JP2000502995A - Compositions and methods for stimulating antibody release by B lymphocytes - Google Patents

Abstract

(57) Abstract: The present invention comprises adding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), or a combination thereof, useful for stimulating antibody release by B cells. Are described. Compositions, pharmaceutical compositions, vaccines, and methods of using vaccine adjuvants are also described. Further, the present invention describes an assay system useful for identifying compounds capable of stimulating antibody release by B cells.

Description

DETAILED DESCRIPTION OF THE INVENTION             B Compositions and methods for stimulating antibody release by lymphocytes Cross-reference of related applications   This application was filed on November 10, 1993, which is incorporated herein by reference. Application No. 08 / 150,510, filed on September 30, 1994, which is a continuation-in-part of Application No. 08 / 150,510 Provisional Application No. 08/4 filed June 6, 1995, a continuation application of 08 / 315,492 No. 67,146 is a continuation-in-part application.                                 Government interests   The invention described herein does not grant any patent rights to the inventors or their agents. It may be manufactured, licensed, and used for government purposes without paying a fee.                                 Field of the invention   The present invention relates to granulocyte macrophage colony stimulating factor (GM-CSF), interleukin Kin-2 (IL-2), interleukin-3 (IL-3) and universal T cell Pairs containing T cell stimulating peptides such as pitope (TCE), alone or in combination Such factors alone, together with the product and interferon-γ (IFN-γ). Or a composition comprising in combination. Composition comprising an antibody by mammalian B lymphocytes Useful for stimulating release. The present invention also stimulates antibody release by B lymphocytes It also relates to an in vitro assay system for identifying the composition.   Stimulation of antibody release by B lymphocytes prevents the harmful effects of infections and diseases, Useful in the context of treatment and / or improvement. This utility is Under normal conditions, and under immunosuppressive or immunocompromised conditions, the immune response of the mammal To adjuvants to strengthen. New compositions also enhance the human immune system It can be used in conjunction with other immunotherapy.                                 Background of the Invention   The human immune system works together to protect the human body against diseases and disorders Consisting of many different types of cells with overlapping functions. The cells of the immune system Has complex, multi-functional and interconnected relationships.   GM-CSF, IL-2, IL-3, and IFN-γ are all cytokines. "Sitekai "B" refers to B and T lymphocyte cells ("B cells" and "T cells") and nutrients. One of the compounds that regulates the response of cells of the immune system, such as Lark killer ("NK") cells Class. "Cytokine" is a specific cell collection when contacted with an inducer. Non-antibody proteins that are released by gangs and act as intercellular mediators First name. "Lymphokines" are released upon contact with certain antigens or other stimuli Released by sensitized lymphocytes, helps to exert cellular or humoral immunity It is a soluble substance.   The terms “cytokine” and “lymphokine” are interchangeable Was. In an attempt to simplify the names of these compounds, the second national conference held in 1979 One group of lymphokine workshop participants may have different groups of leukemia Developing a unified naming system based on the ability of proteins to act as signaling signals between spheres To do so, he proposed the term "interleukin", abbreviated as "IL".   To date, most, if not all, of the 21 Different cytokines have been identified. Each has its own molecular configuration, Perform the function of Many known cytokines have distinct activities in B cells It is shown to be. In vitro, lymphokines IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IFN-γ, and TGF-β (transforming growth factor-β ) Enhances B cell proliferation, immunoglobulin secretion, or otherwise secreted Ig Have been shown to affect subclasses of. Sis examined Depending on the system, the addition of one or more of the above lymphokines can lead to in vivo Increased antibody production or the secreted antibody isotype (ie, IgG, I gM, IgE, IgA, etc.). Reported to affect B cell proliferation Lymphokines reported include IL-1, IL-2, IL-4, and IL-10, and B Those reported to affect cell differentiation and Ig secretion include IL-2, IL-5, Includes IL-6, TGF-β, and IFN-γ.   Any of the cytokines reported to enhance Ig secretion in vitro Bo showed no significant role. Thus, IL-2, IL-5, IL-6 Alternatively, injection of a monoclonal antibody specific to IFN-γ does not It is not suppressed at will.   This suggests that under physiological conditions, B cell differentiation is simply a direct T cell interaction. Other cytokines that are only dependent or not yet known Is suggested.   Antigens that stimulate T cell activation (so-called thymus-dependent antigens or "TD antigens") Immune response to direct interaction between B cells and T cells in order to secrete Ig Depending on this, this is not the case for antigens that cannot induce T cell activation. T thin Cell-independent antigens ("TI antigens") help directly or indirectly T cells Induces high levels of antibody production without. Thus, B cell differentiation and TI anti- The events that control immunoglobulin secretion to the virogen are pathways that have not yet been defined. There must be dependencies. B cell differentiation leading to immunoglobulin secretion The event or control that controls this step, as it is the final event underlying the Defining Itokine designs a method to enhance or suppress the immune response Invaluable to get in.   To enable rapid recognition of the present invention, immunoglobulins, antibodies, lymphocytes, The main known functions of B cells, T cells, and NK cells are described below as background. A brief description is given below. Also reported to affect B cell proliferation or antibody secretion Cytokines, i.e., IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, TGF A brief summary is also provided for the known activities of -β and IFN-γ. GM-CSF and IL- A summary of the three known activities is provided as well. Reference material is basic immunology (Fundamn ental immunology ), 2nd edition, William E. Paul, M.D. (Raven Press, New York 1989); Basic immunology (Fundamnental Immunology), Third Edition, William E. Paul, M.D. (Raven Press, New York)  1993); Interferon: principles and medical applications (Interferon: Principles and M edicalApplications ), Baron et al. (S. Baron) (University of Texas at Galveston) The University of Texas Medical Branch, Galveston, Texas 1992); Cytokine Handbook (The Cytokine Handbook), Angus To Angus Thompson (Academic Press Inc., San Diego, CA 1992) And cytokine handbook (The Cytokine Handbook), Second Edition, Ann Angus Thompson (Academic Press Inc., San Diego, C A 1994), all of which are specifically incorporated herein by reference.   Mammals, including humans, face countless organisms every day. By these organisms The primary component of the immune system that plays an essential role in protecting the host against infectious diseases is humoral antibodies It is. Antibodies are protein molecules, also known as immunoglobulins, that stimulate antibody production It has a strong specificity to the foreign particles that do. For example, systemic infection by "Bacteria A" So, antibodies that bind with high affinity to "Bacteria A" but do not bind to "Bacteria B" Is induced. Similarly, "Bacteria B" is an anti- "Bacteria B" that does not cross-react with "Bacteria A". Induce antibodies.   Immunoglobulin (Ig) consists of a pair of light (L) [low molecular weight] κ chains (κ or λ), And two pairs of heavy (H) chains (γ, α, μ, δ, and ε) Structurally related, consisting of chains, all four linked together by disulfide bonds Is a class of proteins that Both heavy and light chains are involved in antigen binding, and Ig molecules Different regions have regions that are highly variable. In addition, heavy and light chains are non-variable or Includes regions that are stationary. Based on the structural and antigenic properties of the heavy chain, Ig can be an IgG, Classified into IgA, IgM, IgD, and IgE isotypes. IgG based on H chain differences The subclass is called IgG1 or the like.   Lymphocytes include lymph nodes, spleen, thymus, tonsils, Peyer's patches (small intestinal tissue), and White blood cells that are sometimes formed in the body's lymphoid tissues, such as the bone marrow. Individual lymphocytes respond to a limited group of structurally related antigens It is specialized in the respect. This involvement involves the binding of certain antigens to the immune system. An antigen-specific receptor present on the lymphocyte membrane prior to first contact (ie, immune Globulin). The organism reacts to virtually any antigen Different clones, each having a specific antigen-specific receptor Is obtained by having a very large number of lymphocytes. As a result, lymphocytes Is a very heterogeneous cell population.   Lymphocytes interact with each other not only in the specificity of their receptors, but also in their functional properties. Different. Broadly divided into two classes, B lymphocytes and T lymphocytes Lymphocytes are recognized. In addition to these two classes, certain "non-specific "Lymphoid cells that mediate cytotoxic reactions are known. These are natural NK (NK) cells.   B lymphocytes, also known as "B cells," are only partially known. A type of lymphocyte derived from hematopoietic stem cells through a complex series of differentiation events . B cells are precursors to antibody-secreting cells and thus produce immunoglobulins Involved in. B cell surface receptors are antibodies or antibodies that specialize on cell surface expression. Or an immunoglobulin molecule. Newly differentiated B cells are initially the only IgM class Express surface Ig. In connection with B cell maturation, another immunoglobulin The isotype appears.   B cells must first be activated to release antibodies in response to cytokines. I have to. Cross-linking of membrane Ig protein molecules by antigen (cross-linking Di-dependent B cell activation), direct encounter with T cells (helper T cells or eg CD4 0 Helper T cell-related molecules such as ligands), or include encounters with mitogens There are many ways to activate B cells. In such an encounter, the antigen is Presents the epitope recognized by Ig.   Since each B cell has many membrane Ig molecules with the same variable region, the optimal membrane I g-mediated crosslink activation activates high level crosslinkage of cell surface receptors Which contains one or more copies of an epitope recognized by cell surface Ig Is required to be presented by the antigen. Many simple protein antigens have this ability However, such requirements are important for polysaccharides and the surface of microorganisms and DNA. Filled with other antigens having such repetitive epitopes. These antigens include: For many medically important microorganisms, such as pneumococci, streptococci, and meningococci There is a coating polysaccharide.   Cross-linking of membrane Ig has also been shown to lead to B cell loss or inactivation. There is a lot of data. In general, certain types of receptor cross-linkage events Inactivates rather than activates if they occur in the absence of specific stimulus signals It is thought to lead to. Highly repeated epitopes expressed on polysaccharides are probably The magnitude of receptor-mediated stimuli may lead to activation in the absence of costimulation There is.   T lymphocytes, or "T cells," are thymus-derived, long-lived (months to years) ) Immunologically important cells involved in cellular immunity. T cells are defined as "helper T cells. ), "Suppressor T cells", and "killer T cells". A group consisting of T cells involved in delayed type hypersensitivity and related immune phenomena are also Known.   Natural killer cells, or "NK cells," are certain "non-specific" It is a lymphoid cell that mediates the response. Such a non-specific cytotoxic response can be Uses a different recognition system than the system used by B cells to Kill cells. Kill one cell type from another through contact interactions This constitutes one of the main effector effects of the immune system's self defense.   In addition to these cells, other compounds and cytokines play important roles in host defense Plays. One group of cytokines are interleukins.   IL-1 is primarily an inflammatory cytokine, but IL-2 and other cytokines Is primarily a growth factor for lymphocytes. IL-1 is a polypeptide synthesized by monocytes. It is a peptide hormone. IL-1 during inflammation, injury, immune challenge, or infection Is produced, but due to its diverse biological properties, this cytokine is Seems to affect the disease. In animals, IL-1 is associated with hypotension and shock. It is a powerful inducer. IL-1 acts on the hypothalamus to cause fever and bone Acts directly on the skeletal muscle to promote protein catabolism.   IL-2, also known as T cell growth factor, is a T helper and suppressor lymph Lymphokines and peptide hormones produced by both spheres. This Cytokines are T cells, B cells, NK cells, lymphokine-activated killer (LAK) Directly affects cell and monocyte, macrophage, and oligodendrocyte proliferation and differentiation Effect.   IL-3, also known as a multicolony stimulating factor, acts on many target cells in the hematopoietic system. To use. This cytokine is the broadest for any of the hematopoietic growth factors (HPGF) Target specificity and promotes the production and differentiation of hematopoietic stem cells (ie, blood cell precursors) Can stimulate, macrophages, neutrophils, eosinophils, basophils, mast cells, This gives rise to megakaryocytes and erythroid cells.   The association between IL-3 and B cells was unknown before the present invention. Actually as of 1994 Thus, the range of target cells for IL-3 includes cells involved in T and B-lymphocyte lineages. Rare And that IL-3 has an important direct effect on B cell development. There was no strong evidence. J.W.Schrader, "Chapter 5: Interloy Kin-3 ”cytokine handbook (The Cytokine Handbook) Second edition, Anga Anthus Thomson, p. 84 (Academic Press, New York, 1994) .   IL-3 is synthesized by T cells and mast cells, but secretion of IL-3 by B cells Not reported. Several reports indicate that IL-3 is Beta) and moderate increase in Ig secretion by human B cells activated by IL-2 It has been proved that strong can be induced. For example, Xia et al. Recombinant IL-3 is a normal B cell growth factor. ''J. of Inmmunology,148, 491-497 (1992) reported that IL-3 enhances the proliferation of B cell-rich cell populations. Likewise Tadmori et al. (Human recombinant IL-3 stimulates B cell differentiation),J.of I mmunolog y,142, 1950-1955 (1989) show that IL-3 is derived from tonsil cells, including B cells, Or I in a peripheral blood-derived B cell-rich population activated by bacterial antigens. Reported to stimulate gG secretion. In addition, Matsumoto et al. Interleukin-3 activates anti-IgM IgE synthesis in non-atopic human B cells Guidance ",Int.Arch.Allergy Appln.Immunol.89, 24-30 (1989) are human recombinant IL-3 enhances IgE synthesis by normal B cells or a mixture of T and B lymphocytes, -1, IL-2, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and IFN-γ induce IgE synthesis Reported that you could not. Matsumoto also added anti-IL-3 antibodies Factor in T cell supernatant involved in the induction of IgE synthesis He stated that IL-3 could not be conclusively identified.   These results were due to IL-3 mediated enhancement in cell proliferation. These trials In experiments, B cells were not electronically sorted and therefore highly purified Had not been. Thus, the Ig-enhancing effect is high for non-B May reflect the effects of IL-3 on cells and non-T cells It is not possible to determine whether IL-3 is directly acting on B cells from the experiment of (1). Furthermore, in previous experiments, B cells were not fractionated according to size. this As such, a possible role for the pre-activated state of B cells was not addressed.   In further contrast to the present invention, Kimoto et al. Inability to stimulate T or B lymphocyte development in vivo, but not to T cell dependent antigens Enhances the immune response toJ. of Immunology,140, 1889-1894 (1988) A mouse with an osmotic mini-pump loaded with mouse recombinant IL-3 Reported no increase in the total number of B and T cells in the organ. Furthermore, Kimoto et al. (Kimoto) states that IL-3 does not act directly on lymphocytes or their precursors, By acting on Raku accessory cells, humoral immunity against T cell-dependent antigens It suggested that it might enhance the epidemic response.   IL-4 is a glycoprotein also known as B cell stimulating factor 1 (BSF-1) and B cell differentiation factor Quality. This includes B cell proliferation, Ig class switching, T cell proliferation and differentiation, Co-stimulates lophage activation, controls mast cell proliferation, and co-spicks hematopoietic progenitor cells It works to be intense.   B cell growth factor II (BGF-II), T cell surrogate factor, IgA-enhancing factor and eosinophil IL-5, also known as knee stimulator, is produced by T lymphocytes and mast cells Is a glycoprotein. This cytokine is found in eosinophil colonies in the bone marrow. And has two functions as a colony stimulating factor. IL-5 is Specific in vitro antibodies by B cells primed by antigen in vivo Induce production. IL-5 acts as a differentiation factor in vitro, but in vivo It does not seem to act as a differentiation factor.   BSF-II, hybridoma / plasmacytoma growth factor, interferon- β2, and IL-6, also known as hepatocyte stimulating factor, are lymphoid and non-lymphoid. Is a glycoprotein produced by both cells. This cytokine is A, controls acute phase response, and hematopoiesis. IL-6 acts on B cell lines at the mRNA level And induces biosynthesis of secreted Ig. In addition to IL-5, IL-6 is also very strict It has been shown to function as a differentiation factor under conditions. All other known T cell or macrophage-derived factor activates B cells without the addition of growth factors To secrete Ig.   IL-10, also known as a cytokine synthesis inhibitor, is used in T cells, macrophages , And other cell types. This cytokine is found in mast cells and And B cell augmentation or stimulation, plus cytokine synthesis and some microbes object Inhibits some macrophage functions, including activity. IL-10 is an anti-CD40 antibody Or strong proliferation of human B cells activated by antigen receptor cross-linking cause.   In addition to interleukins, other cytokines have been characterized. Ko Ronnie stimulating factor (CSF) is a group factor mainly related to hematopoiesis. They are, Defined as a protein that stimulates the clonal expansion of bone marrow cells in vitro.   Granulocyte-macrophage colony stimulating factor (GM-CSF) Is a glycoprotein growth factor that regulates differentiation. This growth factor, under different conditions, T cells, macrophages, endothelial cells, stromal cells, fibroblasts, mast cells, and Produced by many different cells, including others. The main action of GM-CSF is granule Of survival, differentiation, and proliferation and functional activity in the sphere-macrophage population Including control. Prior to the present invention, GM-CSF stimulates the release of antibodies by B cells There are no reports showing that this can be done.   Finally, another class of cytokines that function in the in vivo immune system is Interferon (IFN). IFN has many other important effects on cells In addition to exerting anti-viral effects, it inhibits virus replication by inhibiting virus replication. It is the main participant in your first line. IFN directly protects cells from infection Does not work. Rather, they stop virus growth in neighboring cells Stimulates the production of proteins that in turn, thus protecting cells from infection.   IFN is divided into three groups, α, β and And γ. IFN-α and IFN-β production is not a specialized cell function Perhaps all cells of the organism are capable of producing these IFNs.   In contrast to IFN-α and IFN-β synthesis that can occur in any cell, IFN-γ Production is a function of T cells and NK cells. All IFN-γ inducers are polyclonal Antigen-specific (mitogen or antigen) or clonally restricted Activate T cells by the method. Human IFN-γ promotes the proliferation of activated human B cells, Synergizes with IL-2 to enhance immunoglobulin light chain synthesis in B cell culture be able to.                                 ****   A brief discussion describing the function of various human immune cells, and IL-1, IL-2, IL-3, IL -4, IL-5, IL-6, IL-10, GM-CSF, and IFN-γ Are extremely diverse. But even with this level of knowledge , It is not possible to fully understand the complexity of the immune system. Many gaps remain Exist. For example, cytokines regulate the responses of cells in the immune system. Thus, except for acting as an intercellular mediator, It is not possible to make a general statement regarding. Thus, in the art, Better understand the immune system and provide additional and better ways to treat immune disorders There still exists a need to do so.                                 Summary of the Invention   The present invention fulfills the need in the art for new and improved immunotherapy. . Novel compositions and methods using IL-2, IL-3, GM-CSF, universal TCE, and IFN-γ Can improve immune disorders and provide new treatments with current immunotherapy adjuvants Make it work.   The present invention relates to GM-CSF and I in an amount effective to stimulate antibody release by B cells. It is intended for compositions comprising L-2 or IL-3, alone or in combination. Departure Another purpose of Ming is that the resulting complex binds to proteins, GM-CS in an amount effective to stimulate antibody release by B cells, which is bound to a polysaccharide A composition comprising F and IL-2 or IL-3, alone or in combination. Book The invention also relates to in vitro T or B cell expansion or in vitro antibody production. By increasing the responsiveness of cells involved in the immune response, as established by In combination with other lymphokines that can enhance immunoreactivity. GM-CSF, IL-2, or IL-3 molecule that retains GM-CSF, IL-2, or IL-3 activity Individually manipulated fragments can also be used in the present invention.   It is another object of the present invention that all of the compounds are effective in stimulating antibody release by B cells. Contains GM-CSF, IL-2, or IL-3 present, alone or in combination with IFN-γ A composition.   Another object of the present invention is to provide non-antigen specific proteins that can further bind to polysaccharides. A composition comprising universally linked universal TCE. In another embodiment, a universal TCE Is directly linked to the polysaccharide.   Another object of the invention comprises the novel composition and a pharmaceutically acceptable carrier It is a pharmaceutical composition.   To stimulate antibody release by B cells, GM-CSF, IL-2, IL-3, IFN-γ, and Utilizing novel compositions comprising the TCE and the universal TCE is also included in the invention. By B cells Stimulation of antibody release can be used, for example, to treat vaccines in normal or immunocompromised states. The mammalian immune response to the species can be enhanced.   Another object of the present invention is to use the novel compositions as adjuvants for vaccines. It is for. For example, to allow rapid stimulation of immune cells present in the blood system Many vaccines are currently administered intravenously or intramuscularly. A new composition, As a fusion protein covalently linked to a carrier molecule, as a mixed fusion protein, or Or any other combination with the vaccine to be administered Thus, the degree of antibody response can be increased at both systemic and local levels.   Another object of the invention is that antibody production is pathogenic, such as in autoimmune diseases Neutralization of GM-CSF, IL-3, and IFN-γ under such conditions.   Production of monoclonal antibodies in vitro or in vivo using compositions Can also be optimized. For example, sensitizing animals in vivo with antigens and compositions can do. Alternatively, anti-stimulation by stimulating or sensitizing lymphocytes in vitro Production of the body can be enhanced in the presence of the novel composition. This is a human monoc It is particularly useful for producing lonal antibodies.   The invention also allows for the identification of compositions useful for stimulating antibody release by B cells. New assay system. This assay system simulating in vivo antibody stimulation includes Dextran-conjugated anti-Ig antibodies and highly purified B cells. Anti-Ig dextra Conjugates provide a mechanism comparable to antigen-induced B cell activation in vivo Activates B cells effectively and polyclonally through membrane Ig You.   Other objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description. Will be apparent or will be understood by practicing the present invention. It is. The accompanying drawings and tables, which are incorporated in and constitute a part of this specification, are , Together with the description, serve to illustrate and explain the principles of the invention.BRIEF DESCRIPTION OF THE FIGURES FIG. : IgM secretion (ng / ml) by B cells in various media was measured. Culture medium Are written along the left side of the drawing. The composition tested was a dextran-conjugated anti-IgD It showed the highest IgM secretion in media containing body, IL-1 and IL-2. In this medium Add supernatant from cell culture, RA5-SN, and GM-CSF, or IL-3 as described in the parent application. I got it. A control composition was also measured. RA5-SN supernatant contains both GM-CSF and IL-3 Showed significant IgM secretion by B cells as compared to the other compositions tested.FIG. : B cells in the presence or absence of various concentrations of IL-3 or GM-CSF Dextran-conjugated anti-IgD antibody (3 ng / ml) + IL-1 (150 U / ml) + IL-2 (150 U / ml) ml). IgM secretion was measured by ELISA 6 days after the start of culture .FIG. : GM-CSF and IL-3 were added at various concentrations, and IgM secretion (ng / ml). When 10 U / ml of GM-CSF and about 3 U / ml of IL-3 are added, about 4650 ng A maximum IgM secretion of / ml was obtained.FIG. : B in the presence or absence of IL-3 (10 U / ml) or GM-CSF (10 U / ml) Cells were dextran-conjugated anti-IgD antibody (3 ng / ml) + IL-1 (150 U / ml) + IL-2 (1 (50 U / ml). In addition, anti-IL-3 (10 μg / ml) and / or GM-CSF (10 μg / ml) antibody was added. Controls without anti-IL-3 or anti-GM-CSF were also prepared. Made. The IgM concentration was measured by ELISA six days after the start of the culture.FIG. : Activity of dextran-conjugated anti-IgD antibody-activated B cells in AF7 supernatant IgH secretion (ng / ml) by the transformed B cells was measured. IgM secretion involves two or more of the following: Measured in various media: IL-1, IL-2, αIL-3, and αGM-CSF.FIG. : In the presence or absence of IL-3 (100 U / ml) and / or GM-CSF (100 U / ml) In the presence of B cells, dextran-conjugated anti-IgD antibody (3 ng / ml) + IL-1 (150 U / ml) Activated by + IL-2 (150 U / ml). Viable cells (exclude trypan blue Cells were counted using a hemocytometer 4 days after the start of culture. In replication culture The IgM concentration was measured by ELISA six days after the start of the culture. Data are from two cultures. Expressed as mean ± standard error of the mean.FIG. : B fine in the presence or absence of IL-3 (100 U / ml) + GM-CSF (100 U / ml) The vesicles were dextran-conjugated anti-IgD antibody (3 ng / ml) + IL-1 (150 U / ml) + IL-2 (150  U / ml). The IgM concentration in the replica culture was compared with that of the control composition at 0 hour by IFN-. The composition to which γ was added and the composition to which IFN-γ was added for 24 hours were analyzed by ELISA. Was measured. Data are expressed as the mean of two cultures ± standard error of the mean.FIG. : Schematic diagram of yeast expression vector.FIG. : Protein before and after purification on nickel agarose column and sizing gel Coomassie blue stained gel containing A-IL2 (PA-IL2) product.FIG. : Anti-IL-2 (left gel) and anti-protein A (PA) (right gel) Immunoblot of protein A-IL2 (PA-IL2). IL2 and PA as controls The protein A was fused in a truncated form of Protein A. Runs at higher molecular weight than product.FIG. :Functional activity of IL2 in fusion products. 5x10 CTLL strains that are IL2 reactive^ThreeH T2 cells are combined with IL2 or protein A-IL2 and thymidine incorporation 48 hours later Was measured.FIG. : A) purified protein A-IL2 (PA-IL2) fusion product; B) protease-free Size exclusion HPLC of unpurified concentrated supernatant containing PA-IL2 from yeast (Beckman SEC) 2000). No purification is performed on the supernatant, and a "clear" with the lower molecular weight cleavage product. "Product" obtained in protease-free transfected yeast It is shown that.FIG. : Purified protein D (lanes 1 and 2) and molecularly engineered universal TCE- Protein D (TCE-protein D, lane 4) and protein A (control 5) was run on SDS PAGE and stained with Coomassie Blue (top panel) ). The second gel was immunoblotted with anti-protein D (lower panel). The figure is Indicates that the protein and TCE-protein D were purified to homogeneity .                           Description of the preferred embodiment   The present invention provides sites that alone and in combination increase antibody secretion by B cells 100-fold. Describe the composition of Cain. The composition stimulates the release of antibodies by B cells An effective amount of IL-2, IL-3, and GM-CSF, alone or in combination. . Together with IFN-γ, all present in effective amounts to stimulate antibody release by B cells The present invention also provides a composition comprising IL-2, IL-3 and GM-CSF alone or in combination. Included I will. More preferably, the composition comprises a CD40 ligand (CD40L), or at least Also comprise another cytokine, or a combination thereof. Also more preferred Alternatively, the composition comprises a CD40 ligand (CD40L), a universal TCE, or at least one other Or a combination thereof. Universal TCE is non-antigen specific Means a strong T cell stimulating peptide that does not have a B cell epitope. strong T cell stimulation, as reflected in T cell proliferation assays known to those of skill in the art, It means that T cells can be strongly stimulated. Peptides are about 8 to 20 amino acids. A molecule of no acid residues, preferably a molecule of 11 to 18 residues. Non-antigen-specific Peptides stimulate T cells with different genetic backgrounds across diverse antigen specificities That means. Not having a B cell epitope means that the epitope is And therefore do not induce antibody production specific for the epitope. Means that The peptide may be chemically linked to the protein, or The fusion protein may be constructed according to well-known methods. Peptides are known to those skilled in the art. According to the method, it may be coupled to polysaccharides including CDAP.   Most preferably, the composition comprises GM-CSF, IL-3, or a combination thereof, IFN-γ, CD40 L, and IL-1 + IL-2. In another most preferred embodiment, the composition comprises , GM-CSF, IL-3, or a combination thereof, IFN-γ, CD40L, universal TCE and IL-1 + IL-2 including. Most preferably, the composition comprises GM-CSF, IL-3, IFN-γ, CD40L, Bound to TCE, IL-1 + IL-2, or protein and further bound to polysaccharides , Including some or all of the above.   The enhancement of Ig secretion mediated by IL-2 or IL-3 or GM-CSF is typically 10 When IL-3 and GM-CSF are used in combination within a range of 5050 fold, enhancement up to 100 fold is induced. Preferably, GM-CSF and IL-3 are present at about 1 to about 10 U / ml in vitro. In vivo amounts are scaled up accordingly, as is well known in the art. . More preferably, GM-CSF and IL-3 are present at about 10 to about 100 U / ml, especially about 100 Present in U / ml.           B Identification of IL-3, GM-CSF and IFN-γ as cell stimulants   Prior to the present invention, GM-CSF or IL-3 acted directly on B cells to release antibodies. Or GM-CSF and IL-3 synergistically and directly on B cells Typically It was not known to act to stimulate antibody release. Besides, GM-CSF is Until then, it was not involved in direct control of mature B cell function. Similarly, IFN-γ Independently stimulates optimal antibody secretion even when added 24 hours after stimulation The activity is further enhanced when IL-3 and GM-CSF are used alone or in combination. I didn't even know that.   Compositions described in parent application 08 / 150,510 include IL-3 and GM-CSF. Was rare. In early experiments, the Ig content of electronically sorted highly purified B cells was Lung response may be significantly lower than B cell-rich populations, including a small number of "non-T non-B" cells It has been determined. Based on these findings, the first application was filed for NK cells and / or NK cells. Vesicle-derived cytokines may enhance Ig secretion in anti-Ig dextran-stimulated B cells It was shown.   The parent application also provides T cell clones (TH1 or TH2) that enhance Ig secretion by B cells. The supernatant from E. was disclosed. Experiments show that this enhancement is IL-1, IL-2, IL-5, IL-6, or Was determined not to be due to IL-10. Surprisingly, the differentiation inducing activity is G It was found to be due to the presence of M-CSF and IL-3. GM-CSF and IL-3 as anti-Ig When added to strand-stimulated cells, Ig secretion was induced. Conversely, anti-GM-CSF and / or Alternatively, the addition of anti-IL-3 reduced the stimulatory activity of the T cell supernatant.   Ig secretion is further added after 24 hours of culture by adding IL-2 to IFN-γ Or by adding IFN-γ + IL-2 There was found. The highest levels of Ig secretion include IL-2, IL-3, and GM-CSF at the start of culture. When added to anti-Ig dextran stimulated cells, and after 1 day of IFN-γ (anti-Ig dextran 24 hours after stimulation with tolan). Immediately after B cell activation I The addition of FN-γ does not enhance the stimulatory effect or impair IL-3 or GM-CSF. Intensification is minimal. Thus, the time to add IFN-γ depends on B cells. It is important to further enhance the release of the antibody. This finding was made prior to the present invention. Had not been reported.   TH1 or TH2-derived supernatant depending on the activity of IL-3 or GM-CSF, or IL-3 + GM-CSF Neutralization of anti-IL-3 or anti-GM-CSF antibodies to determine if their differentiation activity can be explained The effect of adding amount on Ig secretion was analyzed. Each antibody has Ig secretion Mediated undesired suppression, but in combination with anti-IL-3 and anti-GM-CSF, TH1- or TH2-derived Induced over 80% suppression of Ig secretion of anti-Ig dextran stimulated cells in the presence of supernatant .   Findings that IL-3 and GM-CSF may enhance Ig secretion by B cells and anti-IL- The finding that 3+ anti-IL-GM-CSF can suppress Ig secretion was completely unexpected. So IFN-γ added 24 hours after the start of culture enhances the stimulating effect of IL-3 and GM-CSF It was surprising to do it. These findings have not been reported before Was.                               Pharmaceutical composition   Pharmaceutical compositions are also included in the present invention. The composition comprises an effective amount of IL-3 and GM-C. SF, either alone or in combination, and a pharmaceutically acceptable carrier. Pharmacy With an effective amount of IL-3 and GM-CSF, alone or in combination, with a chemically acceptable carrier. A pharmaceutical composition comprising an effective amount of IFN-γ is also included in the present invention.   Treatment includes intravenous, intraperitoneal, intravenous injection, intraarticular, intraventricular, and arachnoid Under, intramuscular, subcutaneous, intranasal, intravaginal, oral, or any other suitable Administration by various administration methods. The composition may also be administered intramuscularly to specific areas. Alternatively, it may be administered locally, such as injection by either subcutaneous or subcutaneous.   For GM-CSF, IL-3, and IFN-γ, any pharmaceutically acceptable carrier Can be used. Carrier is water, petroleum, animal oil, vegetable oil, peanut oil, soybean It can be a sterile liquid, such as an oil, including oils, mineral oils, sesame oil and the like. Intravenous injection If provided, water is the preferred carrier. Saline, water-soluble dextrose and Glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Can be. Suitable pharmaceutical carriers are “Remington's,” which is incorporated by reference. Pharmaceutical Sciences (Remington's Pharmaceutical Sciences) ", 18th Edition (Genaro (A. Ge nnaro), Mack Pub., Easton, Pa., 1990).                     Vaccine adjuvant containing composition   The present invention also includes a vaccine adjuvant comprising the composition of the present invention. Many Vaccines currently stimulate intravenously or intravenously to rapidly stimulate immune cells present in the blood system. Or intramuscularly. By combining the new composition with the drug to be administered, Thus, the extent of the antibody response increases both locally and systemically.   In in vitro assays, anti-IgM or anti-IgD antibodies may be When present in a valent form, it acts efficiently to activate all B cells. This high Level of activation allows the release of antibodies by B cells in conjunction with the use of highly purified B cells Can be identified. However, this reaction is not desirable in vivo. I don't. In patients, the goal is to have very few receptors with specific receptors for the antigen Is to activate only B cells. The specific antigen of the vaccine is Acts to crosslink with the body. In contrast, anti-Ig dextran was used The in vitro model acts to crosslink all antigen receptors.   When used as a vaccine adjuvant, preferably the composition of the present invention comprises Attached to a polyvalent carrier molecule, such as kisstran or a bacterial coat polysaccharide. pneumonia Coating polysaccharides of cocci, streptococci, and meningococci are preferred. GM-CSF, IL-2 or GM-CSF, IL-2 or IL-3 molecule that retains IL-3, GM-CSF or IL-3 activity Engineered fragments, or combinations thereof, can be independently attached to a multivalent carrier . Alternatively, GM-CSF, IL-2, and IL-3 are fused together or fused to another protein To form a fusion protein, which can be similarly bound to a polyvalent carrier. it can. Various other variants of the vaccine will be apparent to those skilled in the art.   This application describes an anti-cytokine that allows for slow but long-term transport of cytokines. And the use of cytokine-cytokine complexes. The complex is a mixture with the vaccine antigen. Or the conjugate can bind to the antigen of the vaccine .   In yet another embodiment, the vaccine adjuvant is CD40L, GM-CSF, I L-3, or one or more cytokines other than IFN-γ, or a combination thereof. C D40L and one or more cytokines, or universal TCE, are also Can be made.                 Compositions used in conjugate vaccines   In order to form a conjugate vaccine, the antigen of the vaccine and the composition of the invention are used. Covalently binds to a polyvalent carrier molecule, such as dextran or bacterial capsular polysaccharide be able to. Pneumococcal, streptococcal, and meningococcal capsular polysaccharides are preferred.   An antigen is a peptide or protein specific for the disease to be vaccinated. You.   CD40 or lower to further optimize the humoral immune response during vaccine administration And one other cytokine, or universal TCE, or a combination thereof as a multivalent carrier Can be combined.   Table III shows a typical vaccine using the composition of the invention. As noted in the table Some vaccines are conjugate vaccines. Methods of conjugation are well known to those skilled in the art, Brunswick et al., Incorporated by reference.J . Immunol.14 0 : 3364 (1988) Heteroligation method; Wong S.S. And crosslink chemistry (Chemistry of Protein Conjugates and Crosslinking) CRC Press, Boston (1991); and Brenkeley et al. Brief overview of the preparation of protein conjugates with haptens and crosslinkers (Br iefSurvey of Methods for Preparing Protein Conjugates With Dyes, Haptens  and Cross-Linking Agents) "Bioconjugate Chemistry,ThreeNo. 1 (1992 1 Month). Preferred covalent attachment methods are those whose disclosure is specifically incorporated herein by reference. No. 07, filed Sep. 23, 1993, entitled “CDAP” binding method Application 08 / 408,7, filed on March 22, 1995, which is a continuation-in-part of / 124,491 No. 08 / 482,661, filed June 7, 1995, which is a continuation-in-part of No. 17 Has been stated.How to use the composition   It is a further object of the present invention to use IL-2, IL- to stimulate antibody release by B cells. 3, and compositions comprising GM-CSF alone or in combination, and with IFN-γ , IL-2, IL-3, and GM-CSF, alone or in combination. .   Suitable hosts for treatment include any suitable mammal. Preferred hosts are Humans, including neonates, adults, and immunodeficient patients.   The composition can be administered by any suitable method of administration. Composition preference The method of administration is subcutaneous, intravenous, intranasal, mucosal, oral, intramuscular, or a combination thereof. Including   The dose of the compound used varies depending on age, individual differences, symptoms, administration method, and the like, It can be easily determined by those skilled in the art. Typical doses of GM-CSF and IL-3 are J. Nemunaitis, "Granulocyte macrophage colony stimulating factor : Review from preclinical development to clinical application (Granulocyte-macrophage-colony-st imulating factor: a review from preclinical development to clinical appl ication) ",Transfusion,33, 70-83 (1993); Lieschke et al., " Granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor (Granulocyt e-Colony-Stimulating Factor and Granulocyte-Macrophage-Colony-Stimulatin g Factor (first half) "The N.Eng J. of Med.327, 28-35 (1992); Lieschke), “Granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor Child (Granulocyte-Colony-Stimulating Factor and Granulocyte-Macrophage-Col ony-Stimulating Factor (second half) "The N.Eng J. of Med.327, 99-106 (1992) Schulz et al., “Recombinant interleukin-3 and granulocyte macrophages. Adjuvant therapy with spore colony stimulating factor ",Pharmc.Ther.52.85-94 (19 92); Hoelzer et al. (Interlo in the treatment of patients with neoplastic disease). Combination of Ikin 3 alone and GM-CSF '',Seminars in Hematology,28, Suppl.2 (April ) 17-24 (1991).Neutralization method using composition   Another object of the present invention is to provide lupus, systemic lupus lupus erythematosus (SLE) , Idiopathic thrombocytopenic purpura (ITP), vasculitis, Graves' disease, allergic reaction GM under conditions where the production of antibodies is pathogenic, as in autoimmune diseases such as -A method to neutralize the activities of CSF, IL-3 and IFN-γ.   For the construction of a neutralizing vaccine, antibodies to the cytokine are generated. Business Are familiar with well-established methods for obtaining specific antibodies. Then get the antibody To the patient. The antibody can be conjugated to a carrier to increase the half-life of the antibody. Wear.Use of compositions to optimize monoclonal antibody production   The compositions can also be used to produce monoclonal antibodies in vitro or in vivo. Raw can be optimized. For example, a solution containing the antigen and composition of interest Animals can be sensitized by injection in vivo. Composition is B fine Administration of a composition conjugated to a specific antigen to stimulate the release of antibodies by the vesicles Antibody production against a specific antigen is optimized.   One of skill in the art, along with such techniques for immunization, will appreciate anti-immunity in light of the disclosure herein. Familiarize yourself with the dosage of antigens and compositions needed to induce the body and stimulate its release Seems to be.   Alternatively, in vitro stimulation or sensitization of lymphocytes for antibody production comprises a novel composition Can be enhanced in the presence of an object. The teaching of such irritation and sensitization is The art, together with the determination of the appropriate amount of antigen and composition to apply in light of the disclosure of the specification, It is within the ordinary skill in the art. The method is particularly useful for producing human monoclonal antibodies. Useful.B Assay system for identifying compositions useful for stimulating the release of antibodies by cells   A further aspect of the invention is a novel in vitro assay that mimics in vivo antibody stimulation System development. This assay uses a composition that stimulates the release of antibodies by B cells. An object can be identified.   Resting B cells do not release antibodies, so first measure them before measuring antibody release. Must be activated. In the present invention, the anti-IgM or anti-IgD antibody is particularly Snapper et al., “Mouse margin zone and filtration, incorporated by reference. In vitro comparative analysis of proliferation, Ig secretion, and Ig class switching by vesicle B cells (C omparative In Vitro Analysis of Proliferation, Ig Secretion, and Ig Clas s Switching by Murine Marginal Zone and Follicular B Cells) "J.of lmm unology .150.2737-2745 (1993), high molecular weight dextran That is, MW = 2.0 × 10⁶) To form a multivalent antigen on a polysaccharide carrier. this The technique is to sustain bivalent anti-Ig molecules at picomolar levels through B cell membrane Ig And highly stimulating multivalent binding that can induce signal transduction repeatedly Convert to things. Anti-Ig dextran conjugates can increase high levels of B cell proliferation, at concentrations as low as 1 pg / ml, a concentration 10,000 times lower than stimulated by g. Intense. B cell activation occurs regardless of antigen specificity. Anti-Ig dextran Does not stimulate antibody release by resting B cells in the absence of cytokines. B fine High levels of Ig secretion are observed when cytokines are added to stimulate antibody release by the vesicles. Can be   Until now, activation of B cells through antigen receptors has been Optimally using anti-immunoglobulin (Ig) antibodies conjugated to such high molecular weight polysaccharides To It was known that it could be. However, the B cell used in the assay The composition that stimulates the release of antibodies by B cells because the vesicles were not highly purified It was not possible to screen things. Fine cells in the B cell supernatant Vesicles are often sufficient to stimulate antibody release by B cells. Secretes in. Therefore, the irritating effect of the investigated composition is less than the added composition Or whether it was due to contaminants.   In contrast, the present invention uses highly purified B cells in the assay system. Preferably, B fine The cells are sorted electronically to obtain highly purified B cells. With highly purified B cells Affects the release of antibodies by B cells activated through multivalent mIg crosslinks It will be possible to measure the stimulating effect of quality. This system was described before the present invention. Had not been. In fact, prior to the discovery of this assay system, the release of antibodies by B cells Composition could not be tested for the only characteristic stimulating activity on .                                 ****   The unexpected effects of the present invention are shown in the following experiments and are depicted in FIGS.   Having generally described the invention, it has been provided herein by way of illustration only and is not limiting. A more complete understanding can be obtained by reference to specific examples that are not intended to Is obtained.Example 1   This example shows that the B cell antibody stimulating activity of the cell supernatant reported in the parent application is one This indicates that it was not due to a known cytokine.   Materials and Methods: Naitonal Cancer Institute (Frederic Female, DBA / 2 mice were used from 7-10 weeks of age. Culture medium used Is 10% fetal bovine serum (Sigma, St. Louis, MO), L-glutamine (2 mM) , 2-mercaptoethanol (0.05 mM), penicillin (50 μg / ml) and strain RPMI1640 (Biofluids) supplemented with peptomycin (50 μg / ml) s), Rockville, MD).   Dextran-conjugated anti-IgD antibody is Hδ^a/ 1 (monoclonal mouse IgG2b (b allo Type)) anti-mouse IgD (a allotype) with high molecular weight dextran (2 × 10⁶ MW ). About 9 dextran-conjugated anti-IgD antibodies were Dex Attached to the trans molecule. FITC-anti-CD3εmAb (2C11) gen) (San Diego, CA).   PE-labeled affinity-purified polyclonal goat anti-mouse IgM antibody was Biotechnology Associates (Southern Biotechnology Associates) -Mingham, AL). Mouse recombinant IL-1 and IL-2 were Dr. Stephanie Vogel (USUHS, Bethesda, MD) and Dr. Maurice Gately (Hoffman La Roche, Nat) Courtesy of Leh, NJ). Recombinant mouse IL-3 and GM-CSF Purchased from Pharmingen. Polyclonal goat anti-mouse IL-3 and GM -All CSF antisera are from R & D Systems (Minneapolis, MN). Purchased.   The functional assay is a 96-well flat bottom coster plate (Costar, Kembu). Ridge, MA). Cultured cells are treated with 6% CO_TwoAt 37 ° C in a humid atmosphere containing × 10^FiveCells / ml were incubated in a total volume of 200 μL.   Polyclonal IgM concentrations were measured by ELISA. Ig used in each assay Quantification was performed by comparison with the M standard curve.   B cell preparation and culture: A B cell-rich population was obtained from splenocytes. Rat anti-Thy-1 , Anti-CD4, and anti-CD8 monoclonal antibodies, followed by monoclonal T cells were removed by anti-rat Igk and complement. Cells are 70, 65, 60, and And 50% Percoll solution (densities 1.086, 1.081, 1.074, and 1.062 g, respectively) / ml) based on the density on a discontinuous Percoll gradient consisting of: High density ( Small, resting) cells were recovered from the 70-65% interface and contained 9090% B cells. Next After staining with FITC-anti-CD3ε + PE-anti-IgM antibody, the EPICS elite cytometer ( (M) IgM + CD on Coulter Corp., Hiala, FL) Highly purified B cells were obtained by electronic cell sorting of 3-cells. sorting Immediate re-analysis of the isolated cells showed consistently> 99% B cells Was. These cells were used for all experiments.   The TH1 mouse cell line was cloned and used for experiments. As described in the parent application, B A composition that stimulates the release of the antibody by the cells was isolated. Determine the identity of the components of the composition Monoclonal antibodies specific to IL-1, IL-2, IL-3, etc. Activity of all known cytokines in addition to Was systematically suppressed.   The composition comprises two independent B cell antibody release stimulants, IL-3 and GM-CSF. Therefore, even if experiments were performed to suppress each of the known cytokines one by one, It did not result in complete suppression of the defined activity.Example 2   This example demonstrates the T cell culture supernatant, GM-CSF, and I identified in the parent application. Measure the relative stimulating effect of L-3.   IgM secretion (ng / ml) by B cells in various media was measured. Diagram of culture medium 1 along the left side. The culture medium contains anti-IgD dextran and IL-1 + IL-2; Anti-IgD dextran and IL-4; soluble CD40 ligand and IL-1 + IL-2; soluble C D40 ligand and IL-4, membrane CD40 ligand; membrane CD40 ligand and IL-1 + IL-2; And membrane CD40 and IL-4. The composition investigated was as described in the parent application. Cell culture supernatants, RA5-SN, GM-CSFN and IL-3. Control for each medium The composition was examined similarly.   As shown in FIG. 1, the compositions tested were dextran-conjugated anti-IgD antibodies, IL-1 and IL-1. And the highest IgM secretion in media containing IL-2 and IL-2. Both GM-CSF and IL-3 RA5-SN supernatants have significant IgM secretion by B cells as compared to the other compositions tested. Indicated.Example 3   This example demonstrates that IL-3 and GM-CSF stimulate Ig secretion by activated B cells. Is shown.   Dextran binding in the presence or absence of various concentrations of IL-3 or GM-CSF B-cell by anti-IgD antibody (3 ng / ml) + IL-1 (150 U / ml) + IL-2 (150 U / ml) Was activated. IgM secretion was measured by ELISA 6 days after the start of culture.   Purified resting B cells that were electronically sorted proliferated, but dextran-conjugated anti-I In response to activation by gD antibody or dextran-conjugated anti-IgD antibody + IL-1 + IL-2, I g could not be secreted. As reported in the parent application, anti-CD3-activated CD4 + T Supernatants obtained from the H1 and TH2 clones were dextran-conjugated anti-IgD antibodies + IL-1 + Induces a strong Ig secretion response by B cells stimulated with IL-2. This stimulus is IL-4 and I L-5 independent.   TH1 and TH2 clones secrete IL-3, GM-CSF, and TNF-α upon activation To determine whether these cytokines induce Ig secretion Was.   Cytokines are titrated with increasing logarithms at a final concentration of 0.1-1000 U / ml. Was. TNF-α had no effect on Ig synthesis. In contrast, IL-3 and GM-CSF Shows Ig above secretion observed only with dextran-conjugated anti-IgD antibody + IL-1 + IL-2 Stimulated a significant enhancement of secretion (FIG. 2). Both IL-3 and GM-CSF are 100 U / ml Stimulated the optimal Ig secretion response and increased 19-fold and 9-fold, respectively. IL-3 or GM -Induction of low but significant amounts of Ig synthesis in response to CSF is still observed at 1-10 U / ml Was done.   In many experiments, the degree of increase in Ig secretion mediated by IL-3 and GM-CSF was , Typically in the range of 10-50 fold (FIG. 3). Dose response or induction of Ig secretion No significant difference between IL-3 and GM-CSF at any of the optimal levels Was.Example 4   This example demonstrates that anti-IL-3 and anti-GM-CSF antibodies react in response to IL-3 and GM-CSF. This indicates that it specifically inhibits the induction of Ig secretion.   Dext in the presence or absence of IL-3 (10 U / ml) or GM-CSF (10 U / ml) By run-conjugated anti-IgD antibody (3 ng / ml) + IL-1 (150 U / ml) + IL-2 (150 U / ml) Activated B cells. In addition, anti-IL-3 (10 μg / ml) and / or anti-GM-CSF ( 10 μg / ml) antibody was added. Control without anti-IL-3 or anti-GM-CSF (10 μg / ml) Was similarly prepared. The IgM concentration was measured by ELISA six days after the start of the culture.   Anti-IL-3 and anti-GM-CSF antibodies characterize the Ig-inducing activity of IL-3 and GM-CSF, respectively. It was differentially and completely inhibited (FIG. 4). These antibodies, alone or in combination, Has no effect on Ig secretion in response to kisstran-conjugated anti-IgD antibody + IL-5 activation (Data not shown). The results also show that the combination of anti-IL-3 and anti-GM-CSF antibodies Let But CD4⁺IL-4 + IL-5 independent Ig induction of supernatant from either TH1 or TH2 clones It has been demonstrated that conductivity can be significantly reduced. IL-3 and GM-CSF are CD40 mediated Inability to induce Ig secretion by activated B cells through signaling pathways Has also been proven.Example 5   This example demonstrates that IL-3 and GM-CSF are activated by dextran-conjugated anti-IgD antibodies. Induces Ig secretion by activated B cells but is activated by unconjugated anti-IgD antibody Does not induce Ig secretion. This example also demonstrates that IL-3 and GM-C This shows that SF acts synergistically with IL-1 + IL-2.   Mlg crosslinkage, which is multivalent but not divalent, is associated with cytokine-mediated Ig content. Co-stimulates secretion and class switching. In this example, IL-3 or GM-CSF In response to costimulation of the corresponding Ig secretion, dextran-conjugated anti-IgD antibodies (Hδ^a/ 1 mAb) Unconjugated anti-IgD antibody (Hδ^a/ 1 mAb).   B cells were first cultured with AF7 supernatant, αIL-4, and αIL-5. AF7 supernatant KLH-specific, Ia derived from C57BL / 6 mice^b-Restriction, confirm CD4 + T cell clone Erect and maintained by standard methods. AF7 is a TH2 clone based on IL-4 production And lacks production of IL-2 and IFN-γ.   Supernatants containing cytokines were obtained from AF7 cell cultures as follows. Tissue culture Incubate the wells at 37 ° C for 3 hours with 10 µg / ml of anti-CD3 mAb (2C11) in PBS. And then washed three times with fresh PBS. Return to dormant state, antigen, APC, and IL AF7 cells on day 7 after stimulation with -2 were applied to anti-CD3-coated plates for various times , 1 × 10⁶Cells / ml, from which cell-free supernatant is obtained and either at -20 ° C or 4 ° C Saved by ore. In the latter case, the supernatant is collected and the cell assay is done within 1-2 weeks. Used for b.   Next, in the presence or absence of IL-1 (150 U / ml) + IL-2 (150 U / ml), Vesicles were conjugated with dextran-conjugated anti-IgD antibody (Hδ^a/ 1-dex, 3 ng / ml) or unbound (Hδ^a/ 1 , 30 μg / ml) with an anti-IgD antibody. Control compositions can be bound or unbound Dextran was not included. IL-3 (100 ml) and / or GM-CSF (100 U / ml ) Was added to prepare a control composition without IL-3 or GM-CSF (FIG. 5). IgM concentration Degree Six days after the start of the culture, the measurement was performed by ELISA.   Conjugated dextran (Hδ^a/ 1-dex), as opposed to unbound dextran (H δ^a/ 1) is ineffective as a costimulator of Ig secretion in the presence of IL-3 or GM-CSF (Table 1). Unbound dextran increases cell size and increases MHC class II Despite being able to induce early B cell activation events, such as increased induction, these Was obtained. IL-3 or GM-CSF alone secretes Ig detectably Resting B cells could not be induced (Table 1). However, IL-3 and GM- When CSF is added during the culture of dextran-conjugated anti-IgD antibody activated cells, extracellular IL-1 + In the absence of IL-2, compared to stimulation with dextran-conjugated anti-IgD antibody alone, A 7.4-fold and 5.4-fold induction of Ig secretion occurred, respectively. Dextran-conjugated anti-IgD anti Addition of IL-1 + IL-2 to cells activated only in the body does not increase Ig secretion anymore Was.   Dextran-conjugated anti-IgD antibody by combining IL-3 or GM-CSF with IL-1 + IL-2 Ig secretion compared to that observed in B cell cultures activated with + IL-1 + IL-2 alone Was enhanced by a factor of 48 and 75. Thus, IL-1 + IL-2 is a dextran-conjugated anti- Induced Ig secretion by resting B cells with IgD antibody activation, strong against IL-3 or GM-CSF Synergistic.                                   Table I Culture medium Stimulation level      Hδ^a/ 1-dex 65      IL-3 <24      GM-CSF <24      Hδ^a/ 1-dcx ＋ IL-3 480      Hδ^a/ 1-dex + GM-CSF 350      Hδ^a/ 1-dex + IL-1 + IL-2 57      Hδ^a/ 1-dex + IL-1 + IL-2 + IL-3 2750      Hδ^a/ 1-dex + IL-1 + IL-2 + GM-CSF 4250      Hδ^a/ -1 <24      Hδ^a/ -1 + IL-1 + IL-2 <24      Hδ^a/ -1 + IL-1 + IL-2 + IL-3 <24      H δ ^a / -1 + IL-1 + IL-2 + GM-CSF <24 Example 6     This example demonstrates that both IL-3 and GM-CSF primarily stimulate B cell differentiation to Ig secretion. It shows that it works through.   To elucidate the mechanism by which IL-3 and GM-CSF enhance Ig secretion, And the effect of GM-CSF on cell hyperproliferation were determined in comparison with Ig secretion.   B cells were isolated in the presence of IL-3 (100 U / ml) and / or GM-CSF (100 U / ml) In the absence of dextran-conjugated anti-IgD antibody (3 ng / ml) + IL-1 (150 U / ml) + IL Activated with -2 (150 U / ml). Surviving cells (cells excluding trypan blue) Counting was performed 4 days after the start of the culture using a ball calculator. IgM concentration in replication culture Six days after the start of the culture, the measurement was performed by ELISA. Data are the mean of two cultures ± flat Expressed as the standard error of the mean.   IL-3 has a significant but less than 2-fold increase in viable cell outgrowth In contrast to stimulating strength, GM-CSF had no significant effect on the degree of cell proliferation ( (Fig. 6).^ThreeStudies evaluating DNA synthesis based on H-thymidine incorporation have Consistent with findings.   Induction of Ig secretion in replication cultures in response to IL-3 and GM-CSF was 13.8-fold and 13.8-fold, respectively. And 13.6 times. These results indicate that the main effects of IL-3 and GM-CSF were Stimulates a strong increase in the average amount of secreted Ig Does not increase the total number of Thus, from this data, IL-3 and And GM-CSF bind to resting B cells activated through multivalent antigen receptor crosslinks. It is strongly suggested that this is a differentiating factor. The combined action of IL-3 and GM-CSF Using the optimal dose of each cytokine, consistently an additional Ig secretion response The above reaction was caused (FIG. 6). These data are for IL-3 and GM-CSF. It indicates that it acts multiplicatively. A similar degree of synergy was due to the absence of IL-1 + IL-2. -3 and GM-CS on cells activated by dextran-conjugated anti-IgD antibody in the presence It was also observed in combination with F (data not shown).Example 7   This example demonstrates the effect on antibody secretion of B cells when CD40L is added to the B cell composition. Prove the stimulating effect.   B cells were transformed with dextran-conjugated anti-IgD antibody (3 ng / ml or 0.3 ng / ml) + IL-1 (150 ng / ml).  (U / ml) + IL-2 (150 U / ml). Control composition, IL-3 + GM-CSF added Composition containing IL-3 + GM-CSF + CD40 ligand (CD40L) The IgM concentration in the replica culture was measured by ELISA (Table II). IL-3 and GM-CSF Was present at 100 U / ml. CD40L is recombinant soluble at a final concentration of 10 μg / ml Sex CD8-CD40 ligand fusion protein. Fusion protein, Connecticut Boehringer Ingelheim Pharmaceuticals, Ridgefield Dr. Cary of Boehringer Ingelheim Pharmaceuticals, Inc. (M.Kehry).  The most significant result (61,250 ng / ml) was with 3 ng / ml of anti-IgD-dex and IL-3 + GM-CSF. + CD40L. However, low concentrations of multivalent activator (anti-IgD-deX At 0.3 ng / ml), significant IgM secretion was also obtained (42,125 ng / ml). This As described above, even at a low concentration of a conjugate, the inclusion of CD40L in A strong antibody release reaction can be obtained.Example 8   This example demonstrates antibody secretion of B cells when IFN-γ is added 24 hours after stimulation of B cells. Shows the stimulating effect on   B cells were isolated in the presence or absence of IL-3 (100 U / ml) + GM-CSF (100 U / ml). Dextran-conjugated anti-IgD antibody (3 ng / ml) + IL-1 (150 U / ml) + IL-2 (150 U / ml ). The IgM concentration in the replica culture was determined by adding the control composition, IFN-γ, for Join The composition obtained and the composition to which IFN-γ was added for 24 hours were measured by ELISA. Specified. Data are expressed as the mean of two cultures ± standard error of the mean (Figure 7).   Composition containing anti-IgD-dex activated B cells and IL-1 + IL-2 does not add IFN-γ In some cases, minimal IgM secretion (885 ng / ml) and even when IFN-γ is added at 0 hours It showed minimal secretion (975 ng / ml). IFN-γ was added 24 hours after B cell stimulation Of course, antibody secretion jumped to 11,250 ng / ml.   In a composition comprising anti-IgD-dex activated B cells, IL-1 + IL-2, and IL-3 + GM-CSF And more significant results were obtained. In medium without IFN-γ, antibody secretion was 21,250  ng / ml. Surprisingly, when IFN-γ was added at 0 hours, antibody secretion was 7, It was suppressed to 250 ng / ml. However, when IFN-γ was added 24 hours after B cell stimulation, A multiplicative response occurred, producing as much as 200,000 ng / ml of antibody secreted.Example 9   This example illustrates exemplary vaccines and vaccine adjuvants using the compositions of the present invention. Offer a bunt.   Table III shows a typical vaccine using the composition of the present invention. As shown in the table, Some vaccines are conjugate vaccines. Conjugation methods are well known to those skilled in the art, Brunswick et al. (Brunswick),J . Immunol.140: 3364 (1988) Heteroligation method; Wong S.S. Ross Link Chemistry (Chemistry of Protein Conjugates and Crosslinking) ", CRCPress, Boston (1991); and Brenkeley et al. Brief Overview of Preparation of Protein Conjugates by Tens and Crosslinking Agents (Brief Survey of Methods for Preparing Protein Conjugates With Dyes, Haptens and  Cross-Linking Agents) ",Bioconjugate Chemistry,ThreeNo. 1 (January 1992) including.   The polyvalent carrier is placed on a polyvalent conjugate used in vitro (ie, anti-IgD-dex). Be replaced. Such carriers include, for example, dextran, or pneumococci, It can be a capsular polysaccharide from bacteria such as streptococci, or meningococci. Polysaccharides are Depending on the intended use of the vaccine, it may or may not be medically appropriate Is also good.   The cytokine can be GM-CSF, IL-3, IFN-γ or a combination thereof.   This example is not intended to be limiting and other types of vaccines Will become apparent to those skilled in the art from consideration of the specification and practice of the present invention. I think that the.                                       Table IIIVaccine structure and components 1. Cytokine^*+ Antigen (mixed); simultaneous administration in aqueous solution, sustained release particles, adjuvant Etc. 2. Cytokine + antigen + polyvalent carrier; cytokine, antigen, or both directly Can be bound to a carrier; ie: 3. Cytokine-antigen; direct covalent bond 4. Cytokine-antigen bound (covalently bound) to a polyvalent carrier, ie: 5. Cytokine-antigen (fusion protein) 6. Cytokine-antigen (fusion protein) bound (covalently bound) to a polyvalent carrier 7. Peptide-cytokine (fusion protein) + antigen 8. Peptide-site, wherein the fusion protein, the antigen or both are bound to a polyvalent carrier Cain (fusion protein) + antigen 9. Cytokine-multivalent carrier, ie: Ten. Antibody complex (ie, IL-3 + anti-IL-3) + antigen 11． Antibody conjugate (ie, IL-3 + anti-IL-3) + antigen + multivalent carrier (cytokine, The antigen, or both, can be bound to the carrier) 12． Anti-cytokine antibody; this is a neutralizing vaccine 13. Anti-cytokine antibody + polyvalent carrier; this is a neutralizing vaccine 14. The vaccines of Examples 1 to 13 are mixed with CD40L or added to a polyvalent carrier. Can be further modified by 15． The vaccines of Examples 1 to 14 are GM-CSF, IL-3 or INF-, such as IL-1 + IL-2. Adding one or more cytokines other than γ, mixed or bound to a polyvalent carrier Can further be modified.^* The cytokine can be GM-CSF, IL-3, IFN-γ or a combination thereof.                                 ****   In summary, the experimental results indicate that IL-3 and / or GM-CSF release antibodies by B cells. These cytokines act synergistically to stimulate It is shown to stimulate the release of antibodies by B cells. This induction is due to anti-IL-3 and And anti-GM-CSF antibody, respectively. IF added 24 hours after culture The stimulatory effect of N-γ is also shown.   The effects of IL-3 and GM-CSF were demonstrated by dextran-conjugated anti-Ig Is enhanced by multivalent antigen receptor cross-linking as mediated by . The effects of IL-3 and GM-CSF also do not induce high levels of membrane Ig crosslinks May be enhanced by antigen. Dextran-conjugated anti-Ig antibody and IL-1 + IL-2 Required for optimal IL-3 and GM-CSF mediated Ig secretion, Both SFs have moderate Ig content due to cells activated with dextran-conjugated anti-Ig antibody alone. Stimulates the renal response. This is because IL-3 and GM-CSF are directly This is the first report to prove that it can work.   In addition, the use of GM-CSF, IL-3, IL-1 + IL-3, and IFN-γ added 24 hours after culture Use resulted in maximal antibody secretion, but a modest increase in antibody secretion was also observed after culture. The addition of IFN-γ to the time was also observed.Example 10   This example illustrates the preparation of a cytokine-protein fusion product. A. Construction of plasmid YEpFlag-1 expression plasmid (Kodak Scientific Cloning and expression of fusion proteins It was used for. YEpFlag-1 vector: ADH2 promoter for regulated expression in yeast Ta , A tryptophan marker for selection in yeast, and a protein in yeast. Contains α-factor for white matter secretion. The vector also contains a multiple cloning site, Also includes the sylin resistance gene, and the Flag peptide sequence. Kpnl and BamHI restriction sites Insertion of fusion protein DNA into Flag peptide sequence and α-factor peptide sequence Fourteen base pairs were removed from the row. To facilitate purification of the fusion protein, six Hiss A idine residue was added to its C-terminus, thereby binding to Ni-NTA resin and Ni-NTA tree The acid can be eluted from the fat. This method subsequently removes the Flag peptide enzymatically. Better than Flag-dependent purification because it does not require You. The 14 base pairs removed from the 3 'end of the α-factor extracellular secretory peptide sequence So returned.   Prior to constructing the fusion proteins, the individual proteins were first individually cloned. Step Rote D, pneumococcal surface protein A (protein A) and pneumolysin (pneum olysin)son), Infect.and Immun.632,696-699 (February 1995); Langerman et al. (Langermann), J. Exp. Med. 180, 2277-2286 (December 1994); And Walker et al. (Walker) Infect.and Immun.55,5,1184-1189 (1987 May 5) ) And cloned according to the method described above. The fusion protein is located on the N-terminal site Caine is linked to six histidine tails on the C-terminus by six glycine residues. Was constructed by binding to an antigen protein containing B. Construction of IL-2 and GM-CSF plasmids Human IL-2 or mouse G containing plasmid M-CSF cDNA was used as a template for PCR. The 5 'primer is a Kpnl restriction site and Contains the base pair of the α-factor removed, as well as the first 18 bases at the N-terminus of the protein . The 3 'primer is the last 18 bases at the C-terminus of the protein, a cytokine It contained three glycine codons between the row and the Smal restriction site. PCR product and Y Digest EpFlag plasmid with Kpnl and Smal restriction endonucleases and Was done. E. coli DH10B competent cells are shaped by the ligation mixture. Quality control, select ampicillin resistant clones and transfer plasmid DNA to several Purified from clone. The presence of IL-2 and GMCSF genes was determined by PCR. Sequences are from Applied BioSystems DNA sequencing Check that the recombinant plasmids are pYIL2 and pYGM. Identification did. C. Pneumococcal surface protein A (PA), protein D, and pneumolysin (Ply) Rasmid construction Genomic DNA from S. pneumonia Rx1 was ligated to PA and Ply It was used as the other mold. Haemophilus influenzae Plasmid containing the cloning gene for Roblin D-binding protein (PD) (pHIC 3 48) as a template for PCR using convenient restriction sites at the ends (SmaI and BamHI). ) To obtain a PD-gene fragment. The 3 'primer is the 6 codons of histidine and the stop codon. Don included. The histidine tail at the C-terminus of the protein is Ni-NTA resin (Qiagen (Qiagen)). The PCR product is expressed and isolated in yeast culture. PYEPFLAG-1 expression vector (Kodak Scientific Sma I and Bam HI of the imaging system (Kodak Scientific Imaging System) Cloned between the sites. Plasmid containing the correct PD-gene copy Identified as PD. The PA gene is cleaved to 888 bp, which is an epitope of T cell activation. (Obtained from Dr. Larry McDaniel of the University of Alabama) Together, they encode that portion of PA that retains its B cell antigenic epitope. P The CR fragment is composed of three glycine ligases as linkers between the SmaI restriction site and the components of the fusion protein. Don, PA sequence (Ply sequence), 6 histidine codons (used for protein purification) Histidine sequence), a stop codon and a BamHI restriction site at the 3 'end. P The CR product was cloned into YEpFlag1 between the SmaI and BamHI sites. After transformation, Clones were selected for ampicillin resistance. The presence of PA or Ply gene is PCR Detected by Plasmids were named pYPA and Ply and cloned Column accuracy is determined by the Applied Biosystems model 373A DNA sequencing Confirmed by sequence analysis on the system. D. Construction of fusion protein plasmid: SmaI / BamHI containing sequences for PA and PLY. Fragments were excised from each plasmid and pYIL2 digested with Smal and BamHI. Or ligated with one of pYGM. New fusion protein expression plasmid And pIL2PA (IL2 + PA), pIL2Ply (IL2 + Ply), pGMPA (G MCSF + PA) and pGMPly (GMCSF + Ply). E. Pan DR Epitope Peptide (PDREP)-Universal for MHC Class II Restricted T Cells " Cloning of single-stranded sequence for epitope Alexander et al. (Alexande r ), Immunity, 1, 751-761 (1994). Has aKFVAAWTLKAAa. Our construct forms part of a fusion protein This PDREP has the same structure, but the terminal L-alanine residue is changed to D-alanine. Has been replaced.   To generate a fragment of DNA containing the sequence of PDREP, two complementary synthetic oligonucleotides The mixture of leotide was heated to 90 ° C. and cooled slowly. Oligonuk, if necessary The 5 'end of the reotide was phosphorylated before annealing.   Agarose gel electrophoresis showed the presence of double-stranded fragment (~ 60 b.p.) . The sense strand is 53b and the antisense strand is 57b. At the 5 'end, the fragment is a Kpnl restriction site Cohesive end corresponding to α leader peptide codon excluded during cloning It had 39 bp for the sequence for reconstitution, PDREP and blunt 3 'end. this Fragments were ligated with Kpnl and Smal digested PYPA. Ampicillin resistance The DNA from the sex clone is analyzed by PCR and sequenced.   To prepare a triple PDREP sequence DNA fragment, a 78-base double-stranded sequence PDREP fragment was Produced by annealing of each synthetic oligonucleotide. Lighesi After several stages of the solution, we have established a complete large gap between the Kpnl and BamHI sites. Alpha leader peptide sequence, three 39 base pair sequences for PDREP, and PA A construct containing the truncated gene was obtained. F. Yeast transformation Starter culture of tryptophan (-) yeast of BJ3505 was performed using YP D medium was cultured overnight at 30 ° C. from 25 ml of the frozen stock. Fresh YP from overnight culture Inoculate 100 ml of D medium and incubate overnight until O.D. at 280 nm is between 0.3 and 0.5 did. The yeast and fusion protein plasmids were then converted to published protocols. Therefore, it was prepared for electroporation. Electroporation set at 1.5 KeV, 200 ohm, 25 uF This was performed using a BioRad gene pulser. Electroporated yeast When seeded on a selective medium plate, transformed colonies appeared in 3 to 5 days. G. Isolation of yeast expressing the fusion protein. Transformed colonies were expanded on expression plates. The fusion protein secreting clones were selected by propagation. The expression plate is On top of the expression medium agar plate, place nitrocellulose and then cellulose acetate plate. I Prepared by mounting Luther. Colonies are grown on plates for 3-5 days After that, the nitrocellulose was removed and immunoblotting was performed. 1x TBS membrane Wash / block with Tween 20 (TBS20) for 2 hours, 1 hour with each anti-protein antibody Incubated. The membrane was washed with 1 × TBS20 for 1 hour and then rabbit anti-mouse I Incubated for 1 hour in gG antibody, then washed for an additional hour with 1 × TBS20. The membrane was incubated for 1 hour in 1 g of alkaline phosphatase-labeled goat anti-rabbit. , Washed with TBS20 for 1 hour and developed with BCIP. Select colonies with positive protein secretion 25 minutes of expansion medium frozen in 60% glycerol after overnight incubation.  ml was inoculated. H. Batch fusion protein production Prepared from Add 10 ml from the starter culture to 1 L of expansion medium and at 30 ° C Incubation was performed with shaking at 240 rpm. 3 days after inoculation The supernatant was collected, and the supernatant was collected by centrifugation at 2000 rpm, and sterilized by filtration. Next The soil was concentrated 5 times, and dialyzed against 10 times the volume of 2 × PBS using an Amicon stirring cell. I. Purification 1 ml of Ni-NTA resin (Qiagen (Q iagen)) for 2 hours on a rotating shaker, The fusion protein containing the six residues of histidine was purified. Medium and Ni-NTA in small 10 In addition to the cm column, Ni-NTA was washed with 1 L of 1 × PBS. Protein is 2.5 M sodium acetate Elution was carried out using lithium, pH 4.5.   Fractions were collected and the O.D. at 280 nm was measured to determine relative protein concentration; Fractions with a D. of greater than 0.1 were pooled and dialyzed against 1 × PBS pH 7.4. Original medium Media samples, concentrated media, and purified protein from And stained with Coomassie blue or immunoblot. Immunoblot Gel for use with Pharmacia's NovaBlot And transferred to nitrocellulose and immunoblotted as described above. protein The control for identification was medium from yeast with the pYEP plasmid without the fusion protein. , And positive controls for IL-2 and PSP-A. Dyed with Coomassie Blue Compare the protein band on the colored gel to the molecular weight marker to determine the size of the fusion protein. Was estimated. Recombinant protein without a histidine tag was obtained on a 5100HR column. Dating By isolation. Purity is determined by SDS PAGE and immunoblotting of each component of the fusion protein. Assay. Samples were also prepared by Beckman Ultrasferigel (Beck man Ultraspherigel) may be determined by analytical HPLC on a 2000 gel filtration column . The cytokine activity of the final product was examined. J. Binding of proteins or cytokine proteins to polysaccharides   Pn14 (SmithKlin or ATCC) is 1-cyano-4-dimethylamido Using nopyridinium tetrafluoroborate (CDAP), I will guide you. Briefly, 30 μl of CDAP (100 mg / ml acetonitrile solution) by adding 30 μl of 0.2 M trimethylamine (TEA) 30 seconds later , 1 ml of Pn14 (10 mg / ml aqueous solution) was activated. At 2 minutes, 0.5M hexane 0.5 ml of a 0.75 M HRPES, pH 7.5 solution of diamine was added. 1.5 hours later, transfer the product to P6 Cartridge (desalted with BioRad, equilibrated with physiological saline and ultrafiltered And desalted again. The amine content was determined by Vidal et al. (Vidal), J.Imm.M ethods, 1986, 86, 155, and polysaccharides were obtained from Monsigny et al.Monsigny ), Anal. Chem. 1988, 175, 525.   The protein was made 5-20 mg / ml in 0.15 M HEPES, pH 7.5, and 0.1 M stock of DMF A 20-fold molar excess of N-succinimidyl S-acetylthioacetate (SATA, Thiolated with Lokem (ProChem, Rockford, IL). 2 in the dark at room temperature After incubating for a period of time, sample using a P6 cartridge (Bio-Rad). Desalted and equilibrated with 10 mM NaAc, 0.1 M NaCl, 2 mM EDTA, pH 5, Concentrate to 10-20 mg / ml using a Centricon 30 device (Amicon) Was.   NH2-Pn14 was combined with 0.1 M N-hydroxysuccinimidyl iodoacetate per 1 mg of Pn14. Acetate (SIA, Prochem, Rockford, IL) with 10 μl of iodoacetylation After hours, desalted and concentrated as above.   Combined thiolated protein and iodoacetylated Ps produce 0.5 M hydroxyl The reaction was started by adding 1/9 volume of a 0.75 M HEPES solution of amine pH 7.5. I let you. After the overnight reaction, the solution is brought to 0.2 mM in mercaptoethanol for 1 hour. And then incubate with 10 mM iodoacetamide for 10 minutes. Be I did it. The conjugate was passed through a S400HR gel filtration column and equilibrated with PBS. High molecular weight material Pool the charge and add a Millex GV filter (Millipore) Filter sterilized by passing through. Conjugates and bio- Assay for polysaccharides was performed using Monciny et al. Monsigny) and assayed as described above. K. Binding of "universal" T-cell epitopes to polysaccharides   Reduce the universal T cell epitope peptide containing N-terminal cysteine with DTT, And equilibrated with 10 mM sodium acetate, pH 5, HEPES buffer, pH 7.5. In the reaction with iodoacetylated polysaccharide. Or even with a one-step process Good. Peptides are synthesized at the N-terminal hydrazide and reacted directly with CDAP-activated polysaccharide at pH5. I responded. In either case, after overnight reaction, the conjugate is dialyzed against physiological saline. And sterilized by filtration. Peptides are quantified from their absolute spectra and Was assayed by the method of Monsigny et al. L. Measuring enhancement of anti-protein and anti-polysaccharide antibody responses   In mice, protein, protein-cytokine or protein-cytokine-multiple A subcutaneous injection of 0.01-10 μg of saccharide was used to demonstrate anti-protein and anti-polysaccharide responses at 14, 28 and Measured after 52 days. Antibody response was determined by ELISA. Obtained enhanced anti The protein and anti-polysaccharide reactions are shown in Table IV.                                   Table IV                    By PspA-IL2-Pn14 vaccine construct                  Induction of enhanced anti-protein and anti-polysaccharide responses M. Vaccines engineered with a universal T cell epitope and conjugated pneumococcal polysaccharide type 14 Of in vitro T cell proliferation in response to in vitro constructs   Mice vaccinated with control or experimental vaccines with T cells from draining lymph nodes And purified. 2.5 x 10 cells^Four~ 2 × 10^FiveCells / well as a source of cells present Itomycin C treated splenocytes 5 × 10^FourThe cells were cultured together. The results are shown in Table V.                                    Table V             For vaccine constructs engineered with universal T cell epitopes (TCE)             In vitro T cell proliferation reactedMice can be used for universal T-cell epitope (TCE), protein D-TCE, or protein D-T Immunization was performed with a CFA solution of CE-pneumococcal polysaccharide type 14. 10 days later, draining lymph nodes are separated And cultured with various stimuli. Thymidine incorporation was measured after 4 days. T thin Cell proliferation was somewhat lower in the group immunized with PD-TCE-Pn14, which is probably It is the result of a chemical bond between PD-TCE and a polysaccharide that can affect key epitopes Conceivable.   Having described the invention sufficiently, departures from the scope of the invention described herein. In fact, it will be apparent to those skilled in the art that many changes and modifications can be made. It appears to be. The appended claims are not limiting.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/535 C07K 14/535 14/54 14/54 17/06 17/06 19/00 19/00 C12N 15/09 C12N 15/00 A [Continued abstract]

Claims (1)

[Claims] 1. Effective amount of granulocyte macrophage colony stimulating factor (GM-CSF), interlo Stimulates the release of antibodies by B cells, including Ikin-3 (IL-3), or a combination thereof Compositions useful for: 2.a) Granulocyte macrophage colony stimulating factor covalently linked to a polyvalent carrier (GM-CSF), interleukin-3 (IL-3), or a combination thereof;   b) Vaccine antigen covalently linked to a polyvalent carrier A conjugate vaccine comprising: 3. GM-CSF and IL-3 are fused together or individually fused to another suitable protein. 3. The combination of claim 2 to form a fusion protein, which is bound to a polyvalent carrier. Vaccine. 4. The vaccine according to claim 2, further comprising interferon-γ (IFN-γ). 5. CD40 ligand, or at least one other than GM-CSF, IL-3, or IFN-γ 3. The vaccine of claim 2, further comprising a cytokine of, or a combination thereof. 6. At least one cytokine is interleukin-1 (IL-1) and interleukin-1. The vaccine according to claim 5, which is a combination of -leukin-2 (IL-2). 7. CD40 ligand, or at least one other than GM-CSF, IL-3, or IFN-γ 6. The cytokine of claim 5, or a combination thereof, is covalently linked to a multivalent carrier. The vaccine as described. 8. Whether the polyvalent carrier is a bacterial coat polysaccharide, dextran, and a genetically engineered vector 9. Any of claims 2, 3, 4, 5, 6, 7, or 8 selected from the group consisting of: The vaccine according to claim 1. 9. The bacterial capsular polysaccharide is derived from pneumococcus, streptococcus, or meningococcus. Item 10. The vaccine according to Item 8. 10.a) Antibodies to granulocyte macrophage colony stimulating factor (GM-CSF);   b) an antibody to interleukin-3 (IL-3); and   c) Antibody to interferon-γ (IFN-γ) A neutralizing vaccine adjuvant comprising one or more antibodies selected from the group consisting of: 11. The method of claim 10, wherein at least one of the one or more antibodies is bound to a multivalent carrier. Adjuvant. 12． Whether the polyvalent carrier is a bacterial coat polysaccharide, dextran, or a genetically engineered vector 12. The adjuvant according to claim 11, wherein the adjuvant is selected from the group consisting of: 13. Wherein the bacterial capsular polysaccharide is derived from pneumococcus, streptococcus, or meningococci. Item 12. The adjuvant according to item 12.