The present invention provides methods of use of various antibody-immunostimulant fusion proteins as adjuvants of antigenic protein vaccinations to elicit humoral and/or cellular immune responses in vaccinated subjects. Compositions which include these fusion proteins and innate and/or exogenous antigenic proteins are also provided.

Description of the Invention

Antibody-Immunostimulant Fusion Proteins as Adjuvants of Protein Vaccination

The present invention is based on the use of antibody-immunostimulant fusion proteins not to directly target specific cells to destroy them, etc. (e.g., tumor cells, or infectious bacteria, etc.) but instead, the present invention is based upon targeting a soluble (or another sate, see, below) form of an antigen. The antigen, along with antibody-immunostimulant fusion protein acting as its adjuvant (e.g., a substance or molecule acting or helping to increase an immune response), elicits an immune response (humoral and/or cellular) within the subject against the antigen (e.g., the disease related antigen such as those present on tumor cells, on infectious organisms, etc.). The antigen to which the antibody immunostimulant fusion protein acts as an adjuvant need not be a soluble antigen, though that is often the case in many embodiments. Other embodiments comprise wherein the antigen to which the antibody-immunostimulant fusion protein acts as an adjuvant include such forms as, but not limited to, an antigen(s) (soluble or insoluble) bound to a matrix such as a bead, etc., an insoluble aggregate of antigens or aggregate of soluble antigens (both of which could also comprise other materials, e.g., to help in aggregation, etc., non-viable cell associated antigens (e.g., also including non-viable organismal associated antigens such as form bacteria, viruses, etc., antigens conjugated with liposomes, etc. Additionally, in yet other embodiments, the antibody-immunostimulant fusion protein which acts as the adjuvant to the antigen may itself be conjugated with, e.g., a liposome, etc. while the antigen is, or is not, so conjugated to a liposome.

The present invention provides methods of use of various antibody-immunostimulant protein fusions as adjuvants for antigenic protein vaccinations and methods of prophylactically and/or therapeutically treating a disease state in a subject. Compositions comprising the fusion proteins and antigens of the invention are also provided.

Furthermore, the immune response elicited by the methods and compositions of the invention are specific against, as explained in more detail below, the disease related antigen present within (or, if used in prophylactic treatment, expected or possibly expected within) the subject or closely antigenically related molecules. Thus, for example, an embodiment of the invention optionally comprises an anti-tumor associated (TAA) antigen antibody-immunostimulant and a soluble disease related antigen (e.g., here the TAA) used as a therapeutic treatment. The immune response elicited by such treatment is optionally against such antigen (or a closely related antigen) present on, e.g., the cell surface of tumors present within the subject.

It will be appreciated that the above, as well as the other sections herein, discusses "antigen," e.g., in terms of an antigen administered to a subject along with an antibody-immunostimulant fusion protein. This usage should be understood to describe e a disease related antigen as described previously unless otherwise stated.

The antibody-immunostimulant fusion proteins herein act as adjuvants to disease related antigens (e.g., tumor antigens presented by or on tumor cells or shed from tumor cells such as HER2/neu, or antigens presented by or on an infectious organism such as a virus, a bacteria (e.g., a protein A antigen from Staphylococcus aureus), a fungus, a prion, a parasite, an autoimmune disorder, etc.). The current invention utilizes the humoral and/or cellular immune response generated by the disease related antigen (and its antibody-immunostimulant adjuvant) as a means of therapeutic and/or prophylactic treatment of the subject against the organism or disease which generated or caused the disease related antigen's presence in the subject.

The subject's immune response is optionally elicited by the antibody fusion proteins binding their respective antigen (i.e., their respective disease-related antigen) to form an antibody-antigen immunocomplex. See, FIG. 2 (see Original Patent). Of course, such optional mechanism of action should not be construed as limiting. Other possible and/or additional mechanisms of action optionally are used by the efficacious methods and compositions of the invention. Optionally, this immunocomplex delivers the disease-related antigen to a dendritic cell (DC) or to another appropriate antigen presenting cell (APC) through the interaction of the antibody-immunostimulant fusion protein with surface receptors on the DC or APC such as GMCSF, IL-2, IL-12 receptors, etc. See, FIG. 2. Depending upon, e.g., the specific immunostimulant molecule used in the fusion proteins (e.g., the specific cytokine, chemokine, etc.) the presentation of the antigen to the DC or APC optionally leads to a potent activation of one or both arms of the immune response, i.e., cellular (T.sub.H1) and humoral (T.sub.H2). Such activation optionally produces a significant immuno-protective activity when the vaccinated subject is challenged with the same (or even, in some embodiments, a closely related) disease related antigen.

Again, it should be noted that the current invention encompasses a myriad of fusions and their uses against a myriad of diseases/conditions. In many examples herein, the anti-HER2/neu antibody fusion, etc. is used as one example, but such should not be construed as limiting. Discussion of HER2/neu protection, etc. is to illustrate the general concepts of the methods and compositions of the invention, namely that use of an antibody-immunostimulant fusion protein as an adjuvant of an antigen vaccination leads to humoral and/or cellular immune response in a subject and thus can be used as a therapeutic and/or prophylactic treatment of the subject for the disease or infection which presents such antigen.

The optional interaction of the antibody-immunostimulant fusions and the disease-related antigen with the APC or DC, as illustrated in FIG. 2 could change the quantity and/or quality of antigen presentation (e.g., from that which would occur with solely the disease related antigen used in treatment), which could result in (depending again upon, e.g., the specific immunostimulant fused with the antibody) a strong T and/or B cell immune response against the disease related antigen. Additionally, the general immunostimulatory activity of many immunostimulants (e.g., of cytokines) which are fused to the antibody fusion proteins of the invention may also optionally contribute (or may optionally) per se to the enhancement of the immune response against the targeted antigen (e.g., IL-2-cell proliferative signal, GMCSF-APC activation and IL-12-deviation to T.sub.H1, etc.). Again, such optional mechanisms of action should not be construed as limiting; the efficaciousness of the methods and compositions of the invention are not limited to only these mechanisms of action.

The elicited immune response (i.e., produced through use of the methods, etc. of the current invention) is against the disease related antigens expressed on the surface of, e.g., cancer cells or infectious agents (humoral immune response) as well as against disease related antigen peptides associated with MHC class I on the surface of tumor cells or infectious agent cells, etc. (cellular immune response). In some embodiments, the current invention additionally elicits humoral and/or cellular immune responses against other closely related antigens (e.g., antigens closely related either structurally or conformationally to the antigen used as the protein vaccination). For example, since HER2/neu has high homology with other growth factor receptors such as epidermal growth factor receptors 1, 2, and 3 (EGF1, EGF2, EGF3), the elicited immune response (humoral and/or cellular) from the invention against HER2/neu is optionally directed not only against the targeted disease related antigen (HER2/neu), but also against other homologous receptors that are expressed on a cancer cell.

In some embodiments, the methods, etc. of the current invention (as well as the toxicological studies, use studies, etc. of the current invention) are carried out in animal models (see, e.g., Examples I and II below), however, the current invention also encompasses embodiments wherein human subjects are utilized (including clinical trials, etc.). In humans, as in other animal subjects, the antibody-immunostimulant fusion proteins serve as an adjuvant of, e.g., a soluble antigen in both, prophylactic or therapeutic vaccinations. Thus, the invention can target patients with specific antigen expressing tumors, e.g., HER2/neu breast cancers, etc. as well as disease-related antigens presented by infectious organisms (viruses, bacteria, etc.) both wherein the tumor/infectious organism, etc. is within a subject (therapeutic) or before such disease/infection arises in a subject (prophylactic). Thus, the applications allowed by the methods and compositions of the invention comprise a broad range of treatments for both human and other animals in protection against numerous disease states, including cancers and infection by microorganisms.

For example, in prophylactic vaccination, patients at high risk to develop tumors (e.g., those tumors that express HER2/neu) are optionally vaccinated with a mixture of antibody-immunostimulant fusion protein and an appropriate tumor antigen (e.g., HER2/neu, etc.). For example, women whose family history indicates a high probability of developing breast cancer are optionally prophylactically treated with an embodiment of the current invention. For example, antibody-immunostimulant fusion proteins comprising an antibody specific for HER2/neu fused with, e.g., IL-2, IL-12, and GMCSF (i.e., in different antibody constructs) are optionally administered to the woman along with an appropriate amount of HER2/neu antigen (see, below). Typically such fusion proteins and antigens are incubated together in order to form the appropriate immuno-complexes before administration to the subject. The use of the invention would thus cause the woman's immune system to develop an immune response against the HER2/neu protein and thus the woman would be better able to more effectively combat any HER2/neu expressing cancers that arose, and would optionally increase her chances of long-term survival.

Again, it should be noted that in other embodiments of the invention, different antibody/immunostimulant combinations are used against different diseases/conditions and thus against different antigens, etc. Thus the current invention is also optionally used to prophylactically treat subjects for exposure to particular viruses, bacteria, etc. For example, the current invention is optionally used to prophylactically treat persons such as health care workers who might be in environments where risk of exposure to particular viruses/bacteria is high. For example, health care workers likely to be exposed to, e.g., S. aureus contamination are optionally prophylactically treated with an anti-protein A antibody-immunostimulant fusion protein and the protein A antigen (see, e.g., Example II below for a similar example with mice). Alternatively, persons likely to encounter, e.g., certain viruses (e.g., such as HIV for sex workers, etc.) are optionally prophylactically treated with an appropriate antibody-immunostimulant fusion specific for an appropriate HIV antigen along with that particular antigen.

In therapeutic treatment vaccinations, patients, e.g., those bearing tumors expressing a particular antigen are vaccinated with a mixture of antigen-specific antibody-immunostimulant fusion protein(s) and the antigen(s) (optionally, the soluble antigen, see above). Again, therapeutic vaccinations are applicable to, e.g., myriad tumor types (and to different antigens presented on the same tumors) and to therapeutic treatment of various infections such as viral, bacterial, etc. So, similarly to a prophylactic treatment (see, above) the antigen targeted can be tumor associated, virus associated, bacterial associated, etc. Therapeutic treatment using the methods and compositions of the invention are especially useful in situations wherein the subject is having difficulty mounting an effective immune response against the disease state. For example, when disease related antigens are not being appropriately interacted with APCs, etc. or when the disease related antigens are recognized as "self" by the immune system, etc.

In some situations, it should be noted, patients will present disease profiles wherein high levels of the specific targeted antigen are present within the patient. For example, some tumors express high circulating levels of soluble antigen (due to, e.g., tumor shedding of the antigen). Such is the case with some HER2/neu expressing tumors; the tumors shed high levels of the antigen. Additionally, in some infections, high levels of a targeted antigen can be present in the patient. Some, e.g., bacterial infections can result in high levels of innately present antigen which is thus able to be targeted. For example, various septicemias can optionally present high levels of soluble antigen in a subject's blood stream. Therefore, in some cases the injection of antibody-immunostimulant fusion protein(s) alone is enough to target the desired antigen. In other words the patient's innate levels of antigen, e.g., soluble HER2/neu, bacterial antigen, etc. are high enough to be targeted by the antibody-immunostimulant fusion proteins and thus trigger the desired immune response. However, even if high levels of innate antigen exist, such patients can also optionally still be injected with a mixture of the antibody-immunostimulant fusion protein(s) and the targeted antigen.

The different antibody-immunostimulant fusion proteins and antigens herein can be used separately or in combination, thus creating an additive or a synergistic effect. In various embodiments of the invention, different immunostimulant domains are optionally used with the same antibody framework (i.e., the same antibody against the same antigen--see, as with the different fusions in Example I, below). Alternatively, and/or additionally, multiple antigens (e.g., two different surface antigens on a bacterial cell, mycoplasm, etc. or two different tumor associated antigens) are optionally used (i.e., the different antigens each have one or more antibody-immunostimulant fusion protein made to target them). Thus, various layers of fine-tuning and specificity are built into the current invention, which thus allow more precise control and targeting of disease treatment in subjects.

Additionally, the methods of the current invention (e.g., as illustrated by treatment with anti-HER2/neu antibody fusion proteins, etc.) are not necessarily a replacement of available therapeutic technologies such as the recombinant antibody Trastuzumab (Herceptin, Genentech, San Francisco, Calif.) treatment. Instead, the current invention is optionally used as an alternative therapy in combination with other treatments (e.g., anti-cancer approaches such as chemotherapy and/or radiotherapy, antibiotics, etc.). For example, in some situations patients with high levels of circulating antigen (e.g., as is seen with tumors that shed ECD.sup.HER2) or with mutated forms of an antigen (e.g., a mutated form of HER2/neu) who do not respond to treatment with Trastuzumab (see, e.g., Baselga et al., 1996 "Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in parities with HER2/neu-overexpressing metastatic breast cancer" J Clin Oncol 14:737-44), optionally can benefit from the methods, etc. of the current invention.

In addition, some embodiments of the current invention are also effective for ex vivo generation of mature dendritic cells. In such case, dendritic cells obtained from subjects are treated (in vitro) with a mixture of antibody-immunostimulant fusion protein(s) and the appropriate soluble (or other format, such as antigen on latex beads, etc.) antigen. Then, the mature and programmed dendritic cells are re-implanted into the patient. This is similar in some aspects to some optional embodiments above, i.e., the antibody-immunostimulant complexes form and interact with an APC, etc., but here, such interaction occurs ex vivo.

Components and Construction of Antibody-Immunostimulant Fusion Proteins and Antigen Vaccines

It will be appreciated that while, e.g., antibody-cytokine fusion proteins of anti-HER2/neu and IL-2, IL-12, and GMCSF were utilized in the examples herein, the current invention encompasses myriad other combinations of immunostimulant molecules and antibodies in the antibody-immunostimulant fusion proteins it uses. In other words, depending upon the specific condition/disease being considered or treated, various combinations of immunostimulant molecules (e.g., cytokines, chemokines, etc.) and antibodies (e.g., different antibody fragments, antibodies of different isotype, and different antibodies with specificity against different antigens) are encompassed within the current invention.

For example, exemplary non-limiting illustrations of antibody-immunostimulant fusion proteins specific for the extracellular domain of the human tumor associated antigen HER2/neu (ECD.sup.HER2) were constructed and used in Example I, etc. (using cytokines in the examples detailed). These antibody fusion proteins were composed of human IgG3 containing the variable region of Trastuzumab (Herceptin, Genentech, San Francisco, Calif.) which was genetically fused to the immunostimulatory cytokines interleukin-2 (IL-2), interleukin-12 (IL-12), or granulocyte-macrophage colony stimulator factor (GMCSF). These recombinant proteins are illustrated in FIG. 1 (see Original Patent).

In addition to the variability of the immunostimulant domain of the fusion proteins utilized herein, the specific antibody domain used also optionally varies. The antibody domains utilized in the examples herein are not to be construed as limiting. For example, different antibodies (e.g., against bacterial antigens, against viral antigens, against different tumor associated antigens, against mycoplasm antigens, against antigens of parasites, prions, autoimmune disorders, etc.) are all optional embodiments of the current invention. This optional variation in antigen specificity allows the methods and compositions of the current invention to be used to treat and/or prevent myriad specific conditions, disease states, etc. Not only is the antigen specificity of the antibody domain variable, but the type of antibody framework which comprises the protein fusion can vary as well. For example the antibody domain of the antibody fusion proteins herein can optionally comprise Fab, Fab', F(ab).sub.2, F(ab').sub.2, Fv, scFv, an antibody fragment, and various combinations thereof, etc.

Antibodies

The current invention utilizes antibody-immunostimulant fusion proteins as adjuvants of protein vaccinations. The antibody immunostimulant fusion proteins used comprise an immunoglobulin molecule (or a portion thereof) that, typically, is specific for the antigen used in the protein vaccination. In typical embodiments, the antibody is specific for a disease related antigen.

The antibody domain of the fusion protein optionally comprises all or part of an immunoglobin molecule and optionally contains all or part of an immunoglobin variable region (i.e., the area of specificity for the disease related antigen) and optionally comprises region(s) encoded by a V gene, and/or a D gene and/or a J gene.

As explained above (see, Definitions, supra) the antibodies used herein optionally comprise F(ab).sub.2, F(ab').sub.2, Fab, Fab', scFv, etc. depending upon the specific requirements of the embodiment. Some embodiments utilize fusion proteins comprising IgG domains. However, other embodiments comprise alternate immunoglobins such as IgM, IgA, IgD, and IgE. Furthermore, all possible isotypes of the various immunoglobins are also encompassed within the current embodiments. Thus, IgG1, IgG2, IgG3, etc. are all possible molecules in the antibody domains of the antibody-immunostimulant fusion proteins used in the invention. In addition to choice in selection of the type of immunoglobin and isotype, different embodiments of the invention comprise various hinge regions (or functional equivalents thereof). Such hinge regions provide flexibility between the different domains of the antibody-immunostimulant fusion proteins. See, e.g., Penichet, et al. 2001 "Antibody-cytokine fusion proteins for the therapy of cancer" J Immunol Methods 248:91-101.

The use of antibody domains fused with various immunostimulants is relatively well known in the art and the use, selection, and construction (or purchase) of appropriate immunoglobins is known to those of skill in the art. See, e.g., Dela Cruz et al., 2000 "Recombinant anti-human HER2/neu IgG3-(GMCSF) fusion protein retains antigen specificity, cytokine function and demonstrates anti-tumor activity" J Immunol 165:5112-21; Penichet et al., 2001, "A recombinant IgG3-(IL-2) fusion protein for the treatment of human HER2/neu expressing tumors" Human Antibodies 10:43-49; Penichet et al., 2001 "Antibody-cytokine fusion proteins for the therapy of cancer" J Immunol Methods 248:91-101 (and the references cited therein); and Peng et al., 1999, "A single-chain IL-12 IgG3 antibody fusion protein retains antibody specificity and Il-12 bioactivity and demonstrates antitumor activity" J Immunol 163:250-8, all of which are incorporated for all purposes herein.

Immunostimulants

Another domain which comprises the antibody-immunostimulant fusion proteins of the invention is the immunostimulant domain. As described above, an immunostimulant molecule (or domain) acts to stimulate or elicit an immune response or an action of the immune system of a subject. Immunostimulant domains that are part of the antibody-immunostimulant fusion protein are typically (but not only) of several broad types. Typically, embodiments include, but are not limited to, cytokines and chemokines. In general, cytokines act to, e.g., stimulate humoral and/or cellular immune responses, while chemokines in general induce immune cell migration and activation. The choice of which immunostimulant to include in a particular embodiment depends upon, e.g., which particular immune response effects are desired, e.g., a humoral response, or a cellular immune response, or both. In typical embodiments both cellular and humoral immune responses against a disease related antigen are desired. Thus, as illustrated in Examples I and II below, multiple fusion proteins with varying immunostimulant domains are optionally used in the methods and compositions of the invention.

It will be appreciated that the discussion herein of immunostimulants comprising the listed molecules (e.g., IL-2, etc.) is not to be taken as limiting. In other words, it is to be understood that various embodiments of the invention comprise different combinations of immunostimulant molecules (e.g., other cytokines, chemokines, etc. besides, or in addition to, those listed herein). Thus, specific cytokines/chemokines, etc. (e.g., various interleukin molecules, interferons, IL-2, IL-10, IL-12, IL-17, IL-18, RANTES, mip1.alpha., mip1B, GMCSF, GCSF, gamma interferon, alpha interferon, etc.) fused in the antibody-fusions herein are not limiting and different specific cytokines, chemokines, immunostimulants, etc. can be utilized for different applications, all of which are part of the current invention herein.

For example, one common immunostimulant domain capable of use in the current invention comprises cytokines. Cytokines comprise a large family of growth factors that are primarily secreted from leukocytes and include, e.g., IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-13, interferons, interleukins, IFNs (interferons), TNF (tumor necrosis factor) and CSFs (colony stimulating factors). Various cytokines can stimulate humoral and/or cellular immune responses in subjects and can active phagocytic cells. Interleukins are one species of cytokines which are secreted by leukocytes and which also affect the various cellular responses/actions of leukocytes (e.g., IL-2, IL-12, etc.). In various embodiments, interleukins are used as an immunostimulant domain in the methods/compositions of the invention. Additionally, in other embodiments of the invention, non-interleukin cytokines comprise the immunostimulant domain of the antibody-immunostimulant fusions. See, e.g., Mire-Sluis 1993 TIBTECH 11:74-77; Colombo et al. 1992 Cancer Res 52:4853-4857; Arai, K. et al, 1990, "Cytokines: coordinators of immune and inflammatory responses" Annu Rev Biochem 59:783+, etc.

More specific examples of possible cytokines used in particular embodiments of the current invention include (but are not limited to) the following.

IL-2. IL-2 is a common immunostimulant used to construct antibody-immunostimulant fusion proteins. See, e.g., Penichet et al. 2001 "Antibody-cytokine fusion proteins for the therapy of cancer" J Immunol Methods 248:91-101, and the references cited therein. IL-2 stimulates T cells to proliferate and to become cytotoxic. Additionally, IL-2 induces NK cells to respond with increased cytotoxicity toward cells (e.g., tumor cells). Additionally, IL-2 increases vascular permeability leading to the efflux of intravascular fluids into extravascular areas.

IL-12. IL-12 is normally released by professional antigen presenting cells and promotes cell-mediated immunity. It does so by inducing naive CD4+ cells to differentiate into T.sub.H1 cells. IL-12 also can enhance the cytotoxicity of NK and CD8+ T cells. The IFN-.gamma. produced by T and NK cells that are stimulated by IL-12 can lead to other immune actions as well. IL-12 can exist as single chain or double chain (heterodimers) variants. Either permutation of IL-12 is optionally used herein as an immunostimulant domain in the antibody-immunostimulants used herein.

GMCSF. GMCSF is associated with growth and differentiation of hematopoietic cells and is a potent immunostimulator with pleiotropic effects (e.g., augmentation of antigen presentation in numerous cells). Additionally, it is involved in increased expression of MHC II on monocytes and adhesion molecules on granulocytes and monocytes. Furthermore, GMCSF is involved in the amplification of T cell proliferation. In certain embodiments of the current invention, GMCSF comprises the immunostimulant domain in the antibody-immunostimulant fusions used in the invention.

In other common embodiments of the invention, the immunostimulant domain comprises a chemokine (or a fragment thereof). Chemokines, e.g., selectively attract various leukocytes to specific locations and can induce not only cell migration but also activation. Chemokines are typically classified into alpha, beta, and gamma sub-types. Their classification is divided according to the configuration of the first cysteine residues at the amino terminus of the protein. Different classifications of chemokines act to attract different classes of inflammatory cells. Thus, use of such different chemokines in the fusion proteins used in the current invention can result in different immune responses activated in a subject that is treated with such fusion proteins. Chemokines capable of use in the fusion proteins used in the current invention include (but are not limited to) C-X-C group chemokines, IL-8, mip2.alpha., mip2.beta., PF4, platelet basic protein, hIP10, C-C family chemokines, LD78, Act-2, MCAF, 1309, RANTES, TCA3, IP-10, C chemokines, lymphotactin, CX3C (or c-x3-c) chemokines, fractalkine, etc.

Other embodiments of the invention comprise antibody-immunostimulant fusion proteins comprising immunostimulants other than cytokines or chemokines. For example, antibody-immunostimulant fusion proteins optionally comprise KLH (keyhole limpet hemocyanin) or other such immunogenic compounds, as well as "super antigens" which cause direct stimulation of T cells and/or B cells without direct antigen presentation. Super antigens and compounds such as KLH (as well as their use, etc.) are well known by those in the art. See, e.g., Johnson, et al. "Superantigens in human disease" Scientific American April 1992, p. 92-101, and Sekaly, R. (ed.) "Bacterial Superantigens" Seminars in Immunol. Vol. 5, 1993.

Again, the actual specific immunostimulant molecule in various embodiments of the fusion proteins used in the invention (whether comprising a cytokine, chemokine, etc.) will depend upon, e.g., the specific disease state/condition, the specific antigen targeted, the specific action desired (e.g., elicitation of a humoral immune response, a cellular immune response, or both), etc.

Construction

The construction of antibody-immunostimulant fusion proteins is well known to those versed in the art. For example, Penichet et al. 2001 "A recombinant IgG3-(IL-2) fusion protein for the treatment of human Her2/neu expressing tumors" Hum Antibodies 10:43+; Peng, 1999, supra; and Dela Cruz, 2000, supra all describe antibody-immunostimulant fusion proteins and their construction. Numerous other sources are replete throughout the literature.

The specific antibody-immunostimulant fusion proteins utilized in the current invention are optionally obtained or created by any method known in the art (including purchase from commercial sources). For example, nucleic acid sequences encoding the appropriate antibody framework (see, above) are optionally cloned and ligated into appropriate vectors (e.g., expression vectors for, e.g., prokaryotic or eukaryotic organisms). Additionally, nucleic acid sequences encoding the appropriate immunostimulant molecule are optionally cloned into the same vector in the appropriate orientation and location so that expression from the vector produces an antibody-immunostimulant fusion protein. Some optional embodiments also require post-expression modification, e.g., assembly of antibody subunits, etc. The techniques and art for the above (and similar) manipulations are well known to those skilled in the art. Pertinent instructions are found in, e.g., Sambrook et al., Molecular Cloning--A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 1999). In some alternate embodiments, the antibody domain and the immunostimulant domain are assembled post-expression through, e.g., chemical means.

Administration of Antibody-Immunostimulants as Adjuvants of Protein Vaccination

Compositions

The antibody-immunostimulant fusion proteins and/or protein vaccinations (e.g., the disease related antigens) are optionally administered to subjects in need of treatment (either therapeutically or prophylactically) in any appropriate sterile pharmaceutical carrier. Such pharmaceutical carrier acts to maintain the solubility and action of the fusion proteins and antigens. In some embodiments, it may be desired to administer additional components in conjunction with the fusion proteins/antigens. For example, in some treatment regimes, chemotherapeutic agents, antibiotics, additional antibody fusion proteins comprising growth factors, etc. are all optionally included with the compositions of the invention.

In typically embodiments, preparations for administration to subjects include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Some embodiments include non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oils), organic esters (e.g., ethyl oleate) and other solvents known to those of skill in the art. Physiologically acceptable carriers (or excipients) are optionally used in certain embodiments of the invention. Examples of such include, e.g., saline, PBS, Ringer's solution, lactated Ringer's solution, etc. Additionally, preservatives and additives are optionally added to the compositions to help ensure stability and sterility. For example, antibiotics and other bacteriocides, antioxidants, chelating agents, and the like are all optionally present in various embodiments of the compositions herein.

In the preparation of the compositions herein, typical embodiments include wherein the antibody-immunostimulant fusion proteins and the specific disease related antigen are incubated together for specific periods of time (e.g., in order for the appropriate immunocomplexes to form between the antigen and the fusion protein) before the composition is administered to the subject. Typical embodiments include wherein such incubations are done at 4.degree. C., e.g. overnight. However, other embodiments include wherein the incubation temperatures and times vary. For example, the compositions may be incubated at a variety of lengths and temperatures. The determination of the conditions/temperatures is determined based upon, e.g., the specific antigen involved, the specific antibody fusion proteins involved, the affinity between the antigen and the antibodies, etc. In some embodiments of the invention, the ratio of the number of molecules of an antibody-immunostimulant fusion protein and the number of molecules of an appropriate antigen are roughly or approximately equal (e.g., 1:1). However, in other embodiments the ratio is optionally not 1:1. For example, some embodiments optionally comprise wherein the number of molecules of antibody-fusion proteins is greater than the number of molecule of antigen or wherein the number of molecules of antigen are greater than the number of molecules of antibody fusion protein. In some embodiments the number of antigen molecules is great enough to totally saturate the number of antibody fusion protein molecules. In other words, all available antibodies will have antigen immunocomplexed to them. In other embodiments, the antigen is limiting (e.g., there is more than enough antibody so that all available antigen is immunocomplexed with the antibody). In other embodiments, the various amounts of the disease related antigen and the antibody-immunostimulant fusion protein are allocated so that an equal molarity (or an approximately equal molarity) exists between the components. In some typical embodiments, the amount of each component is allocated so that the binding unit equivalents of each component are equal or roughly/approximately equal. See, Example I, below.

In some embodiments, the various constituents of the compositions come pre-measured and/or prepackaged and/or ready for use without additional measurement, etc. The present invention also optionally comprises kits for conducting/using the methods and/or the compositions of the invention. In particular, these kits optionally include, e.g., appropriate antibody-immunostimulant fusion proteins (and optionally mixtures of a number of such proteins for performing synergistic treatments, see, above), and optionally appropriate disease related antigen(s) as well). Additionally, such kits can also comprise appropriate excipients (e.g., pharmaceutically acceptable excipients) for performing therapeutic and/or prophylactic treatments of the invention. Such kits optionally contain additional components for the assembly and/or use of the compositions of the invention including, but not limited to, e.g., diluents, adjuvants, etc.

The compositions described herein are optionally packaged to include all (or almost all) necessary components for performing the methods of the invention or for using the compositions of the invention (optionally including, e.g., written instructions for the use of the methods/compositions of the invention). For example, the kits can optionally include such components as, e.g., buffers, reagents, serum proteins, antibodies, substrates, etc. In the case of prepackaged reagents, the kits optionally include pre-measured or pre-dosed amounts that are ready to incorporate into the methods without measurement, e.g., pre-measured fluid aliquots, or pre-weighed or pre-measured solid reagents that can be easily reconstituted by the end-user of the kit.

Such kits also typically include appropriate instructions for performing the methods of the invention and/or using the compositions of the invention. In some embodiments, the components of the kits/packages are provided in a stabilized form, so as to prevent degradation or other loss during prolonged storage, e.g., from leakage. A number of stabilizing processes/agents are widely used for reagents, etc. that are to be stored, such as the inclusion of chemical stabilizers (i.e., enzymatic inhibitors, microbicides/bacteriostats, anticoagulants), etc.

In some embodiments, the multiple compositions are used to treat the subject. For example, multiple dosages of the fusion protein/antigen mixture are optionally given to a subject over a prescribed time period. Ranges for such are optionally highly variable depending upon, e.g., the subject's response to treatment, any toxicities and/or or adverse reactions to treatment, etc. and are optionally adjusted to suit each individual treatment regime/subject. Additionally, the fusion protein is optionally given to the subject in a separate composition than the antigen mixture. For example, the antigen composition is optionally administered to the subject prior to, approximately concurrently to, or after the fusion protein composition is administered to the subject. Furthermore, as mentioned herein, some disease states/conditions present situations wherein a separate administration of disease related antigen is not given. For example, some HER2/neu expressing tumors shed large amounts of the HER2/neu antigen. In optional embodiments, the current invention utilizes such shed antigen by optionally using such to form immunocomplexes with the fusion proteins administered. Again, such optional mechanism of action should not be construed as limiting upon the efficaciousness of the methods and compositions of the current invention.

In some embodiments herein, the invention comprises a composition of an antibody-immunostimulant fusion protein wherein the fusion protein comprises an effective adjuvant of a disease related antigen. In some embodiments, the composition also includes the disease related antigen. Additional embodiments encompass wherein the antibody-immunostimulant fusion protein has antibody specificity against the disease related antigen. The immunostimulant domain of the fusion proteins in these compositions optionally comprises a cytokine (or a sequence or subsequence thereof), a chemokine (or a sequence or subsequence thereof), or an immunostimulant other than a chemokine or cytokine. Examples of such immunostimulant domains (e.g., as are included in optional embodiments of the compositions herein) include, but are not limited to, e.g., cytokines, chemokines, interleukins, interferons, C-X-C chemokines, C-C family chemokines, C chemokines, CX3C chemokines, super antigens, growth factors, IL-1, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IL-18, RANTES, mip1.alpha., mip1.beta., GMCSF, GCSF, gamma interferon, alpha interferon, TNF, CSFs, mip2.alpha., mip2.beta., PF4, platelet basic protein, hIP10, LD78, Act-2, MCAF, 1309, TCA3, IP-10, lymphotactin, fractalkine, KLH, and fragments thereof of any of the above. Additionally, any of the above embodiments optionally also has a linker (other embodiments optionally do not have linkers). Linker regions or domains are optionally between, e.g., the immunostimulant domain and the antibody domain in the fusion proteins, etc.

The antibody domain of the fusion proteins in the compositions of the invention optionally includes an antibody specific for, but not limited to, e.g., a HER2/neu antigen, a tumor antigen, a bacterial antigen, a viral antigen, a mycoplasm antigen, a fungal antigen, a prion antigen, an autoimmune disorder antigen, or an antigen from a parasite (e.g., an infectious mammalian parasite). In other embodiments, such fusion proteins comprise antibody domains specific for antigens other than tumor antigens. Furthermore, in yet other embodiments, the antibody-immunostimulant fusion proteins in the compositions of the invention comprise an antibody fragment, or an Fab domain, an Fab' domain, an F(ab').sub.2 domain, an F(ab).sub.2domain, an scFv domain, IgG, IgA, IgE, IgM, IgD, IgG1, IgG2, or IgG3.

Also, in some embodiments of the compositions of the invention, the antigen comprises, e.g., a soluble antigen, a soluble antigen bound to a matrix, an insoluble antigen bound to a matrix, an insoluble aggregate of antigens, a nonviable cell-associated antigen, or a nonviable organism-associated antigen, or an antigen conjugated with a liposome. Additionally, such antigen can comprise, e.g., HER2/neu (or HER2/neu shed from a tumor cell) or fragments thereof. Additionally, the antigen in such compositions optionally comprises: an antigen other than a tumor antigen, an antigen arising from a subject, an antigen arising from a disease state within the subject, an antigen arising from a disease related organism within a subject (e.g., a disease state caused by one or more of a tumor, a bacteria, a virus, a mycoplasm, a fungus, a prion, an autoimmune disorder, or an infectious parasite such as an infectious parasite of a mammal, etc.). The antigen can also comprise a tumor antigen, a bacterial antigen, a viral antigen, a mycoplasm antigen, a prion antigen, an autoimmune disorder related antigen, or an infectious parasite antigen. In some embodiments herein, the antigen is an exogenous antigen (which is optionally substantially identical to an antigen arising from a subject, or from a disease state within a subject or from a disease related organism within the subject).

In other embodiments of the compositions herein, the number of antigen molecules and the number of fusion protein molecules are optionally approximately 1:1. In other embodiments, they are optionally in ratios wherein the number of antigen molecules is greater than or lesser than the number of fusion protein molecules, or wherein the number of fusion proteins is substantially saturated by the number of antigen molecules, or wherein the number of antigen molecules is substantially saturated by the number of fusion protein molecules.

The compositions of the invention are optionally incubated for a specific period of time and under specific conditions (e.g., overnight at 4.degree. C., etc. or for even brief periods of time such as 1 second or less, etc.). The compositions of the invention also optionally comprise an excipient (e.g., a pharmaceutically acceptable excipient).

Examples I and II below, give several non-limiting examples of the compositions and administration of the compositions of the invention. See, below. It will be appreciated that different combinations of antibodies and immunostimulants will optionally require different administration profiles (e.g., certain immunostimulant domains optionally need a specific buffer, etc.). Additionally, some treatment regimes optionally will include additional therapeutic and/or prophylactic components (e.g., antibiotics and the like).

Administration

In typical embodiments, the antibody-immunostimulant fusion proteins and the antigen vaccinations are injected parenterally, (e.g., intravenously, intraperitoneally, intramuscularly, or subcutaneously) in a subject. In other embodiments, the compositions of the invention are delivered via non-injection means, see, below. Typically, the dosage ranges for such administration are large enough to elicit the desired effect in the subject (e.g., elicitation of humoral and/or cellular immune responses against the disease related antigen and/or, e.g., positive anti-tumor or anti-infection activity). The dosages given are optionally optimized for the individual subject based upon, e.g., the subject's age, gender, species, and weight, as well the extent or presence of the disease state to be treated (either therapeutically or prophylactically). For example, the dosage of the fusion protein/antigen compositions given can range from less than 0.1 mg/kg subject weight to 200 mg/kg subject weight or more. The dosage given depends upon, e.g., the specific subject (age, weight, general health, gender, species, etc.), the presence and/or progression or stage of a disease state, the specific antigen, the specific antibody fusion protein, and the specific immunostimulant. For example, some optional immunostimulants present toxicities in higher doses (thus, more composition does not necessarily equal more benefit). Thus, the administration is optionally tailored for each subject. Doses are optionally given in a series. In other words, multiple doses are optionally given over a course of treatment. The dosage course is optionally modified during the treatment based upon the subject's response. For example, if a subject does not response satisfactorily within a specific time period, the dosage and/or timing of dosages is optionally increased or altered.

Again, Examples I and II below, give non-limiting examples of dosage (amounts and timing) schedules using the compositions of the invention. Such treatment schedules, again, are solely examples tailored for use with the mice, etc. in the Examples, and are not to be taken as limiting.

The present invention also includes methods of therapeutically or prophylactically treating a disease or disorder, eliciting an immune response (humoral and/or cellular) in a subject and administering an immunological composition by administering in vivo or ex vivo one or more nucleic acids or polypeptides/fusion proteins/antigens of the invention as described herein (or compositions comprising a pharmaceutically acceptable excipient and one or more such nucleic acids or polypeptides and/or fusion proteins and/or antigens) to a subject, including, e.g., a mammal, including, e.g., a human, primate, mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, sheep; or a non-mammalian vertebrate such as a bird (e.g., a chicken or duck) or a fish, or invertebrate.

In one optional aspect of the invention, in ex vivo methods, one or more cells or a population of cells of interest of the subject (e.g., dendritic cells, antigen presenting cells, etc.) are obtained or removed from the subject and contacted with an amount of a fusion protein and antigen of the invention that is effective in prophylactically or therapeutically treating a disease, disorder, or other condition. The contacted cells are then returned or delivered to the subject to the site from which they were obtained or to another site (e.g., via intramuscular injection, etc.) of interest in the subject to be treated. The methods/compositions of the invention optionally elicit an effective immune response whether such cells are delivered to a site of need (e.g., a tumor or infection site) or to a site unrelated to such (e.g., a distant body part, etc.). If desired, the contacted cells may be deposited, injected, grafted, etc. onto a tissue, organ, or system site (including, e.g., tumor cells, tumor tissue sample, organ cells, blood cells, cells of the skin, lung, heart, muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, mouth, tongue, etc) of interest in the subject using standard and well-known depositing, injection and grafting techniques or, e.g., delivered to the blood or lymph system using standard delivery or transfusion techniques.

The invention also optionally provides in vivo methods in which one or more cells or a population of cells of interest of the subject are contacted directly or indirectly with an amount of an antibody fusion protein and/or antigen of the invention effective in prophylactically or therapeutically treating a disease, disorder, or other condition. In either format, the antibody fusion protein and/or antigen is optionally administered or transferred to the cells (e.g., tumor cells, tumor tissue sample, infection site (such as an abscess, etc.) organ cells, blood cells, cells of the skin, lung, heart, muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, mouth, tongue, etc.) by any of a variety of formats, including topical administration, injection (e.g., by using a needle or syringe), or vaccine or gene gun delivery, pushing into a tissue, organ, or skin site. The molecules can be delivered, for example, intramuscularly, intradermally, subdermally, subcutaneously, orally, intraperitoneally, intrathecally, intravenously, or placed within a cavity of the body (including, e.g., during surgery), or by inhalation or vaginal or rectal administration. In more typical embodiments, the antibody fusion protein and/or antigen of the invention are optionally administered or transferred to a site that is not directly in need of treatment, etc. For example, in typical embodiments, the antibody fusion protein and/or antigen of the invention are injected (e.g., see, above), e.g., intramuscularly or intravenously at a site distant from, e.g. a tumor, infection site, etc. (e.g., injection into the flank of an animal when the tumors to be combated are in the lungs, etc.). The immune response is still generated by the antibody-immunostimulant fusion proteins/antigen compositions of the invention.

In another optional aspect, the invention provides ex vivo methods in which one or more cells of interest or a population of cells of interest of the subject (e.g., tumor cells, tumor tissue sample, organ cells, blood cells, cells of the skin, lung, heart, muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, mouth, tongue, etc.) are obtained or removed from the subject and transformed by contacting said one or more cells or population of cells with a polynucleotide construct comprising a target nucleic acid sequence encoding antibody-immunostimulant fusion proteins and/or antigen used in the invention, as biologically active molecules that are effective in prophylactically or therapeutically treating the disease, disorder, or other condition. The one or more cells or population of cells is contacted with a sufficient amount of the polynucleotide construct (e.g., encoding antibody-immunostimulant fusion proteins and/or antigen) and a promoter controlling expression of said nucleic acid sequence such that uptake of the polynucleotide construct (and promoter) into the cell(s) occurs and sufficient expression of the target nucleic acid sequence of the invention results to produce an amount of the biologically active molecules effective to prophylactically or therapeutically treat the disease, disorder, or condition. The polynucleotide construct may include a promoter sequence (e.g., CMV promoter sequence) that controls expression of the nucleic acid sequence of the invention and/or, if desired, one or more additional nucleotide sequences encoding at least one or more of another molecule of the invention, such as a cytokine, adjuvant, or co-stimulatory molecule, or other polypeptide, etc. of interest, etc.

Following transfection, the transformed cells optionally are returned, delivered, or transferred to the subject to the tissue site or system from which they were obtained or to another site (e.g., tumor cells, tumor tissue sample, organ cells, blood cells, cells of the skin, lung, heart, muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, mouth, tongue, etc.) in the subject. If desired, the cells may be grafted onto a tissue, skin, organ, or body system of interest in the subject using standard and well-known grafting techniques or delivered to the blood or lymphatic system using standard delivery or transfusion techniques. Such delivery, administration, or transfer of transformed cells is typically made by using one or more of the routes or modes of administration described above. Expression of the target nucleic acid occurs naturally or can be induced and an amount of the encoded antibody-immunostimulant fusion proteins and/or antigen is expressed sufficient and effective to treat the disease or condition. The site of expression of the compositions, etc. need not be at or near the site of need in the subject. As explained throughout, the antibody-immunostimulant fusion proteins and/or antigens in the compositions of the invention do not necessarily need to come into direct contact with, e.g., a tumor cell, infectious organism, etc. in order to elicit an immune response against such, e.g., tumor or infection.

In another optional aspect, the invention provides in vivo methods in which one or more cells of interest or a population of cells of the subject (e.g., including those cells and cells systems and subjects described above) are transformed in the body of the subject by contacting the cell(s) or population of cells with (or administering or transferring to the cell(s) or population of cells using one or more of the routes or modes of administration described above) a polynucleotide construct comprising a nucleic acid sequence that encodes a biologically active antibody-immunostimulant fusion protein and/or antigen used in the invention that is effective in prophylactically or therapeutically treating the disease, disorder, or other condition.

The polynucleotide construct optionally can be administered or transferred to cell(s) by first directly contacting cells using one or more of the routes or modes of administration described above with a sufficient amount of the polynucleotide construct comprising the nucleic acid sequence encoding the biologically active molecules, and a promoter controlling expression of the nucleic acid sequence, such that uptake of the polynucleotide construct (and promoter) into the cell(s) occurs and sufficient expression of the nucleic acid sequence of the invention results to produce an amount of the biologically active antibody fusion protein and/or antigen effective to prophylactically or therapeutically treat the disease or disorder. Expression of the target nucleic acid occurs naturally or can be induced such that an amount of the encoded antibody fusion protein and/or antigen is expressed sufficient and effective to treat the disease or condition by eliciting the appropriate immune response. The polynucleotide construct may include a promoter sequence (e.g., CMV promoter sequence) that controls expression of the nucleic acid sequence and/or, if desired, one or more additional nucleotide sequences encoding at least one or more of another molecule used in the invention, a cytokine, adjuvant, or co-stimulatory molecule, or other such molecules of interest.

In each of the in vivo and ex vivo treatment methods as described above, a composition comprising an excipient and the antibody fusion protein and/or antigen or nucleic acid encoding such as used in the invention can be administered or delivered. In one aspect, a composition comprising a pharmaceutically acceptable excipient and such molecules or nucleic acid as used in the invention is administered or delivered to the subject as described above in an amount effective to treat the disease or disorder.

In another aspect, in each in vivo and ex vivo treatment method described above, the amount of polynucleotide administered to the cell(s) or subject can be an amount sufficient that uptake of said polynucleotide into one or more cells of the subject occurs and sufficient expression of said nucleic acid sequence results to produce an amount of the biologically active molecules effective to enhance or elicit an immune response in the subject. In another aspect, for each such method, the amount of molecules administered to cell(s) or subject can be an amount sufficient to enhance or elicit an immune response in the subject.

In yet another aspect, in an in vivo or ex vivo treatment method in which a polynucleotide construct (or composition comprising a polynucleotide construct) is used, the expression of the polynucleotide construct can be induced by using an inducible on-and-off gene expression system. Examples of such on-and-off gene expression systems include the Tet-On.TM. Gene Expression System and Tet-Off.TM. Gene Expression System, respectively. Other controllable or inducible on-and-off gene expression systems are known to those of ordinary skill in the art. With such system, expression of the target nucleic of the polynucleotide construct can be regulated in a precise, reversible, and quantitative manner. Gene expression of the target nucleic acid can be induced, for example, after the stable transfected cells containing the polynucleotide construct comprising the target nucleic acid are delivered or transferred to or made to contact a tissue site, organ or system of interest. Such systems are of particular benefit in treatment methods and formats in which it is advantageous to delay or precisely control expression of the target nucleic acid (e.g., to allow time for completion of surgery and/or healing following surgery; to allow time for the polynucleotide construct comprising the target nucleic acid to reach the site, cells, system, or tissue for expression; to allow time for the graft containing cells transformed with the construct to become incorporated into the tissue or organ onto or into which it has been spliced or attached, etc.)

In some embodiments, the invention comprises a method of administering an immunological composition by providing an antibody-immunostimulant fusion protein and administering the fusion protein to a subject wherein the fusion protein comprises an effective adjuvant to a disease related antigen and wherein the fusion protein and the antigen in combination elicit an immune response in a subject. Furthermore, some embodiments involve the administration of such fusion protein along with providing a disease related antigen (e.g., administering the fusion protein and the antigen to a subject wherein the fusion protein is an effective adjuvant of the antigen). In some embodiments, the fusion protein comprises a cytokine (or a sequence or subsequence thereof), a chemokine (or a sequence or subsequence thereof), or an immunostimulant other than a chemokine or cytokine. In other embodiments, the methods of the invention use fusion proteins comprising an immunostimulant domain such as (but not limited to), e.g., cytokines, chemokines, interleukins, interferons, C-X-C chemokines, C-C family chemokines, C chemokines, CX3C chemokines, super antigens, growth factors, IL-1, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IL-18, RANTES, mip1.alpha., mip1.beta., GMCSF, GCSF, gamma interferon, alpha interferon, TNF, CSFs, mip2.alpha., mip2.beta., PF4, platelet basic protein, hIP10, LD78, Act-2, MCAF, 1309, TCA3, IP-10, lymphotactin, fractalkine, KLH, and fragments thereof of any of the above.

The antibody domain of the fusion proteins used in the embodiments of the methods of the invention are optionally specific for, e.g., HER2/neu antigen, a tumor antigen, a bacterial antigen, a viral antigen, a mycoplasm antigen, a fungal antigen, a prion antigen, an autoimmune disorder related antigen, an infectious parasite antigen (e.g., a parasite of a mammal). In other embodiments the antibody domain is specific for antigen comprising an antigen other than a tumor antigen. The antibody domain of the fusion proteins in such embodiments of the invention, are optionally (but are not limited to), e.g., an antibody fragment, an Fab domain, an Fab' domain, an F(ab').sub.2 domain, an F(ab).sub.2domain, an scFv domain, IgG, IgA, IgE, IgM, IgD, IgG1, IgG2, or IgG3. In some embodiments of these methods, the fusion protein has antibody specificity for the antigen.

These methods herein also encompass embodiments wherein the antigen comprises, e.g., a tumor antigen, a bacterial antigen, a viral antigen, a mycoplasm antigen, a prion antigen, an autoimmune disorder related antigen, a parasite antigen (e.g., one infecting a mammal), an antigen other than a tumor antigen, an antigen arising from the subject, an antigen arising form a disease state within the subject, or an antigen from a disease related organism within the subject. The disease state within the subject that optionally gives rise to such antigens, optionally is caused by, e.g., a tumor, a bacteria, a virus, a mycoplasm, a fungus, a prion, an autoimmune disorder, or a parasite (e.g., one infecting a mammal). The antigens in such embodiments of the invention are also optionally exogenous antigens, which can optionally be substantially identical to a disease related antigen arising from a subject, arising from a disease state within a subject, or arising from a disease related organism within a subject. Such exogenous antigen is optionally administered prior to administration of the antibody-immunostimulant fusion proteins, or optionally after the fusion proteins are administered to the subject, or approximately concurrently with the fusion proteins to the subject. Prior to the optional concurrent administration the antigen and the fusion protein can be incubated for a specific time period and under specific conditions (e.g., from 1 second or almost instantaneous incubation up to overnight or longer; at, e.g., 4.degree. C., etc.). The antigen used in such embodiments of the invention also optionally comprises, e.g., HER2/neu, HER2/neu shed from tumor cells, or fragments of such HER2/neu. In some embodiments, the methods comprise wherein the number of antigen molecules and the number of fusion protein molecules are optionally approximately 1:1. In other embodiments, the number of antigen molecules and the number of fusion protein molecules are optionally in ratios wherein the number of antigen molecules is greater than or lesser than the number of fusion protein molecules, or wherein the number of fusion proteins is substantially saturated by the number of antigen molecules, or wherein the number of antigen molecules is substantially saturated by the number of fusion protein molecules. In other embodiments of these methods, more than one fusion protein is optionally used. Such multiple fusion proteins can comprise different immunostimulant domains (e.g., such as ones chosen from (but not limited to) non-cytokine/non-chemokine molecules, cytokines, chemokines, interleukins, interferons, C-X-C chemokines, C-C family chemokines, C chemokines, CX3C chemokines, super antigens, growth factors, IL-1, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IL-18, RANTES, mip1.alpha., mip1.beta., GMCSF, GCSF, gamma interferon, alpha interferon, TNF, CSFs, mip2.alpha., mip2.beta., PF4, platelet basic protein, hIP10, LD78, Act-2, MCAF, 1309, TCA3, IP-10, lymphotactin, fractalkine, KLH, and fragments thereof of any of the above. Furthermore, the multiple fusion proteins in these methods optionally have different specificity. The optional multiple fusion proteins can be specific for, e.g., different antigens on a single molecule, different antigens on a single cell, different antigens on a single tumor, or different antigens on a single organism (e.g., a virus, bacteria, fungus, mycoplasm, prion, parasite), etc. The methods of administering an immunological composition also include embodiments wherein such administration elicits an immune response in a subject.

In yet other embodiments, the current invention also includes methods of prophylactically and/or therapeutically treating a disease state in a subject. Such methods include administering an effective amount of an antibody-immunostimulant fusion protein to the subject, wherein the fusion protein comprises an effective adjuvant of a disease related antigen (e.g., one arising from the subject, arising from a disease state within the subject, or arising from a disease related organism within the subject) and wherein the administration elicits an immune response within the subject against the disease related antigen (or closely related antigens). Such method of prophylactically and/or therapeutically treating a disease state also optionally includes administering to the subject an effective amount of an antibody-immunostimulant fusion protein and administering a disease related antigen wherein the fusion protein comprises an effective adjuvant of the disease related antigen.

Use of the Invention to Elicit Immune Responses Against HER2/Neu Tumor Antigen and Staphylococcus Protein A Antigen

One possibility to overcome problems presented in therapeutic and/or prophylactic treatment of some diseases/conditions/etc. (such as microorganism infections that have no effective drug treatment, e.g., multiple drug resistant bacteria, etc., or such as certain cancers, e.g., HER2/neu presenting cancers) is immunization with specific proteins to cause a strong immune response against the expressing tumors or infectious agents, etc. For example, as explained in more detail below, some embodiments of the invention involve treatment (e.g., injection/vaccination) of cancer patients with an appropriate antigen in hope of eliciting an immune response in the patient against the tumor cells. Additionally, such treatment (e.g., vaccination with appropriate antigens) is a common approach in eliciting an immune response against certain types of infectious organisms (e.g., viruses, etc.) Traditional vaccination strategies against infectious organisms are well known to those in the art.

The current invention, uses antibody-immunostimulant fusion proteins as adjuvants of protein vaccinations (see, e.g., Example I below detailing HER2/neu protein and Example II detailing Staphylococcus aureus protein A) as an effective means to elicit both humoral and/or cellular immune responses in subjects against disease related antigens from e.g., tumors, infectious agents such as viruses, etc. For example, as shown in Example I, below antibody-immunostimulant fusion proteins comprising anti-HER2/neu IgG3-(IL-2), anti-HER2/neu IgG3-(IL-12), and anti-HER2/neu IgG3-(GMCSF)) were used as adjuvants (i.e. immunoenhancers) of a soluble form of an antigen used as a protein vaccination (again, herein illustrated by HER2/neu). Of course, in other embodiments, different antigens are selected for use. See, above.

The current invention does not use the antibody-immunostimulant fusion proteins for direct targeting of, e.g., a tumor or an infectious agent, instead the antibody fusions, in conjunction with the antigen, are used to elicit a humoral and/or cellular immune response against the specific antigen (and, thus, against the tumor or infectious organism). It is important to stress that in this approach, direct targeting of a tumor or direct targeting of an infectious agent by the antibody fusion proteins is not a requirement to trigger an antitumor activity or immune activity against the infectious agent. For example, mixing an antibody-immunostimulant fusion protein with its specific antigen (e.g., extracellular domain of HER2/neu (ECD.sup.HER2) in Example I) is enough to elicit a potent cellular and humoral immune response that results in an strong antitumor activity (i.e., the fusion protein of the invention stimulates an endogenous humoral/cellular immune response).

It will be appreciated that, as explained throughout, not only can different immunostimulant, antibody combinations be used against different diseases/conditions, but that in various embodiments, different combinations of antibody-immunostimulant fusion proteins can be used in conjunction with each other. For example, in some treatment regimens different antibody-immunostimulant fusions can be administered to a subject in the same course of treatment (e.g., as was done with the IgG3-IL-2, IgG3IL-12, etc. in Example I below) to produce a synergistic effect in stimulating an immune response. Additionally, in some optional embodiments, different antigens on the same tumor or infectious agent are targeted in the same course of treatment. For example, two or more surface antigens on an infectious bacterium are optionally targeted by two or more different antibody-immunostimulant fusion proteins of the invention.

As will become apparent upon examination of the following, the use of the methods, compositions, etc. of the current invention allow for time saving in the treatment of subjects. Quick responses and actions can be of utmost importance in treatment of many conditions (e.g., in treatment of late stage cancers, advanced bacterial infections, etc.). For example, the use of antibody fusion proteins as an adjuvant of an antigen vaccine takes advantage of the high affinity of an antibody for its antigen. Thus use of the invention is a straightforward way to combine a disease related antigen with an immunostimulant (e.g., a cytokine or other immunostimulatory molecule), thus, avoiding the need to construct antibody fusion proteins consisting of an antigen genetically fused to an immunostimulant (e.g., a cytokine or other immunostimulants). Such fusions can be cumbersome and sometimes can lead to the decrease or loss of activity of one or both of the covalently conjugated partners (i.e., loss or decrease of activity of the antibody or of the immunostimulant). In addition, the use of antibody-immunostimulant fusion protein as in the invention is the only way to target circulating antigens (e.g., shed soluble HER2/neu in vivo, soluble antigens from infectious microorganisms, etc.). Of course, once again, it will be appreciated that the benefits of the use of the current invention in treating HER2/neu presenting cancers and Staphylococcus aureus infections (as used as examples herein) accrues to treatment of many other disease states, infections, cancers, etc. as will be apparent from the information herein.

All of the exemplary proteins used herein to illustrate the properties of the invention (e.g., anti-HER2/neu IgG3-(IL-2), anti-HER2/neu IgG3-(IL-12), anti-HER2/neu IgG3-(GMCSF)) were determined to be properly assembled and secreted. Thus, the fusion proteins migrated on SDS-PAGE with the expected molecular weight under both reducing and non-reducing conditions. Furthermore, they bound the appropriate antigen and carried out ligand and antibody-related activities. More importantly, direct treatment (e.g., intravenous (i.v.) injection or other methods of application) with the exemplary antibody fusion proteins resulted in significant antitumor activity in murine tumor models expressing human HER2/neu under conditions in which the antibody alone (anti-HER2/neu IgG3 containing the same variable region) failed to confer protection (see, below, and Peng et al., 1999; Dela Cruz et al., 2000; Penichet et al., 2001, all supra).

One non-limiting example of the current invention includes using the antibody-cytokine fusion proteins (anti-HER2/neu IgG3-(IL-2), anti-HER2/neu IgG3-(IL-12), and anti-HER2/neu IgG3-(GMCSF)) as immunoenhancers for ECD.sup.HER2 vaccination in animal models (see, below for a more detailed protocol description). It will be appreciated that ECD.sup.HER2 comprises the extracellular domain of HER2neu (e.g., the domain shed by tumor cells; recombinant versions used in examples herein equate to such shed extracellular domain in subjects). In brief, mice were vaccinated with either human ECD.sup.HER2, ECD.sup.HER2 in combination with anti-HER2/neu antibody, or ECD.sup.HER2 with each anti-HER2/neu antibody-cytokine fusion protein (separately). After a booster, mice were challenged with a syngeneic carcinoma which expressed the rat HER2/neu protein (TUBO). There was a significant retardation of tumor growth rate and an increase in long-term survivors in those mice vaccinated with ECD.sup.HER2 plus all three antibody-cytokine fusion proteins as compared to the mice in the control groups (i.e., PBS, ECD.sup.HER2 or ECD.sup.HER2 plus anti-HER2/neu antibody). Increased ECD.sup.HER2 specific antibody titer was detected in mice vaccinated with the ECD.sup.HER2 plus antibody-immunostimulant fusion proteins as compared to the control groups. The group that was vaccinated with ECD.sup.HER2 plus antibody-(GMCSF) showed the highest antibody titer. Immune sera from the mice showed significant in vitro anti-proliferative activity against SK-BR-3 (a human breast cancer which overexpresses HER2/neu). The level of inhibition of SK-BR-3 correlated with the level of anti-ECD.sup.HER2 antibody. In addition, mice vaccinated with ECD.sup.HER2 plus antibody-immunostimulant fusion proteins produced increased level of ECD.sup.HER2 specific IgG2a antibodies, indicating that a T.sub.H1 type immune response was elicited. When incubated with soluble ECD.sup.HER2, splenocytes from mice vaccinated with ECD.sup.HER2 plus antibody-(GMCSF) fusion proteins demonstrated significant stimulation and IFN-.gamma. secretion as compared with the other groups. These results indicate that both humoral and cell-mediated responses are elicited by the compositions of the current invention and, thus contribute to the observed anti-tumor activity. The current results also indicate that anti-HER2/neu antibody-cytokine fusion proteins can be effective prophylactic and therapeutic agents against HER2/neu expressing tumors in patients (see, below). Once again, it is to be appreciated that the discussion of anti-HER2/neu antibody-immunostimulant fusion proteins is used as an illustration of the general class of antibody-immunostimulant fusion proteins that are used as adjuvants of protein vaccinations in the current invention.

In certain examples herein murine GMCSF and murine IL-12 were used because human GMCSF and human IL-12 are not active in mice. Using murine GMCSF and IL-12 in the fusion proteins examples herein allowed testing of the invention in murine models. Such constructions should not be taken to be limiting, and thus, the invention is applicable to other animal systems (e.g., human, etc.) and other animal molecules (e.g., human GMCSF, human IL-12, etc.). Additionally, in the illustrations herein, human IgG3 was used, however, any immunoglobulin isotype can be used (see, above). Moreover, the concepts of the invention can be directly applied to other kinds of antibody frameworks, including scFv, etc. See, above.
 

Claim 1 of 19 Claims

1. An ex vivo pharmaceutical composition comprising: an antibody-immunostimulant fusion protein having an antibody domain and an immunostimulant domain, a disease-related antigen, one or more antigen presenting cells, and a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient, wherein the fusion protein acts as an effective adjuvant of the disease-related antigen; wherein the antibody-immunostimulant fusion protein comprises antibody specificity against the antigen; wherein the antibody domain comprises an intact antibody, comprising two light chains and two heavy chains, or an antibody fragment, which fragment is selected from the group consisting of an Fab domain, an Fab' domain, an F(ab').sub.2 domain, an scFv domain, and an F(ab).sub.2 domain; and, wherein the immunostimulant domain comprises an immunostimulant selected from the group consisting of IL-2, IL-12, and GM-CSF.
 

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