpha-2 macroglobulin complex
EP1286693A4 (fr) * 2000-06-02 2005-07-13 Univ Connecticut Health Ct Complexes d'alpha (2) macroglobuline et de molecules antigeniques a utiliser en immunotherapie
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US7179462B2 (en) 2000-06-02 2007-02-20 University Of Connecticut Health Center α (2) macroglobulin receptor as a heat shock protein receptor and uses thereof
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US8029808B2 (en) 2002-04-25 2011-10-04 University Of Connecticut Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19645559A1 (de) * 1996-11-05 1998-05-07 Immuno Ag Pharmazeutische Antigen-/Antikörperpräparation
US6403092B1 (en) 1998-04-01 2002-06-11 Duke University Immune response modulator alpha-2 macroglobulin complex
AU754988B2 (en) * 1998-04-01 2002-11-28 Duke University Immune response modulator alpha-2 macroglobulin complex
US7186515B1 (en) 2000-06-02 2007-03-06 University Of Connecticut Health Center Alpha(2) macroglobulin receptor as a heat shock protein receptor and uses thereof
US7179462B2 (en) 2000-06-02 2007-02-20 University Of Connecticut Health Center α (2) macroglobulin receptor as a heat shock protein receptor and uses thereof
EP1286693A4 (fr) * 2000-06-02 2005-07-13 Univ Connecticut Health Ct Complexes d'alpha (2) macroglobuline et de molecules antigeniques a utiliser en immunotherapie
US7449557B2 (en) 2000-06-02 2008-11-11 University Of Connecticut Health Center Complexes of alpha (2) macroglobulin and antigenic molecules for immunotherapy
US7666581B2 (en) 2001-08-20 2010-02-23 University Of Connecticut Health Center Methods for preparing compositions comprising heat shock proteins useful for the treatment of cancer and infectious disease
US6984389B2 (en) 2002-04-25 2006-01-10 University Of Connecticut Health Center Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
US8029808B2 (en) 2002-04-25 2011-10-04 University Of Connecticut Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
US8591890B2 (en) 2002-04-25 2013-11-26 University Of Connecticut Health Center Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
US9248172B2 (en) 2002-04-25 2016-02-02 University Of Connecticut Health Center Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
US9352019B2 (en) 2002-04-25 2016-05-31 University Of Connecticut Health Center Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
US8877204B2 (en) 2003-02-20 2014-11-04 University Of Connecticut Health Center Methods and compositions for the treatment of cancer and infectious disease using alpha (2) macroglobulin-antigenic molecule complexes
US9566348B2 (en) 2003-02-20 2017-02-14 University Of Connecticut Health Center Methods and compositions for the treatment of cancer and infectious disease using alpha(2) macroglobulin-antigenic molecule complexes

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