HK1141251B - A method for enhancing t cell response - Google Patents

Description

Methods for enhancing T cell responses

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application serial No. 60/901,980, filed on 15/2/2007, which is incorporated herein by reference in its entirety.

Background

Embodiments of the invention relate to the fields of immunology and vaccine development. Embodiments of the invention disclosed herein relate to methods and compositions for enhancing immunization and vaccination. More specifically, embodiments of the invention relate to methods of increasing T cell response stimulation. Some embodiments of the invention have further application as vaccination strategies in the treatment of diseases such as infectious diseases or cancer.

Background

Active attenuated vaccines typically induce a strong and long-lasting immune response after one injection, and many of these types of viral vaccines have a potency of over 90% (Nossal, G.vaccines in Fundamental Immunology) (Paul, W.E. eds.) 1387-1425; Lippincot-Raven publisher, Philadelphia, 1999, which is incorporated herein by reference in its entirety). In contrast, vaccines consisting of killed microorganisms, toxins, subunit vaccines including peptide vaccines, or naked DNA vaccines are rather less potent and booster immunizations are necessary. While live vaccines produce increased antigen doses that elicit strong immune responses, non-replicating vaccines produce reduced antigen patterns that, as demonstrated in the examples herein, are rather weak stimulators of T cells.

There is a continuing need to develop immune models that enhance T cell responses against diseases such as, but not limited to, infectious diseases or cancer. Accordingly, embodiments of the invention disclosed herein relate to immunotherapeutic methods comprising increasing antigenic stimulation during immunization, independent of cumulative total antigen dose, thereby enhancing immunogenicity. Accordingly, embodiments of the invention disclosed herein provide for revisions to current immunization models and provide methods and compositions for designing and applying vaccines and immunotherapies.

Summary of The Invention

Embodiments of the invention disclosed herein relate to optimizing CD8⁺Methods and compositions for T cell responses. Accordingly, some embodiments of the invention relate to methods of stimulating a class I MHC-restricted T cell response in a mammal;the method comprises administering to the mammal a plurality of sequential doses of an immunogenic composition, wherein each dose after an initial dose is greater than the immediately preceding dose.

In some embodiments, the continuous dose increases as a linear function of the starting dose. In another embodiment, the continuous dose increases as an exponential function of the initial dose. The exponential function is defined by ≥ 2^n-1Is defined by the exponential factor of (c). In other embodiments, the exponential factor is 5^n-1。

In some embodiments, the immunogenic composition comprises an immunogen plus an immunopotentiator or biological response modifier. The immunopotentiator or biological response modifier may be, for example, but not limited to, cytokines, chemokines PAMP, TLR-ligands, immunostimulatory sequences, CpG-containing DNA, dsRNA, recognition receptor for endocytic Patterns (PRR) ligands, LPS, quillaja saponin (quillaja saponin), tocarol, and the like.

The plurality of doses may be 2 or more doses. In some embodiments, the plurality of doses comprises 2-6 doses. In other embodiments, the plurality of doses comprises more than 6 doses. In some embodiments of the invention, multiple doses may be affected by the half-life (t) of the immunogen_1/2) Influence. For example, to achieve similar results, an immunogen having a relatively short half-life may require more frequent administration, and thus a greater number of doses, than an immunogen having a relatively long half-life.

In some embodiments, the last dose may be administered within 6 days of the first dose. In some embodiments, the last dose may be administered within 7, 8, 9, 10 or more days after the first dose.

Embodiments of the invention relate to methods in which an enhanced response is obtained as compared to immunization using the same cumulative dose without a linear or exponential increase in dose over time. The enhanced response may include an increased number of reactive T cells. In some embodiments, the enhanced response may include increased production of immunostimulatory cytokines. The cytokine may be, for example, IL-2 or IFN- γ. In some embodiments, the enhanced response may include an increase in cytolytic activity. In some embodiments, the enhanced response may include a delay in peak production of immunosuppressive cytokines. The immunosuppressive cytokine may be, for example, IL-10.

Embodiments of the invention relate to methods of administering an immunogenic composition to a mammal by direct delivery to the lymphatic system. For example, the method of administering the immunogenic composition to a mammal can be by intranodal delivery.

In some embodiments of the invention, the immunogenic composition can be administered to the mammal subcutaneously, intramuscularly, intradermally, transdermally, transmucosally, nasally, bronchially, orally, rectally, and the like.

In some embodiments, the immunogen may be provided as a protein, peptide, polypeptide, naked DNA vaccine, RNA vaccine, synthetic epitope, mimotope, and the like, but is preferably, but not limited to, these.

The immunogen stimulates a response against an antigen associated with the disease to be treated or protected against. The antigen can be, for example, but not limited to, viral antigens, bacterial antigens, fungal antigens, differentiation antigens, tumor antigens, embryonic antigens, antigens of oncogenes and mutant tumor suppressor genes, unique tumor antigens resulting from chromosomal translocations, and the like and/or derivatives thereof. The antigen may be a self-antigen.

In some embodiments, the immunopotentiator may be a TLR-ligand. The TLR-ligand may be CpG-containing DNA. In some embodiments, the immunopotentiator may be a double-stranded RNA, e.g., multiple IC.

Some embodiments of the invention disclosed herein relate to a panel of immunogenic compositions, wherein the panel includes an immunogen plus an immunizationAn enhancer or biological response modifier, wherein the doses of each member of said group are related as an exponential series. In some embodiments, the dose is exponentially ordered by ≧ 2^n-1Is defined by the exponential factor of (c). In some embodiments, the exponential series of doses consists of 5^n-1Is defined by the exponential factor of (c).

Other embodiments of the invention relate to kits comprising a set of immunogenic compositions comprising an antigen and an immunopotentiator or biological response modifier and instructions for administering the compositions to a subject in need thereof.

The immunopotentiator or biological response modifier can be, for example, but is not limited to, cytokines, chemokines PAMP, TLR-ligands, immunostimulatory sequences, CpG-containing DNA, dsRNA, recognition receptor for endocytic Patterns (PRR) ligands, LPS, quillaja saponin, tocarol, and the like. In some embodiments, the immunogen and the immunopotentiator or biological response modifier may be contained separately in different containers or in the same container.

In some embodiments of the invention, the kit may comprise two or more doses of the immunogenic composition, each in a different suitable container. The suitable container may be, for example, but not limited to, a syringe, ampoule, vial, or the like, or combinations thereof.

Embodiments of the invention relate to a set of syringes comprising successively increasing doses of an immunogenic composition, wherein in each syringe of the set of syringes each dose after an initial dose is greater than the immediately preceding dose, wherein the immunogenic composition comprises an immunogen, and an immunopotentiator or biological response modifier, thereby enhancing a T cell response in a subject.

In other embodiments, the immunogenic composition comprises a cell. The cell may be a tumor cell or an antigen presenting cell, but is not limited thereto. In other embodiments, the antigen presenting cell may be a dendritic cell. In other embodiments, the immunogenic composition comprises a cell.

In other embodiments, a larger dose includes an increased number of cells. In another embodiment, a larger dose comprises an increased number of epitope-MHC complexes on the cell surface.

Some embodiments relate to a set of vials comprising sequentially increasing doses of an immunogenic composition, wherein in each vial of the set of vials, each dose following an initial dose is greater than the immediately preceding dose, wherein the immunogenic composition comprises an immunogen, and an immunopotentiator or biological response modifier, thereby enhancing a T cell response in a subject.

Some embodiments relate to the use of multiple consecutive doses of an immunogenic composition for stimulating a class I MHC-restricted T cell response, wherein each dose after an initial dose is greater than the immediately preceding dose. In some embodiments, stimulation of MHC class I restricted T cell responses is used to treat neoplastic disease or to treat infectious disease or both. In some embodiments, stimulation of MHC class I-restricted T cell responses is used for the prevention of neoplastic disease or for the prevention of infectious disease, or both.

Some embodiments relate to the use of a plurality of consecutive doses of an immunogenic composition comprising an immunogen and an immunopotentiator or biological response modifier, wherein each dose after an initial dose is greater than the immediately preceding dose, for the manufacture of a medicament. Some embodiments relate to the use of a panel of immunogenic compositions comprising an immunogen plus an immunopotentiator or biological response modifier in the manufacture of a medicament, wherein the doses of each member of the panel are related as an exponential series. Preferably, the medicament stimulates MHC class I-restricted T cell responses in the mammal. Thus, the medicament may be for the treatment of neoplastic diseases or for the treatment of infectious diseases or both. In some embodiments, the medicament is for preventing a neoplastic disease or for preventing an infectious disease, or both.

Other objects and features will be in part apparent and in part pointed out hereinafter.

Brief Description of Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of embodiments of the invention disclosed herein. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 illustrates data indicating that exponentially increasing doses of gp33 and CpG enhance CD8⁺T cell response.

FIG. 2 is a bar graph indicating CD8⁺Enhancement of T cell responses is independent of T cell help.

FIG. 3 shows data indicating that 4 days of antigen stimulation was for CD8⁺T cell induction is optimal.

FIG. 4 illustrates data indicating exponential increasing doses of gp33 and CpG to enhance antiviral CD8⁺T cell response.

Figure 5 shows data indicating that antigenic kinetics do not affect DC activation.

Fig. 6A illustrates flow cytometry data indicating that exponential immunization favors continued T cell proliferation.

Fig. 6B illustrates flow cytometry data indicating that exponential immunization favors continued T cell proliferation.

Figure 7 shows data indicating that exponential immunization with peptide-loaded dendritic cells induced strong T cell and anti-tumor responses.

FIG. 8 illustrates data indicating in vitro exponential stimulation of CD8⁺T cells enhance IL-2 production and cytotoxicity.

Detailed Description

The immune system has evolved to respond optimally to pathogens (Janeway, C.A., Jr. applying the enzymatoevaluation and regression in immunology. Immunization can be optimized by exploiting the characteristics of the pathogen and enhancing the efficacy of baccines. For example, to enhance phagocytosis and antigen presentation, vaccines can be delivered in the form of particles having a size comparable to that of the pathogen, such as emulsions, microparticles, immunostimulatory complexes (iscom), liposomes, viral particles, and virus-like particles, to enhance phagocytosis and antigen presentation (O' Hagan, D.T. & valiant, n.m. nat Rev Drug Discov (review of natural Drug discovery) 2, 727-35, 2003, the entire contents of which are incorporated herein by reference). In addition, pathogen-associated molecular patterns (PAMPs) that stimulate the immune system via Pattern Recognition Receptors (PRRs), including toll-like receptors (TLRs) (Johansen, P., et al, Clin Exp Allergy 35, 1591-1598, 2005 b; O' Hagan, D.T. & Valiane, N.M.Nat Rev Drug Discov (review of Natural Drug discovery) 2, 727-35, 2003; Krieg, A.M., Annu Rev Immunol (review of Immunology) 20, 709-60, 2002, each of which is incorporated herein by reference in its entirety), can be used as adjuvants to activate antigen presenting cells and enhance the immune response to vaccines. One critical hallmark of pathogens is replication. Pathogen replication exposes the immune system to increasing amounts of antigens and immunostimulatory PAMPs over time.

The current paradigm of immunology is that the intensity and quality of the T cell response can be controlled by the dose and location of the antigen and the costimulatory signal. Strategies to increase the efficiency of vaccination may be aimed at increasing the duration of antigen presentation (Lofthouse, S.Adv Drug Deliv Rev (Drug delivery progress review) 54, 863-70, 2002; Ehrenhenhofer, C. & Opdebeeck, J.P., Vet Parasitol59, 263-73, 1995; Guery, J.C., et al, J Exp Med (J.Exp. Med. 183, 485-97, 1996; Zhu, G., et al, Nat Biotechnol (Nature Biotechnology) 18, 52-7, 2000; Boulevych, O.Y., et al, J. Immunol (J.Immunol) 174, 4812-20, 2005; Levitsky, V., et al, J. Exp (Experimental medicine) 915, 183, 1996; van der Burg., S.H., 12412-20, 2005; Rimun. J.M. Im. J.201, J.Immunol., R. J.M.201; Ringe. Immunol. J.201, J.J.),55, J.M.201, J.Immunol., 3308, J.M.201, J.,55, 301-6, 1999; blanchet, j.s. et al, J Immunol (journal of immunology) 167, 5852-61, 2001; brinckerhaff, l.h. et al, Int J Cancer (journal of international Cancer) 83, 326-34; 1999; ayyoub, m, et al, J Biol Chem (journal of biochemistry) 274, 10227-34, 1999; stemmer, c, et al, J Biol Chem (journal of biochemistry) 274, 5550-6, 1999; o' Hagan, D.T. & valiant, n.m., Nat Rev Drug Discov (review for natural Drug discovery) 2, 727-35, 2003, each of which is incorporated herein by reference in its entirety.

Methods for optimizing T cell induction remain a challenge in the art. As is well known in the art, the theory of "depot" immunization assumes that the slow leakage of antigen into tissues over an extended period of time correlates with the immunogenic efficacy of the vaccine. Currently, this antigen depot example is the basis of most adjuvant development programs. However, the present disclosure shows that it would be far more effective to administer the vaccine in a dose escalating fashion over consecutive days or closely spaced days rather than in a long acting formulation or a single bolus (bolus) administration. Daily antigen stimulation with exponentially increasing doses is shown herein to enhance CD8 when compared to single bolus or multiple invariant dose administrations⁺T cell response. As used herein, stimulating an MHC class I-restricted T cell response includes, but is not limited to, inducing, priming, initiating, prolonging, maintaining, amplifying, enhancing or potentiating the response.

It is well known in the art that live attenuated vaccines generally induce strong and long lasting immune responses after one injection, and that many viral vaccines of this type have an efficiency of greater than 90% (Nossal, G.vaccines in Fundamental Immunology) (Paul, W.E. eds.) 1387-1425; Lippincot-Raven publisher, Philadelphia, 1999, which is incorporated herein by reference in its entirety). In contrast, vaccines consisting of killed microorganisms, toxins, subunit vaccines including peptide vaccines, or naked DNA vaccines are quite inefficient and booster immunizations are necessary. Although live vaccines produce increased antigen doses that elicit strong immune responses, non-replicating vaccines produce reduced antigen patterns, i.e., as demonstrated in the examples herein, which are relatively weak stimulations of T cells.

Replication-incompetent (replication-incompetent) vaccines are known to be safer than live vaccines. Embodiments of the present invention challenge the trend of vaccine development using replication incompetent vaccines without considering the dose kinetics of antigen stimulation. In addition, embodiments of the present invention provide immunotherapeutic approaches to enhance T cell responses against diseases such as, but not limited to, infectious diseases or cancer.

Embodiments of the invention disclosed herein aim to address the deficiencies in the field of vaccine design and in the implementation of immunotherapy by manipulating antigen stimulation kinetics as a key parameter of immunogenicity. As disclosed herein, linearly or exponentially increasing immunogenic stimulation induces significantly stronger CD8 as compared to stimulation provided at a constant level⁺T cell response. Immunogens administered as a single pulse (single shot) or as multiple reduced doses induce the weakest immune response. The chemical explanation for the findings disclosed herein may be that the most potent CD8 is required for pathogens that replicate and thus produce increased amounts of antigen⁺T cell response. In contrast, a uniform or reduced amount of antigen indicates that the nonpathogenic stimulus or infection is adequately controlled by innate or already acquired immunity.

While not wishing to be bound by this theory, it is believed that mediating increased sensitivity to riskCandidate for antigen presenting cells, and in particular for CD8⁺T cell-induced Dendritic Cells (DCs). This is supported by the following example, since although different vaccination protocols differ in the absolute number of DCs or level of DC activation in the lymph nodes, they also differ in the time taken to reach a peak in DC activation and number. On the exponentially increasing vaccination mode, the peak of DC activation was delayed by 3 days compared to bolus vaccination. For both vaccination protocols, the peak of DC activation occurred one day after administration of the maximum vaccine dose. This observation may suggest that an optimized vaccination schedule would use a single injection of CpG the day before a single peptide injection, so that the peptide is presented on the most active DCs. However, the current data show that the immunization schedule, as well as the exponentially increasing doses of CpG after a single peptide dose, is significantly less immunogenic than the parallel exponentially increasing doses of CpG and peptide.

To examine whether the dose kinetics of the antigen independent of the total dose (cumulative dose) of the course of the treatment is an individual immunogenicity parameter, mice were immunized with a fixed cumulative dose of an antigenic peptide, such as gp33, and an immunopotentiator or Biological Response Modifier (BRM), such as cytosine-guanine oligodeoxynucleotides (CpG ODNs). Different kinetics were performed, i.e. immunization with exponentially increasing or decreasing doses, constant daily doses or single bolus immunization. Class I MHC-binding peptides were chosen as antigens because their short in vivo half-life allows for dramatic antigen kinetics (Falo et al, Proc Natl Acad Sci USA 89, 8347-8350, 1992; Widmann et al, J Immunol 147, 3745-3751, 1991, each of which is incorporated herein by reference in its entirety). Since mice were immunized with the same total dose of vaccine, specific T cell induction could be monitored as a function of the kinetics of peptide and brm (cpg) administration.

As disclosed in the examples herein and elsewhere, fixed cumulative vaccine doses including peptides and brm (cpg) were administered through different schedules to generate unique dose kinetics for antigen stimulation. With uniform or constant daily antigenStimulation with exponentially increasing antigen elicits CD8 compared to single bolus administration of vaccine⁺Significantly stronger stimulation of T cells and enhanced long-term immunity against viral infections and tumors. The same phenomenon is observed when T cells are stimulated in vitro.

Thus, some embodiments relate to methods and compositions for linear or exponential increase of MHC class I CD8⁺Antigenic stimulation of T cell responses, with results exceeding those described in the art. The data show that increased antigenic stimulation is independent of the enhanced immunogenicity of the antigen dose. Thus, the present invention provides novel methods of enhancing immunogenicity, thereby improving vaccine development.

Embodiments of the invention provide arrays of immunogenic compositions comprising an immunogen plus an immunopotentiator or BRM. Some embodiments include co-administering an antigen and an immunopotentiator to achieve an enhanced immune (CTL) response by providing the antigen and immunopotentiator in an exponentially increasing manner.

The immunogenicity of an antigen can be determined by a number of parameters including the antigen dose (Mitchison, N.A., Proc R Soc Lond Biol Sci 161, 275-92, 1964; Weigle, W.O., Adv Immunol (immunological Advance) 16, 61-122, 1973; Nossal, G.J., Annu Rev Immunol (annual review in immunology) 1, 33-62, 1983, each of which is incorporated herein by reference in its entirety); localization of antigens (Zinkernagel, R.M., Semin Immunol 12, 163-71; discission (discussion) 257. cohnner 344, 2000; Zinkernagel, R.M. & Hengartner, H., Science 293, 251-3, 2001, each of which is incorporated herein by reference in its entirety); particulate or soluble nature of the antigen (O' Hagan, D.T. & Valiane, N.M. nat Rev drug discovery review) 2, 727-35, 2003; Bachmann, M.F., et al, Science 262, 1448-51, 1993, each of which is incorporated herein by reference in its entirety); and whether an antigen is present with costimulatory signals (Janeway, C.A., Jr.Appling the enzymatothover evolution and revolution in immunology). Cold Spring Harb Symp Quant Biol 54Pt 1, 1-13, 1989; Germain, R.N., Nat Med (Nature medicine) 10, 1307-20, 2004; Matzinger, P., AnnuRev Immunol (annual review of immunology) 12, 991-.

It is also believed that the more similar the immunogen and immunization scheme is to an infection by a pathogenic pathogen, the more immunogenic it is. High antigen doses of antigen in lymphoid organs and the presence of antigen, both corresponding to the widespread replication of pathogenic pathogens, induce strong immune responses. Particulate antigens structurally similar to viruses or bacteria induce a stronger immune response than soluble antigens. Furthermore, the coexistence of antigens with pathogen components such as, for example, bacterial DNA, lipopolysaccharides or viral RNA strongly enhances the immune response. As taught herein, exponentially increasing antigenic stimulation can also be recognized by the immune system as a pathogen-associated pattern, driving a strong immune response.

Thus, in view of the foregoing, an antigen intended for use in embodiments of the invention is one that stimulates the immune system of a subject having a malignant tumor or an infectious disease to attack the tumor or pathogen, thereby inhibiting its growth or eliminating it, thereby treating or curing the disease. In some cases, the antigen may be matched to a particular disease found in an animal treated to induce a CTL response (also referred to as a cell-mediated immune response), i.e., a cytotoxic response of the immune system that results in lysis of the target cells (e.g., malignant tumor cells or pathogen-infected cells). As understood by one of ordinary skill in the art, increased cytolytic activity can be a measure of the number of target cells that are killed or lysed in the presence of the immunogenic composition relative to the absence of the immunogenic composition. The method of determining or measuring the number of killed or lysed target cells may be any method known to one of ordinary skill in the art including, but not limited to, chromium release assays, tetramer assays, and the like.

As used herein, stimulating an MHC class I-restricted T cell response includes, but is not limited to, inducing, priming, initiating, prolonging, maintaining, amplifying, enhancing or boosting the response.

Antigens intended for use in the methods disclosed herein include, but are not limited to, proteins, peptides, polypeptides and derivatives thereof, and non-peptide macromolecules. The derivatives can be prepared by any method known to those of ordinary skill in the art and can be assayed by any means known to those of ordinary skill in the art. Thus, in some embodiments, antigens for use in the present invention may include tumor antigens such as, but not limited to, differentiation antigens, embryonic antigens, cancer-testis antigens, antigens of oncogenes and mutated tumor suppressor genes, unique tumor antigens resulting from chromosomal translocations, viral antigens, and other antigens that are currently or will become apparent to those skilled in the art in the future. Antigens for use in the disclosed methods and compositions also include those found in infectious disease organisms, such as structural and non-structural viral proteins. Potential target microorganisms intended for use in the disclosed compositions and methods include, but are not limited to, hepatitis viruses (e.g., B, C, and δ), herpes viruses, HIV, HTLV, HPV, EBV, and the like. The general term for these antigens recognized or targeted by an immune response is the Target Associated Antigen (TAA).

Protein antigens that can be used in the disclosed methods and compositions include, but are not limited to: differentiation antigens such as, for example, MART-1/MelanA (MART-I), gp100(Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multiple lineage antigens such as, for example, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p 15; overexpressed embryonic antigens such as, for example, CEA; overexpressed oncogenes and mutated tumor suppressor genes such as, for example, p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations such as, for example, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as, for example, epstein barr virus antigen EBVA and Human Papilloma Virus (HPV) antigens E6 and E7. Other protein antigens may include, for example, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, C-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β -catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha fetoprotein, β -HCG, BCA225, BTAA, CA 125, CA 15-3\ CA 27.29 BCAA, CA 195, CA242, CA-50, CAM43, CD68\ KP1, CO-029, Ga-5, G250, Ga733 CAM, HTMA-6354, gp CAM 175-MG, EpM-50, MOV-24, MOC-18, MOC-35, CAC-7, CG6390, CAC-7, CG6335, CG120, CG35, CGI-9, CG35, CAMCA, CAC-7, CAC-9, TAAL6, TAG72, TLP, and TPS. These protein-based antigens are known and are available to the skilled person in the art either from the literature or commercially.

In other cases of the invention, peptide antigens of 8-15 amino acids in length are contemplated. The peptide may be an epitope of a larger antigen, i.e. it is a peptide having an amino acid sequence corresponding to a site on a larger molecule which is presented by an MHC/HLA molecule and which can be recognized by, for example, an antigen receptor or a T cell receptor. These smaller peptides are available to those skilled in the art and can be obtained, for example, by the following teachings: U.S. patent nos. 5,747,269 and 5,698,396; AND PCT application No. PCT/EP95/02593 (published as WO 96/01429) entitled METHOD OF IDENTIFYING AND DPRODUCING ANTIGEN PEPTIDES AND USE THEREOF ASVACCINES (METHODs OF IDENTIFYING AND producing antigenic peptides AND their USE as vaccines) filed on 4.7.1995 AND PCT application No. PCT/DE96/00351 (published as WO96/27008) filed on 26.2.1996 entitled AGENT TREE ATING TUMOURS AND OTHER HYPERPLASIA (AGENTs FOR TREATING tumors AND OTHER HYPERPLASIAs), each OF which is incorporated herein by reference in its entirety. Additional methods of epitope discovery are described in U.S. patent nos. 6,037,135 and 6,861,234, the entire contents of each of which are incorporated herein by reference.

Although it is generally ultimately determined that the molecule specifically recognized by the T cell is a peptide, it is noted that in practice in an immunogenic formulation, the form of antigen administered in an immunogenic form need not itself be a peptide. When administered, the epitope peptide may be present in a longer polypeptide, whether as a complete protein antigen or some fragment thereof or some engineered sequence. Included in the engineered sequences will be polyepitopes (polyepitopes) and epitopes such as antibodies or viral capsid proteins that bind in the vector sequence. The longer polypeptides may include EPITOPE CLUSTERS such as those described in U.S. patent application No. 09/561,571, filed on 28/4/2000 with U.S. patent application No. 09/561,571, entitled "EPITOPE CLUSTERS," which is incorporated herein by reference in its entirety. The epitope peptide or longer polypeptide comprised therein may be a component of a microorganism (e.g., virus, bacteria, protozoa, etc.) or a mammalian cell (e.g., tumor cell or antigen presenting cell) or a fully or partially purified lysate of any of the foregoing. They can be used as complexes with other proteins, such as heat shock proteins. Epitope peptides can also be covalently modified, such as by lipidation (lipidation) or by preparing components of synthetic compounds such as dendrimers, Multiple Antigen Peptide Systems (MAPS), and polyoximes, or can be incorporated in liposomes or microspheres, and the like. As used in the present disclosure, the term "polypeptide antigen" encompasses all such possibilities and combinations. The invention includes that the antigen may be a natural component of a microorganism or a mammalian cell. The antigens may also be expressed by the microorganism or mammalian cells by recombinant DNA techniques or, especially in the case of antigen presenting cells, by pulsing the cells with polypeptide antigens or epitope peptides prior to administration. In addition, the antigen that may be administered is encoded by nucleic acids that are subsequently expressed by the APCs. Finally, while classical class I MHC molecules present peptide antigens, there are additional class I molecules adapted to present non-peptide macromolecules, particularly components of microbial cell walls, including but not limited to lipids and glycolipids. The terms antigen, immunogen and epitope, as used in the present disclosure, may also include the macromolecule. Furthermore, nucleic acid-based vaccines may encode one or more enzymes necessary for the synthesis of the macromolecule and thereby confer antigen expression on the APC.

Novel peptides identified by the method disclosed in U.S. patent application serial No. 09/560,465, filed on 28.4.2000 and entitled "EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTINGCELLS (EPITOPE SYNCHRONIZATION in antigen presenting cells)" (the entire contents of which are incorporated herein by reference), which may be apparent to those skilled in the art at present or in the future, and are used in the embodiments of the invention disclosed herein, are also intended.

Additional peptides and peptide analogs are disclosed that can be used in the embodiments of the invention disclosed herein, which are disclosed, for example, in the following: U.S. provisional application No. 60/581,001 filed on 17.6.2004, and U.S. patent application No. 11/156,253 filed on 17.6.2005, both entitled SSX-2 PEPTIDE ANALOGS (SSX-2 PEPTIDE ANALOGS); and U.S. provisional application No. 60/580,962 filed on 17.6.2004 and U.S. patent application No. 11/155,929 filed on 17.6.2005, both entitled NY-ESO peptide analogs; U.S. patent application No. 09/999,186, filed on 7/11/2001, entitled METHODS OF Commerciliazing AN ANTIGEN (method OF COMMERCIALIZING antigens); U.S. patent application No. 11/323,572 (published as US 2006/0165711a1), filed on 29.12.2005, entitled METHODS TO elict, ENHANCE AND SUSTAIN IMMUNE RESPONSES AGAINST MHCCLASS I-RESTRICTED EPITOPES, FOR PROPHYLACTIC or THERAPEUTIC PURPOSES (METHODS FOR eliciting, enhancing AND maintaining an IMMUNE response against class I MHC-RESTRICTED EPITOPES FOR PROPHYLACTIC or therapeutic PURPOSES); and U.S. patent application No. 11/323,520, filed on 29.12.2005, entitled METHOD DS TO BYPASS CD4⁺CELLS INTHE Induction OF AN IMMUNE RESPONSE (bypassing CD4 in the INDUCTION OF IMMUNE RESPONSE)⁺Methods of cells), each of which is incorporated herein by reference in its entirety. The principle of selection of beneficial epitopes for immunotherapy is disclosed in the following: for example, U.S. patent application No. 09/560,465, filed 28.4.2000, U.S. patent application No. 10/026,066 (published as US2003/0215425A1), filed 7.12.2001, and U.S. patent application No. 10/005,905, filed 7.11.2001, all entitled EPITOPEPSYNCHRONIZATION IN ANTIGEN PRESENTING CELLS (epitope synchronization in antigen presenting cells); U.S. patent application No. 09/561,571, filed on 28.4.2000 and entitled EPITOPE CLUSTERS (EPITOPE cluster); U.S. patent application No. 10/094,699 (now U.S. patent 7,252,824), filed on 7/3/2002 and entitled ANTI-neovascular system preparation FOR CANCER; U.S. patent application No. 10/117,937 (published as US2003/0220239a1), filed 4/2002, U.S. patent application No. 10/657,022 (published as US 2004/0180354a1), filed 5/9/2003, and PCT application No. PCT/US2003/027706 (published as WO 04/022709a2), filed 5/9/2003, all entitled epitop SEQUENCES; and U.S. patent No. 6,861,234; each of which is incorporated herein by reference in its entirety.

In some aspects of the invention, vaccine plasmids may be used. The overall design of vaccine plasmids is disclosed, for example, in: U.S. patent application No. 09/561,572, filed on 28.4.2000 and entitled EXPRESSION vector ENCODING target-ASSOCIATED EPITOPES, having the sequence EXPRESSION vector for EXPRESSION of the target-ASSOCIATED epitope; U.S. patent application No. 10/292,413 (published as US 2003/0228634a1), filed on 7/11/2002 and entitled EXPRESSION VECTORS ENCODING target-ASSOCIATED EPITOPES and METHODS FOR designing them; U.S. patent application No. 10/225,568 (published as US 2003/0138808), filed on 8/20.2002 and PCT application No. PCT/US2003/026231 (published as publication No. WO 2004/018666), filed on 19.8.2003, both entitled EXPRESSION vectors encoding EPITOPES OF the dengue-ASSOCIATED antigen; and U.S. Pat. No. 6,709,844, entitled "AVOIDANCE OFUNDESIRABLE REPLICATION INTERMEDIATES IN PLASMINDPROPAGEN" (to avoid undesirable replicative intermediates in plasmid propagation), the entire contents of each of which are incorporated herein by reference.

Embodiments of the invention are also intended for specific combinations of antigens of particular benefit in the directed immune response against specific cancers as disclosed in: FOR example, U.S. temporal number 60/479,554, which was filed on 17.6.2003, and U.S. patent application number 10/871,708 (published as US 2005/0118186), which was filed on 17.6.2004, U.S. patent application number 11/323,049 (published as US 2006/0159694a1), which was filed on 29.12.2005, and PCT patent application number PCT/US2004/019571, which was filed on 17.6.2004, all entitled "combination OF TUMOR-ASSOCIATED antigens in vaccines FOR VARIOUS TYPES OF cancer," each OF which is incorporated herein by reference in its entirety.

Epitopes referred to herein and well known to those skilled in the art are defined as portions of antigens that interact with antigen receptors of the immune system; in this case, the antigen is partially presented by MHC molecules for T Cell Receptor (TCR) recognition. Immunogens are molecules capable of stimulating an immune response. Immunogens contemplated by the present invention as disclosed herein may include, in a non-limiting manner, polypeptides or nucleic acids encoding polypeptides, wherein the polypeptides are capable of stimulating an immune response. The immunogen may be identical to the corresponding TAA or fragment thereof, but need not be. Immunogens may include, but are not necessarily limited to, epitope peptides presented on the cell surface and peptides that are non-covalently bound (complexed) to the binding cleft (cleft) of MHC class I, such that they can interact with the T Cell Receptor (TCR). In addition, the immunogen may comprise epitope-MHC complexes or cells expressing said complexes on their surface.

Mimotopes, as referred to herein and as known to those skilled in the art, are defined as compounds that mimic the structure of an epitope and elicit the same or cross-reactive immune response. Synthetic epitopes referred to herein and as known to those skilled in the art are chemically synthesized non-natural epitope molecules. Methods for synthesizing proteins, peptides, etc. are well known in the art.

Immunopotentiator and Biological Response Modifier (BRMS)

Embodiments of the invention disclosed herein include methods of enhancing a T cell immune response by administering an immunogenic composition comprising an immunogen plus an immunopotentiator or other Biological Response Modifier (BRM). BRMs may act in an immunosuppressive or immunostimulatory manner to modulate an immune response, e.g., by promoting an effector response or inhibiting a T regulatory response. Immunopotentiators or BRMs as used herein may refer to any molecule that modulates the activity of the immune system or its cells by interactions other than with antigen receptors. As used herein, BRMs may also include natural or synthetic small organic molecules that exert immunomodulatory effects by stimulating innate immunity.

Preferred immune enhancing BRMs that may be used in embodiments of the present invention are molecules that elicit cytokine or chemokine production, such as, but not limited to, ligands for Toll-like receptors (TLRs), peptidoglycans, LPS or analogs, imiquimod (imiquimod), unmethylated CpG oligodeoxynucleotides (CpGODNs), dsRNAs, such as bacterial dsDNA (which contains CpG motifs) and synthetic dsRNA (polyI: C), on APC and innate immune cells that bind to TLR9 and TLR3, respectively, and the like. It is noted that these BRMs are potent immunomodulators associated with safety issues in systemic delivery. CpG, in particular (TLR-9 ligand), has shown widespread experimental utility as well as clinical potential as an adjuvant by allowing efficient maturation of antigen presenting cells and subsequent activation of antigen-specific lymphocytes (Krieg, A.M., Annu Rev Immunol (annual review of immunology) 20: 709-. One way to avoid these safety issues is to use intralymphatic (intralymphatic) administration as disclosed in: for example, U.S. patent application No. 11/321,967 (published as us 2006/0153844a1), filed 29.12.2005 AND entitled METHODS TO TRIGGER, mainin, AND manageability components BY target induced immune RESPONSES, METHODS for eliciting, maintaining AND manipulating immune RESPONSES BY TARGETED ADMINISTRATION of a biological RESPONSE modifier TO a LYMPHOID organ, is incorporated herein BY reference in its entirety.

As used herein, the term BRM may refer to any molecule that modulates the activity of the immune system or its cells by interactions other than with antigen receptors. BRMs are also often applied to complex biologics that include one or more active entities, regardless of whether the active ingredients of the mixture have been defined. Examples of complex biologics useful as BRMs include OK 432, PSK, AIL, lentinan, and the like. In some embodiments of the invention, the active ingredients of the mixture are defined. In other embodiments of the invention, BRMs derived from complex biologics are at least partially purified or substantially purified, such as, for example, OK-PSA (Okamoto et al, Journal of the National Cancer Institute, 95: 316-. In preferred embodiments, the BRM is a defined molecular composition. BRMs include immunopotentiating adjuvants that activate papcs or T cells, including, for example: TLR ligands, recognition of endocytic pattern receptor (PRR) ligands, quillaja saponins, tocarol, cytokines, and the like. Some preferred adjuvants are disclosed, for example, in Marciani, d.j. drug Discovery Today 8: 934-943, 2003, the entire contents of which are incorporated herein by reference.

In some embodiments involving administration of cells as an immunogen, the BRM may be a molecule expressed by the cells. In one aspect, the BRM molecule may be naturally expressed by the cell, either constitutively or in response to some biological stimulus. In another aspect, expression is dependent on recombinant DNA or other genetic engineering techniques.

One class of BRMs consists primarily of small organic natural or synthetic molecules that exert an immunomodulatory effect by stimulating innate immunity. Macrophages, dendritic cells and other cells have been shown to carry so-called Toll-like receptors (TLRs) that recognize pathogen-associated molecular patterns (PAMPs) on microorganisms (Thoma-Uszynski, S. et al, Science 291: 1544-. Small molecules that bind TLRs, such as newly generated pure synthetic antiviral imidazoquinolines, e.g., imiquimod and resiquimod, have also been considered and have been found to stimulate the cellular pathway of immunity by binding TLRs 7 and 8 (Hemmi, H. et al, Nat Immunol (natural immunology) 3: 196-200, 2002; Dummer, R. et al, Dermatology (Dermatology) 207: 116-.

Such BRMs are used in preferred embodiments, and interact directly with receptors for detecting microbial components. However, molecules that act downstream of the signaling pathway may also be used. Thus, antibodies that bind co-stimulatory molecules (such as, for example, anti-CD 40, CTLA-4, anti-OX 40, etc.) can be used as BRMs in embodiments of the invention. Similarly, in other embodiments, BRMs used in embodiments of the present invention may include, for example, IL-2, IL-4, TGF- β, IL-10, IFN- γ, and the like; or molecules that cause their production. In addition, other BRMs as contemplated herein may include cytokines, such as, for example, IL-12, IL-18, GM-CSF, flt3 ligand (flt3L), interferon, TNF- α, and the like; or chemokines such as IL-8, MIP-3 α, MIP-1 α, MCP-1, MCP-3, RANTES, and the like.

Adjuvants are molecules and preparations that increase the immunogenicity of an antigen. They may have immunopotentiating activity as described above, but may also have properties that alter the physical state of the immunogen in place of or in addition to said activity. The effect of the adjuvant is not antigen-specific. However, if they are administered with purified antigens, they can be used to selectively promote a response to the antigens. For example, when protein antigens are precipitated by alum, the immune response is increased. Emulsification of the antigen also extends the duration of antigen presentation. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions. Exemplary adjuvants that are often preferred include, but are not limited to, Freund's complete adjuvant (non-specific stimulator of the immune response comprising killed Mycobacterium tuberculosis), Freund's incomplete adjuvant, and aluminum hydroxide adjuvant.

One currently used method to make peptides more immunogenic is to inject them in the context of specialized Antigen Presenting Cells (APCs) such as Dendritic Cells (DCs) (Steinmann, r.m., ann rev Immuno l 9, 271-96, 1991, the entire contents of which are incorporated herein by reference). DCs are potent APCs of the immune system. Other adjuvants which may also be used include MDP compounds such as, for example, thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI is also intended, which comprises three components extracted from bacteria, MPL, trehalose mycolate (TDM) and Cell Wall Skeleton (CWS) in a 2% squalene/tween 80 emulsion. Amphiphiles and surfactants, for example saponins and derivatives such as QS21(Cambridge Biotech) form another group of adjuvants intended for use in embodiments of the invention. Nonionic block copolymer surfactants may also be used (Rabinovich et al, 1994, which is incorporated herein by reference in its entirety).

Administration of immunogenic compositions of the invention

Embodiments of the invention disclosed herein relate to methods of administering an immunogenic composition comprising an immunogen plus an immunopotentiator or BRM to a subject, thereby inducing or enhancing an antigen-specific T cell response. In a preferred embodiment, the immunogenic composition is provided to the subject in an exponentially increasing manner. In another embodiment, the immunogenic composition is provided to the subject in a linearly increasing manner.

The immunogenic composition can be delivered to a subject by any method of delivering a composition known to those of skill in the art according to the methods disclosed herein. Thus, administration of the immunogenic composition of the invention to a subject can be intradermal, intraperitoneal, intramuscular, mucosal and intranodal to lymphoid organs (e.g., within lymph nodes), but is not limited to these. For example, in some embodiments, administration of the immunogenic composition can be performed by transdermal, transmucosal, nasal, bronchial, oral, rectal, and/or subcutaneous means. In some embodiments, administration of the immunogenic composition can include direct delivery into the lymphatic system. In other embodiments, administration of the immunogenic composition may consist of direct delivery into the lymphatic system. As is well known to those skilled in the art, the human lymphatic system includes lymph, lymphocytes, lymphatic vessels, lymph nodes, tonsils, spleen, thymus, and bone marrow.

In some embodiments, it may be desirable to administer or deliver an effective amount of an immunogenic composition comprising an immunogen plus an immunopotentiator or BRM intranodally to a subject thereby eliciting an enhanced T cell response. In some embodiments, the enhanced response comprises a linearly increasing T cell response stimulation. In some embodiments, the enhanced response comprises an exponentially increasing T cell response to the stimulus. Intra-nodal administration is disclosed in: for example, U.S. patent nos. 6,994,851 and 6,977,074; PCT patent publication Nos. WO/9902183A 2; and U.S. patent publication No. 20050079152, each of which is incorporated herein by reference in its entirety. The integration OF diagnostic techniques and immunological METHODS for assessing and monitoring immunoreactivity is discussed more fully in the following items, such as U.S. patent application No. 11/155,928, filed on 17.6.2005 and entitled "improved diagnostic OF ACTIVE immunological BY integrating diagnostic and THERAPEUTIC METHODS", the entire contents OF which are incorporated herein BY reference.

Applicator device

The immunogenic compositions disclosed herein may be delivered by bolus injection with a hypodermic syringe (hypodermic syring) as shown in the examples below, or other similar functional devices known in the art for vaccination. Other methods of delivery/administration may include infusion, e.g., via an immunogen delivery vehicle, such as, for example, a pump, subcutaneous or direct delivery into the lymphatic system. In a preferred embodiment, the delivery vehicle is external to the animal but comprises means (e.g., a needle or catheter) for delivering the antigen into the body, preferably to a lymphatic organ or high lymphatic flow region. An advantage of the immunogen delivery vehicle is that it avoids multiple injections.

A delivery device/carrier (external device) located outside the patient's body, including a reservoir to hold the immunogenic composition, a programmable pump to pump the composition out of the reservoir, a delivery channel or conduit to deliver the composition, and means to introduce the composition into the patient's body to ultimately reach the lymphatic system. In a preferred embodiment, the pump may be programmed to increase the volume infused to provide the increased immunogen concentration required. In an alternative embodiment, the reservoir of the pump is filled with a composition comprising successively increasing concentrations of immunogen. Preferably, the reservoir for the immunogenic composition is large enough to deliver the required amount of immunogen over time, and it is easily refillable or replaceable without requiring the user to reinsert the tool for introducing the immunogenic composition into the lymphatic system. Further discussion of external pumps for immunization includes the use of exemplary pumps, such as those discussed in U.S. Pat. No. 6,997,074 entitled "Method of Inducing CTL responses," which is incorporated herein by reference in its entirety.

Treatment and dosing regimens

In general, embodiments of the invention are useful for treating a subject having a disease for which the immune system of the subject elicits a (mount) cell-mediated response to a disease-associated antigen in order to attack the disease. The type of disease may be, for example, a malignant tumor or an infectious disease caused by bacteria, viruses, protozoa, parasites or any microbial pathogen that enters the cell and is attacked by, for example, cytotoxic T lymphocytes. In terms of therapeutic modalities, the methods are well suited for persistent or chronic conditions, but are not necessarily limited thereto. Furthermore, the present invention is useful for immunizing a subject who may be at risk of developing an infectious disease or tumor.

Dosage regimens and schedules for administering immunogenic compositions comprising an immunogen plus an immunopotentiator or BRM may be used in the treatment of diseases and/or conditions for which the present invention is intended. In some embodiments, the immunogenic compositions disclosed herein can be administered as a plurality of consecutive doses, wherein each dose after an initial dose is an incremental dose. The continuously increasing dose may be provided as a linearly or exponentially increasing dose. As used herein, linearly increasing doses refer to a series of doses equal to nd_iIn which d_iIs the starting dose and n is the index of the series, such that the dose series is d_i，2d_i，3d_i，…nd_i. By exponentially increasing dose, it is meant that a series of doses equals x^n-1d_iWherein x > 1, such that the dosage series is d_i，xd_i，x²d_i，x³d_i，…x^n-1d_i. Thus, if x is 2, each dose is twice the dose immediately preceding it in the series; if x is 5, each dose is 5 times the immediately preceding dose in the series. Thus, in a preferred embodiment, the immunogenic composition of the invention may be administered as a plurality of consecutive doses, wherein each dose is given an initial dose of the index factor x^n-1The double supply was performed. The plurality of doses may be 2, 3, 4, 5,6 or more doses as desired. In cases where the initial dose administered may be a very low dose that produces an immune response, many doses (i.e., 7, 8, 9, 10, 12, 15 or more doses) may be administered to a subject to obtain a more immunogenic doseAn effective dose response.

In some embodiments of the invention, the immunogenic composition comprises a cell comprising an antigen or immunogenic portion thereof. In some of these embodiments, the cell functions as an antigen presenting cell, expresses and processes the antigen, or is pulsed (pulse) with the antigen or an epitope peptide or other immunogenic portion of the antigen. The cells may naturally express an antigen (or immunogen), such as TuAA-expressing cancer cells, or may be manipulated to do so, such as dendritic cells transfected with mRNA encoding the immunogen. For example, the cell may be a cancer or tumor cell or an antigen presenting cell, but is not limited thereto. The tumor cell can be bladder cell, breast cell, lung cell, colon cell, prostate cell, liver cell, pancreas cell, stomach cell, testis cell, brain cell, ovary cell, lymphocyte, skin cell, brain cell, bone cell, soft tissue cell, etc. The antigen presenting cell may be, for example, a dendritic cell. The dose in these embodiments may be increased by continuously increasing the number of cells administered relative to the cells administered in the immediately preceding dose or by continuously increasing the number of epitope-MHC complexes on the cell surface relative to the epitope-MHC complexes on the cell surface in the immediately preceding dose, wherein the epitope is from the target antigen, or both. The number of epitope-MHC complexes on the cell surface can be most easily manipulated by pulsing with different concentrations of the epitope.

Thus, administration is carried out in any manner compatible with dosage formulations and in such amounts as are therapeutically or prophylactically effective. An effective amount or dose of an immunogenic composition of the invention is the amount required to provide the desired response in the subject being treated, including but not limited to: prevention, alleviation, reversal, stabilization, or other amelioration of the disease or disorder, its progression, or its symptoms. The dosage and dosage schedule of the immunogenic composition may vary depending on the subject, on a subject basis, taking into account factors such as the weight and age of the subject, the type of disease and/or condition to be treated, the severity of the disease or condition, previous or concurrent therapeutic interventions, the ability of the individual's immune system to respond, the degree of protection required, the manner of administration, etc., all of which may be readily determined by the practitioner.

The compositions used herein may include various "unit doses". A unit dose is defined as comprising a predetermined amount of the therapeutic composition calculated to produce the desired response, i.e., the appropriate route and treatment regimen, associated with its administration. The amounts to be administered and the particular route and formulation are within the skill of those in the clinical arts. The subject to be treated, in particular the condition and the required protection of the subject, are also important. The unit dose need not be administered as a single injection, but may comprise a continuous infusion over a set period of time.

In other embodiments of the invention, multiple doses of the immunogenic composition are intended to be administered within about 24-48 hours of each other, within about 12-24 hours of each other, but most preferably within about 6-12 hours of each other, with an interval of about 24 hours between doses being most preferred. In some embodiments, it may be desirable to administer multiple doses of the immunogenic composition of the invention at intervals of several days, with (lapse) days (e.g., 1,2, 3, 4, 5,6, or 7) passing between subsequent administrations. For example, the initial dose may be administered at a low dose followed by a second low or high dose, which may be administered 1,2, 3 or more days after the initial dose; then, a third dose may be administered 1,2, 3 or more days after the second dose; next, the fourth dose may be administered 1,2, 3 or more days after the third dose, and the like. In some embodiments, consecutive doses may be provided at intervals that are affected by the half-life of the antigen. The half-life of an antigen is the time at which 50% of the antigen is metabolized or eliminated by the normal biological processes of a subject. Thus, the technical or clinical practitioner will determine the time period for administration of multiple doses of the immunogenic composition and the time elapsed between subsequent administrations to optimize CD8⁺T cell immune response.

The interval between administrations of the immunogenic composition of the invention may range from minutes to days depending on the dosing regimen and the effectiveness of the administered dose. However, it is intended that the last dose be administered within days of the first dose so as to increase the number of reactive T cells, which over time corresponds to a linear or exponential increase in the dose. In various embodiments, the time interval between the first and last dose may be less than 7 days, preferably it may be 4 or 5 days, and more preferably the last dose may be administered within 6 days of the first dose. Thus, the last dose to be administered depends not only on the number of days of administration and the effectiveness of the initial dose, but also on the increase in the number of T cells that generate an immune response. Over time, the immune response elicited will decline and the procedure can be repeated to prolong or reestablish immunity.

The subject to which the immunogenic compositions of the invention may be administered as a therapeutic agent may include humans and animals of various ages, such as, but not limited to, cows, sheep, pigs, goats, and domestic pets such as dogs, cats, rabbits, hamsters, mice, rats, and the like. The immunogenic compositions of the invention may be used primarily to treat humans in need of inducing, sustaining or exponentially stimulating a specific immunogenic response in the treatment of a disease or disorder, such as cancer or an infectious disease.

Reagent kit

Any of the compositions described herein may be assembled together in a kit. In non-limiting examples, one or more agents or reagents for delivering an immunogenic composition can be provided in a kit alone or in combination with additional reagents to treat a disease or condition caused by an infectious disease or cancer. However, these ingredients are not meant to be limiting. The kit will provide suitable container means for storing and dispensing the medicament or reagent.

Kits typically comprise, in suitable container means, an immunogenic composition comprising a pharmaceutical formulation of an immunogen plus an immunopotentiator or BRM for administration to a subject, together with instructions for administration. The kit may have a single container means and/or it may have separate container means for additional compounds such as immunologically/therapeutically effective formulations of therapeutic agents for treating diseases or conditions caused by infectious diseases or cancer. The kit may additionally comprise several doses of the immunogenic composition in suitable container means, each in separate container means. The several doses of the immunogenic composition may be two or more sequentially increasing doses of the immunogenic composition, wherein each subsequent dose is greater than the immediately preceding dose. In some embodiments, the kit comprises two or more doses of the immunogenic composition, each dose in a suitable separate container means. For example, the kit may contain 2, 3, 4, 5,6, 7 or more doses of the immunogenic composition, each dose in a suitable separate container means. In other embodiments, the kit may include several doses of the immunogen, or immunopotentiator or BRM, each in separate container means.

In case the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, wherein sterile aqueous solutions are particularly preferred. The composition may also be formulated as an injectable composition, in which case the container means may itself be a syringe, pipette and/or other such device from which the formulation may be delivered or injected into a subject, and/or even applied and/or mixed with other components of the kit. In some embodiments, the components of the kit may be provided as a dry powder. When the ingredients (e.g., reagents) are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in another container means.

The increased dose used according to the present invention may be provided by administering an even larger volume of the same concentration, or a larger concentration of the same volume, or some combination thereof. Thus, in various embodiments, a kit may comprise separately packaged doses; or one or more multi-dose containers from which an increased volume can be dispensed and administered; or one or more multi-dose containers from which the container volumes are dispensed, with successively fewer dilutions and administration of fixed volumes; or similar assembly and application as will suggest themselves to those skilled in the art.

The container means will typically comprise at least one vial, ampoule, test tube, flask, bottle, syringe and/or other container means containing the immunogen and/or immunopotentiator or BRM. The kit may further comprise second container means for containing a sterile pharmaceutically acceptable buffer and/or other diluent. Kits of the invention will also typically include means for containing materials for carrying out the methods of the invention, as well as any other reagent containers that are tightly closed for commercial sale. The container may comprise an injection or blow moulded plastics container in which the required vial is held. Regardless of the number or type of containers, the kits of the invention may further comprise or be packaged with an apparatus for aiding in the injection/administration of an immunogenic composition comprising an immunogen, plus an immunopotentiator or BRM, in a subject. The instrument may be a syringe, a pump, and/or any such medically approved delivery tool.

In some embodiments, a set of syringes is provided comprising increasing doses of an immunogenic composition, wherein each dose after an initial dose is greater than the immediately preceding dose. In some embodiments, a set of vials is provided comprising increasing doses of an immunogenic composition, wherein each dose after an initial dose is greater than the immediately preceding dose. The increased dose may be continuously increased in a linear manner or in an exponential manner.

Having described the invention in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the invention defined in the appended claims. Further, it is to be understood that all examples in this disclosure are provided as non-limiting examples.

Examples

The following non-limiting examples are provided to further illustrate the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute examples of modes of operation for which they may be considered. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1: materials and methods

Mice.6-12 weeks old C57BL/6 mice were purchased from Harlan (Horst, the Netherlands). TCR318 transgenic mice were obtained from Cytos Biotechnology AG (Cytos Biotechnology AG) (Schlieren, Switzerland) which express a T cell receptor specific for the peptide gp33(aa33-41) represented at H-2^bImmunodominant epitopes in the mouse that are located in glycoproteins of the lymphocytic choriomeningitis virus (LCMV) (Pircher, H., et al 1989.Nature (Nature) 342: 559-. HHD transgenic mice expressing HLA A2.1 were originally obtained from MannKind Corporation (MannKind Corporation) (Valencia, CA; Pascolo, S. et al, J Exp Med 185, 2043-. Mice were bred and maintained in a specific pathogen free facility at the university hospital, zurich, according to the rules of the switzerland veterinary regulatory agency.

LCMV isolate WE was obtained from the Institute of Experimental Immunology, University Hospital, Zurich, Switzerland (the Institute of Experimental Immunology, University Hospital, Zurich, Switzerland). LCMV titers were determined on MC-57 fibroblasts using a focus formation assay (focus for mingassay) (Battegay, M. et al, J Virolmethods (J. Virol methods) 33, 191-198; 1991, the entire contents of which are incorporated herein by reference). Recombinant vaccinia virus (Bachmann, M.F., et al, Eur J Immunol (J European Immunol) 24, 2228-plus 2236, 1994, incorporated herein by reference in its entirety) expressing LCMV glycoprotein (vacc-gp) was grown and spotted on BSC40 cells (Kundig, T.M., et al, J Virol (J Virol) 67, 3680-plus 3683; 1993, incorporated herein by reference in its entirety). LCMV glycoprotein peptides gp33(aa 33-41; KAVYNFATM, SEQ ID NO: 3) and gp61(aa 61-80; GLNGPDIYKGVYQFKSVEFD, SEQ ID NO: 4) and VSV peptide np52(SDLRGYVYQGLKSG, SEQ ID NO: 5) were purchased from EMC Microcollections (Tubingen, Germany). Influenza matrix peptide (GILGFVFTL, SEQ ID NO: 6) was purchased from Neosystems (Strasbourg, France). The HPV16E 7(aa 49-57; RAHYNIVTF, SEQ ID NO: 7) peptide used was synthesized at Mannkang Corporation (Mannkid Corporation) (Valencia, CA) to > 99% purity. Phosphorothioate modified CG-rich oligodeoxynucleotide 1668 (5'-TCCATG ACG TTC CTG AAT AAT-3', SEQ ID NO: 8) was synthesized by Microsynth (Balgach, Switzerland).

Immunization schedules different immunization schedules were designed (s1-s6) to deliver a fixed cumulative dose of 125 μ g gp33(KAVYNFATM, SEQ ID NO: 3) peptide or influenza matrix peptide (GILGFVFTL, SEQ ID NO: 6; Falk, K. et al Immunology 82, 337-342, 1994, the entire contents of which are incorporated herein by reference) and 12.5nmol CpG 1668 (Table 1) over a period of one to four days. Note that tables 3(s3) and 4(s4) are scheduled to follow an exponential decrease or increase pattern, respectively, over a 5-fold dilution step. Immunization with influenza matrix peptide was performed with the same cumulative dose of 125 μ g and according to the same schedule.

Adoptive transfer experiment 1X 10⁶TCR-transgenic T cells were resuspended in 250 μ Ι pbs and injected into the tail vein of sex-matched C57BL/6 mice in order to increase the frequency of precursor T cells and facilitate assessment of immune response. One day later, recipient animals were treated with different agents mixed with cytosine-guanine oligodeoxynucleotides (CpG ODN) in the neck region as shown in Table 1An amount of gp33 peptide was inoculated subcutaneously. Alternatively, mice were infected intravenously with strain LCMV-WE (250 pfu). Immunization with influenza matrix peptide was performed according to the same schedule with the same cumulative dose of 125 μ g.

For FACS analysis of surface antigens, single cell suspensions of blood, spleen or lymph nodes without RBCs were prepared. Cells were incubated on ice for five minutes with anti-CD 16/CD32 for Fc-receptor blocking and stained with PE-labeled gp33I MHC class tetramer (gp33/H-2Db) for 15 minutes at 37 ℃ followed by 20 minutes on ice with other surface antigens. All staining was performed in PBS/FCS 2% with 0.01% sodium azide. For intracellular staining of IFN-. gamma.the single-cell suspension was incubated with 2X 10 cells in complete medium^-6M gp33 peptide and 10. mu.g/ml Brefeldin A (Sigma, Buchs, Switzerland) were cultured in vitro for four hours. Lymphocytes were then surface stained as above, fixed in protein-free PBS/PFA 1% for 10 minutes, permeabilized on ice in PBS/NP 400.1% for 3 minutes, and finally incubated with anti-IFN- γ antibody in PBS/FCS 2% for 35 minutes on ice. Samples were captured on FACSCalibur and analyzed using either CellQuest software from BD Biosciences (San Jose, CA) OR FlowJo software from TreeStar Inc (TreeStar, Ashland, OR). All other antibodies were purchased from BD Pharmingen (San Diego, CA).

Evaluation of antiviral Immunity in vivo vaccinated female C57BL/6 mice were infected 1.5X 10 intraperitoneally⁶pfu vacc-gp. Five days later, ovaries were isolated and tested as described by Kundig, t.m. et al in J Virol (journal of virology) 67, 3680-3; 1993, vaccinia titers were determined on BSC40 cells as described above. Alternatively, mice were infected with 250pfu LCMV-WE and viral titers in the spleen were determined on MC57 cells (Battegay et al, 1991, supra).

Cytotoxicity assays and cytokine secretion assays 1X 10⁵Transgenic gp 33-specific T cells were irradiated in 24-well plates with syngeneic feeder cells (2X 10)⁶Cells/well; 2000rads) were cultured together for six days and pulsed with the indicated amount of gp33 peptide. Followed byThe effector cells were then resuspended in 300. mu.l fresh medium and diluted three-fold. Use 10 of EL-4 cells^-6M gp33 peptide was pulsed and at five hours⁵¹Cr release assay was used as a target cell (Bachmann, M.F. et al, Eur J Immunol (Eur J Immunol) 24, 2228-36, 1994, supra). Radioactivity in cell culture supernatants was measured using a Cobra II meter (Canberra Packard, Downers growth, IL). Non-radioactive culture supernatants were evaluated daily for IFN-. gamma.IL-2 and IL-10 concentrations. Cytokine analysis was performed using bead-multiplex-assay and flow cytometry.

Bone marrow-derived dendritic cells were prepared and seeded, bone marrow cells were isolated from femurs of young C57BL/6 mice at 2X 10⁶Cells were plated in 100-mm dishes, 10ml supplemented medium with 50ng/ml rmGM-CSF and 25ng/ml rmIL-4 (R)&D System (R)&D Systems), Minneapolis, MN). On day seven, cells were harvested and DCs were purified by positive selection using anti-CD 11c microbeads (Miltenyi Biotec, Bergisch Gladbach, germany). Purified cells were seeded in six-well plates and stimulated with 2 μ M CpG ODN 1668 overnight. DC phenotypes were assessed by flow cytometry using a panel of labeled mabs to CD80, CD86, CD40, CD11C, and a mouse germline antibody cocktail (CD3e, CD11B, CD45R/B220, Ly-76, Ly-6G, Ly-6C). All antibodies were obtained from BD Pharmingen. Subsequently, DCs were pulsed with HPV E7(aa49-57) peptide (RAHYNIVTF, SEQ ID NO: 7) at 10. mu.g/ml for two hours at 37 ℃. DCs were washed three times with PBS before bilaterally administering 25 μ LDC to the inguinal lymph nodes of anesthetized C57/B6 mice (Johansen et al 2005a. Eur J Immunol (European journal of immunology) 35: 568-574, the entire contents of which are incorporated herein by reference). Groups of ten mice received a single bolus injection of DCs (1.11X 10) on day 1 (s1)⁵) (s1), or by injecting an exponential or increasing number of DCs on days 1, 3 and 6 (10)³，10⁴And 10⁵)(s4)。

PBMCs were isolated from mice (n-10) on day 17 and monocytes were isolated from RBCs after density centrifugation (mammalian lymphocyte separation)Liquid (Lympholyte Mammal), Cedarlane laboratories). Quantification of E7 by staining cells with H-2Db HPV16E 7(RAHYNIVTF, SEQ ID NO: 7) -PE MHC tetramer (Beckman Coulter) and FITC-conjugated anti-CD 8a (Ly-2) (BD Pharmingen) mAb for one hour at 40 deg.C_49-57Specific CTL responses. Data were collected using FACSCalibur and analyzed using CellQuest software.

For quantification of IFN- γ producing cells, spleens were isolated at day 21 and single cell suspensions (n-7) were prepared. Monocytes were isolated by density centrifugation and resuspended in serum-free HL-1 complete medium, which contained nonessential amino acids, sodium pyruvate, glutamine pen-strep, beta-mercaptoethanol, and HEPES. Triplicates of 2.5X 10 in 96 well filter plates (Multi-screen IP membrane 96-well plates, Millipore)⁵Splenocytes were incubated with 10. mu.g/well of HPV16E7 peptide at 37 ℃ for 72 hours. ELISpot readers and software from AID (Strassberg, germany) were used to quantify ELISpot's after 24 hour development using coatings from U-Cytech biosciences (urtrecht, the netherlands) and detection of IFN- γ antibodies.

Tumor protection study on day 21 (15 days after the last injection of DCs), 10 of tumor cell line C3.43 transformed with HPV⁵Three mice from each vaccinated group and seven naive C57/B6 mice (Feltkamp et al 1993, Eur J Immunol (J European Immunol) 23: 2242-. Cells were administered subcutaneously in the left flank. Tumor progression was monitored by calibre measurements (mm) and calculated tumor volume.

Statistical analysis a student's t-test of putative equivariables was performed and data significance was considered with unpaired two-tailed p-values below 0.05. Data for nonparametric or abnormal distributions were analyzed using the Mann Whitney U test or by Kurskal-Wallis ANOVA. Comparison of Kaplan-Meier survival curves was performed using the log rank test (log rank test).

Example 2: exponentially increasing antigen stimulation enhances CD8⁺T cell response

It was investigated whether T cell responses could be enhanced by increasing antigen stimulation. In the first experiment, 1X 10 was set⁶Transgenic gp 33-specific T cells were transferred into C57BL/6 wild-type recipient mice to increase precursor T cell frequency and facilitate evaluation of immune responses.

Using different vaccination protocols disclosed in fig. 1D and table 1, the same cumulative dose of gp33 peptide (125 μ g gp33 and 12.5nmol CpG total) mixed with CpGODN was used to immunize all mice: s1) bolus injection on day 0; s2) four equivalent doses over four days; s3) dose reduction over four days; and s4) increasing the dose over four days. Furthermore, groups of mice were immunized with a single dose of CpG followed by an exponentially increasing dose of gp33 peptide (s5), or with a single dose of gp33 followed by an exponentially increasing dose of CpG (s 6). Mice infected intravenously with 250pfu LCMV virus at day 0 served as positive controls. At day 6 (FIG. 1A), day 12 (FIG. 1B) and day 8 (FIG. 1C), CD8 was quantified by intracellular IFN-. gamma.staining of blood lymphocytes⁺T cell responses, the lymphocytes were re-stimulated in vitro with gp33 peptide. Figure 1B depicts a representative FACS example analyzed at day 12.

CpG ODNs were chosen as adjuvants because they strongly enhance CD8⁺T cell responses (Krieg, A.M., Annu Rev Immunol (annual review of immunology) 20, 709-60, 2002; Schwarz, K. et al, Eur J Immunol (J. Eur. Immunol) 33, 1465-70, 2003, each of which is incorporated herein by reference in its entirety). Phosphorothioate stabilised ODN was cleared from plasma with a half-life of 30-60 minutes (Farman, c.a).&Kornbrust, D.J., ToxicolPathol (toxicology and pathology) 31Suppl, 119-22, 2003, the entire contents of which are incorporated herein by reference). However, CpG ODN are relatively stable in tissue with a half-life of 48 hours (Mutwiri, G.K., et al, J Control Release 97, 1-17, 2004, all incorporated herein by referenceHerein incorporated by reference). In addition, it is mentioned in the literature that serum proteases degrade free peptides to below detectable levels within 60 minutes (Falo, L.D., Jr., et al, Proc Natl Acad SciUSA (Proc. Natl. Acad. Sci. USA) 89, 8347-50, 1992; Widmann, C., et al, J Immunol (J. Immunol) 147, 3745-51, 1991, supra).

The data show that it results in CD8 of comparable magnitude to LCMV wild-type infection⁺Immunization of a T cell response requires that both gp33 and CpG be administered in an exponentially increasing manner. Immunization with a daily uniform dose of gp33 and CpG induced the second most intense CD8⁺T cell responses, however, were significantly weaker than dose-escalation stimulation (p 0.0001 on day 6). If any of the vaccine components were delivered as a single dose, the immune potency was significantly reduced, but significantly compared to the control used for the first time in the experiment (2.23 ± 0.84% versus 0.19 ± 0.12% p ═ 0.02 on day six).

Similar observations were made in wild type mice that were first used in the experiment that did not receive TCR transgenic cells (fig. 1C). C57BL/6 mice immunized with exponentially increasing vaccine (gp33 and CpG) doses showed significantly enhanced CD8 compared to other vaccination regimens⁺T cell induction (2.1. + -. 0.4%) and the other vaccination protocol induced barely detectable specific CD8⁺T cell frequency. No test group showed a measurable immune response on day four (data not shown). These results indicate that, independent of the overall dose, the kinetics of vaccination is a key parameter for immunogenicity.

No test group showed a measurable immune response on day four (data not shown). Overall, these results show that independent of the overall dose, the kinetics of the antigen and the adjuvant are key parameters for immunogenicity.

In the drawings: s 1: single doses of gp33 peptide and CpG; s 2: equal doses of gp33 peptide and CpG; s 3: exponentially decreasing doses of gp33 peptide and CpG; s 4: exponential increasing doses of gp33 peptide and CpG; s 5: an exponentially increasing dose of gp33 peptide and an initial bolus dose of CpG; s 6: an initial single dose of gp33 peptide and an exponentially increasing dose of CpG; first used in the experiment: untreated mice; LCMV: mice immunized intravenously with 250pfu LCMV on day 0. Values represent mean and SEM of four mice per group. One representative experiment of three similar experiments is shown.

Figure 1B is a representative FACS example analyzed at day 12. And (3) upper group: restimulation with gp33 peptide, the following group: control staining without gp33 restimulation (lower panel).

Example 3: CD8⁺Enhancement of T cell responses independent of T cell help

And CD8⁺The role of T cells in eliciting relevant T-help is well known in the art. Th epitopes may be on functional CD8⁺T cell immunity is critical (Johansen et al, Eur JImmunol (journal of European immunology), 34, 91-97, 2004; Shedlock and Shen, Science, 300, 337-. On the other hand, when the precursor frequency is high, CD8⁺The Th dependence of T cell responses is small (Mintern et al, J Immunol., J.Immunol., 168, 977 @, 980, 2002, incorporated herein by reference in its entirety), particularly when strong immunogens, such as LCMV gp33, are used. Furthermore, the route of administration may also affect the need for T-help (Bour et al, J Immunol. (J. Immunol.), 160, 5522-. Therefore, the Th-dependence of CTL is very dependent on the experimental condition settings. Based on this hypothesis, the inventors examined CD8 by vaccination with exponentially increasing vaccine doses⁺Whether the enhancement of T cell responses is independent of T cell help.

Mice were immunized with exponentially increasing vaccine doses using a mixture of LCMV gp33 class I (aa33-41) peptide and LCMV gp61 class II (aa61-80) Th epitopes from LCMV as described in the protocol above (see table 1).CD8 was observed by vaccination with exponentially increasing vaccine doses⁺Enhancement of T cell responses was independent of T cell help, as obtained for CD8 in mice immunized with the protocol described above⁺The same effect of dose kinetics of T cell responses (data not shown).

To examine whether the exponential immunity observed above can be achieved with other peptides, the assay was extended to another peptide derived from the influenza matrix protein, which binds to human HLA A2.1I-class molecules (Falk, K. et al, Immunology): 82, 337-42, 1994, supra). Transgenic mice expressing HLA A2.1 were immunized subcutaneously with influenza matrix peptide (GILGFVFTL, SEQ ID NO: 1) and CpG ODN (HHD; Pascolo, S. Pen, J.Exp.Med. (J.Exp.Med.) (J.Med.) -185, 2043-51, 1997, supra). The immunization schedule is described in table 1 (above). The vaccine was administered as a single bolus (125 μ g peptide and 12.5nmol CpG, s1) or the same total dose was administered in a dose escalating fashion over four days (s 4). Freund's incomplete adjuvant (IFA), a mineral oil that slowly releases antigen, was used as a positive control (Miconnet, I. et al, J Immunol (J. Immunol.), 168, 1212-8, 2002; Speiser, D.E. et al, J Clin.invest. (J. Clin. Res. 115, 739-46, 2005; Aichele, P. et al, J exp. Med. (J. Experil. Med.) (182, 261-6; 1995, the entire contents of each of which are incorporated herein by reference). Eight days later on CD8 after in vitro restimulation of blood lymphocytes with peptides⁺T cells were analyzed for IFN-. gamma.production (mean. + -. SEM; n-3-4). An exponentially increasing vaccine dose was observed to produce more frequent IFN- γ producing cells (6.2% + -1.5) than treatment with a single dose of peptide and CpG (0.6% + -0.2) or emulsified peptide and CpG in IFA (2.5% + -1.9); (FIG. 2).

Example 4: four or more days of antigen administration induced the greatest CD8⁺T cell response

As shown in the examples above, exponentially increasing antigen over a four day period induced a significantly more intense T cell response than a bolus injection or an equivalent daily vaccine dose. Experiments were performed to check if further extension of antigen presentation would further enhance the response. Groups of C57BL/6 mice were immunized subcutaneously with the same total dose of gp33 peptide and CpG (125 μ g p33 and 12.5nmol CpG) by injection of the dose as a bolus or over four, six or eight days (fig. 3A), but peaked at day 0 (bolus), three, five or seven days following different exponential kinetics.

At various time points of the last injection, blood lymphocytes were isolated and restimulated in vitro with gp33 peptide for determination of CD44 expression and intracellular IFN- γ by flow cytometry. As shown in figure 3B, injection of specific CD8 resulting in a significant frequency of significant enhancement in the peak of immune response over four, six, or eight days compared to a single bolus injection⁺T cells, the peak being four to seven days after the last injection. FACS Density blots depict the frequency of CD44hi and IFN- γ producing CD 8-positive lymphocytes measured by FACS at the peak of the immune response, the numbers showing IFN- γ producing CD44hi CD8⁺Average percentage of T cells. Two similar experiments showed one of them (n-3-4).

IFN-gamma producing CD44hi CD8⁺The average percentage of T cells was also described as a function of time (fig. 3C). Two similar experiments showed one of them (n-3-4). The antigen kinetics that peaked at day four induced significantly stronger CTL responses compared to shorter or longer antigen patterns that induced significantly weaker responses. Moreover, there was no statistical difference in the number of dormant memory cells measured four weeks after the last injection. The biological cause of these observations may be CD8⁺It takes several days for T cells to proliferate and differentiate into effector cells, and it is difficult for the immune system to compete even with pathogens overwhelming the host infected within one or two days. On the other hand, when they are constantly being resisted by the CTL, the pathogens that replicate over an extended period of time cause more damage.

Example 5: exponentially increased antigenic stimulation enhances protective antiviral responses

To further clarify the work of the above observationsRelatedly, female wild-type C57BL/6 mice were immunized with fixed cumulative doses of gp33 peptide and CpG according to various protocols (s1-s 4 as shown in table 1), followed by challenge with LCMV or recombinant vaccinia virus expressing LCMV glycoprotein (vacc-gp) at a time point when the T cell response was already in the systolic or memory phase (Kaech, s.m., et al, Nat Rev Immunol (natural immunology review) 2, 251-62, 2002, the entire contents of which are incorporated herein by reference). Protection against both viruses exclusively relies on CD8⁺T cells (Binder, D. and Kundig, T.M., J Immunol (J. Immunol) 146, 4301-7, 1991; Kundig, T.M. et al, Proc. Natl. Acad. Sci., USA (Proc. Natl. Acad. Sci., USA) 93, 9716-23, 1996, each of which is incorporated herein by reference in its entirety).

Mice (n-4) were immunized with exponentially increasing amounts (s4) or bolus injections (s1) of gp33 peptide and CpG as described above (table 1). Negative control mice were not treated (naive) and positive control mice were infected with LCMV (250 pfu). Mice were bled at day 10 and day 30 for analysis of gp 33-specific effector or memory CTLs using gp 33-MHC-tetramer and flow cytometry (fig. 4A) or IFN- γ producing CD8 following in vitro gp33 restimulation at day 30⁺T cells (fig. 4B). Fig. 4A depicts gp 33-tetramer positive CD44hi expression at days 10 and 30, first used for experiments from left to right, s1, s4 and LCMV. According to the above results, exponentially increasing doses of peptide (gp33) and CpG induced IFN-. gamma.producing effects and memory cells (FIG. 4B) and gp 33-tetramer positive memory (CD 44) significantly more frequently than single immunizations^hi) Cells (fig. 4A). On day 30, all mice were stimulated by intraperitoneal injection of 250pfu LCMV. Four days later, virus titers were measured in the spleen. On day 30, mice were challenged intraperitoneally with 250pfu LCMV. Four or five days later, the spleen or ovaries were collected for LCMV determination. Although the bolus (s1) -immunized mice did not receive significant protection against viral replication (fig. 4C), the exponentially increasing vaccination induced significant protection against LCMV titers of approximately 10 to 20 fold (p < 0.01) when compared to mice used for the experiment or bolus-immunization for the first time.

In another set of experiments, C57BL/6 mice were immunized using a different protocol, followed by 1.5X 10 on day 8 (FIG. 4D) or 24 (FIG. 4E)⁶pfu of recombinant vaccinia virus (vacc-gp) was stimulated intravenously. Five days later, vacc-gp replication was measured in the ovaries (FIGS. 4D and 4E). Likewise, only mice immunized in a dose-escalating manner were able to exhibit significant protective CD8⁺T cell responses, on average, inhibit viral replication by two to three orders of magnitude better than other peptide immunization protocols.

These results, therefore, demonstrate the biological relevance of the kinetics of antigen presentation during immunization.

Example 6: the number of activated APCs is not dependent on the type of stimulation kinetics

To test whether the pharmacokinetics affected the activation status and the number of activated APCs, C57BL/6 mice were immunized with gp33 peptide and CpG according to immunization protocol s1 (bolus injection) and s4 (exponentially increasing dose) as described in fig. 1-3 and table 1. The vaccine is administered subcutaneously in the groin area. After one, four, six and eight days, the inguinal lymph nodes were removed and their single cell suspensions were analyzed by flow cytometry for the expression of the DC marker CD11c, as well as CD86 and MHC class II marker I-Ab (fig. 5A). The results are shown as the expression of mean fluorescence relative to the control first used in the experiment (day 0). The results suggest that the different kinetics neither critically affect the number of DCs in draining lymph nodes nor that their status is monitored by MHC class II (I-Ab) and CD86 expression (fig. 5A). However, although the DC numbers and the activated peaks were comparable, they were spaced 2-3 days apart in time. DC activation reached its maximum one day after the maximum CpG dose, independent of the type of immuno-kinetics.

Thus, exponentially increasing vaccination was for CD8⁺T cell induction was optimal only because the antigenic peptide was delivered at the time point where DCs were most active, a possibility that was validated. If this is true, administration of a high dose of peptide in a bolus one day after the bolus injection of CpG or one day after the last administration of a CpG dose in an exponentially increasing manner over four days should result in a match with administration of the peptide in a bolus one day after the last CpG dosePeptide and CpG equivalent CD8 administered in a dose escalation manner⁺T cell response. In a separate experiment, mice were immunized with gp33 peptide and CpG according to a modified protocol (fig. 5B). One group received CpG boluses on day three and gp33 peptide boluses on day four. One group received exponentially higher CpG doses on days 0 to 3 followed by a rapid injection of gp33 peptide on day four. The last group received exponentially increasing doses of gp33 peptide and CpG as described above for one to four days (s 4). Measurement of IFN-. gamma.CD 8 production in peripheral blood on day 10⁺Frequency of T cells. The results show the mean and SEM (n-3) for one of two equivalent experiments). As is evident from fig. 5B, pre-stimulation of APCs with CpG resulted in a gp 33-specific immune response that was significantly lower than the response generated by gp33 and CpG administered together in an exponential increase pattern (p ═ 0.016).

Example 7: exponentially increased antigen stimulation supports prolonged T cell stimulation

To test how the immuno-kinetics affected CD8⁺Proliferation of T cells, mice were injected with a single dose (s1), equivalent daily dose (s2) or with exponentially increasing doses (s4) of gp33 peptide and CpG as described above and in table 1. One group of mice was not treated as a negative control. To monitor proliferation, all mice received 10 intravenously from transgenic TCR318 mice one day before the first immunization⁷Or 1.5X 10⁶CFSE-labeled splenocytes. At different time points, lymphocytes were isolated by tail bleeds and analyzed by flow cytometry for CD8 expression and CFSE staining. The p-value shows the CFSE-labeled CD8 for a CFSE-tag that has entered fragmentation⁺T cells, statistical difference between the s1 and s4 schedules. The results show one of two comparable experiments. Bolus injection of gp33 peptide and CpG elicited CFSE-labeled CD8 three days post immunization⁺T cells divide (fig. 6A and 6B). Proliferation could be detected two days later (fig. 6B). On day 5, precursor cells still entered division, albeit to a lesser extent than on day 3, and by day 7, CFSE-labeled cells had stopped entering new divisions. In contrast, exponentially increasing stimulation significantly prolonged T cell proliferation. The cells entering division can be in the early stageThree days post priming, assays were performed, although a complete immunization protocol was not yet received, and the split was extended to day five and day seven (fig. 6B). Even on day 9, proliferation was observed (not shown). Moreover, the split index, i.e. the average number of splits performed, s4 was significantly higher than s2 (p < 0.05 by Mann Whitney).

Example 8: exponentially increasing numbers of peptide-pulsed DCs enhanced CD8⁺T cell response

To investigate the contribution of different numbers of APCs, C57BL/6 mice were immunized with the same total number of peptide-pulsed DCs, but using different kinetics. Bone marrow derived DCs were loaded with HPV E7(aa49-57, RAHYNIVTF, SEQ ID NO: 2) peptide and a total of 1.11X 10 on day 1⁵Individual cells were injected as boluses into the groin node (s1), or on day 1 (10)³Cells), day 3 (10)⁴Cells), and day 6 (10)⁵Cells) are administered in a gradual increase (s4) pattern with the same total number of cells. In addition, the vaccine was administered intralymphatically to ensure a constant total number of DCs available for T cell priming. Mice used for the first time in the experiment were used as negative controls. On days 17 and 22, peripheral blood was analyzed by flow cytometry for E7-tetramer positive CD8⁺T cell frequency (FIG. 7A (■)). Values represent mean and SEM (n ═ 10). IFN- γ elispot assays from spleens (n ═ 7) (fig. 7A (□). values represent mean values and SEM (n ═ 7). on day 21, three vaccinated mice and ten mice used for the first time in the experiment were stimulated with the HPV-transformed tumor cell line C3.43 (fig. 7B). tumor progression was monitored by calibre measurements (mm) to calculate tumor volume. survival after stimulation was studied in C57BL/6 mice immunized with DCs loaded with VSV np52 peptide injected through s.c (n ═ 4) (fig. 7C). logrank test of Kaplan Meier curve: s4 ≠ s 2: p ≠ 0.0084; s2 ≠ s 1: p ≠ 0.0082; s1 ≠ used for the first time: p ═ 0.401.

The exponentially increasing dose (s4) again induced a higher number of antigen-specific CD8 than the bolus injection vaccine (s1)⁺T cells. Positive for MHC-E7-tetramer (FIG. 7A (■)) and IFN-. gamma.production (FIG. 7A (□)) measured on days 17 and 22, respectively)CD8⁺This is evident in terms of the frequency of T cells. With measured CD8⁺The magnitude of the T cell response was consistent and mice vaccinated with the dose escalation protocol were resistant to stimulation with HPV transformed tumor cell line C3.43 (fig. 7B). In contrast, mice immunized with only a single bolus were not protected.

By the same token, mice immunized with the (s4) regimen of DCs loaded with VSV np52 peptide showed improved survival following stimulation with transfected mouse lymphoma cells EL-4 to express VSV nucleoprotein (fig. 7C). C57BL/6 mice were immunized by s.c injections of DCs loaded with VSV np52 peptide (n-4). 1.11X 10 doses were administered as boluses on day one (s1) or as equivalent (s2) or as dose escalating doses on days 1, 3, 6 (s4)⁵DCs. Mice used for the first time in the experiment were used as controls. On day 14, dose 10 was administered⁶EL-4N.1 cells i.p stimulated all mice (Kundig et al, J Immunol (J Immunol.). 150, 4450. sup. 4456, 1993, supra). The data demonstrate that the survival of mice immunized with (s4) was also significantly better than mice immunized with DCs administered in uniform numbers on three days according to the (s2) protocol (p ═ 0.084).

Thus, exponentially increasing inoculations proved to be more immunogenic than bolus inoculations. Since these experiments maintained DC activation at the same level throughout the course of immunization and the total amount of DCs was the same, this confirms that the kinetics of appearance of activated peptide-presenting DCs determines CD8⁺The intensity of the T cell response. Thus, the inventors concluded that: synchronization of DC numbers with specific T cell frequencies increases the ultimate explosive capacity of T cell responses. Although the low frequency of specific T cells during early responses can be effectively stimulated with DC pulsed with a small amount of antigen, it is important to restimulate the high frequency of specific T cells with high amounts of DCs during later primary responses.

Example 9: exponentially increasing antigen stimulation enhances IL-2 production in T cells: effect of antigen kinetics on the level of T cell clones

The inventors next investigated whether the observations in the above examples can be explained at the level of T cell clones, or whether they are the result of an in vivo T cell selection process involving differential affinity, antibody avidity and functional T cell clonotypes.

Will be 1 × 10⁵TCR-transgenic T cells with 2X 10⁶Irradiated syngeneic splenocytes, which act as feeder cells, were co-cultured. Expressing T cells recognizing transgenic T cell receptor for gp33 at D^bIn vitro stimulation with the same total dose of antigen corresponding to a different pattern, i.e. 10 on day 0^-9Boluses of M gp33(■); in 4 days, respectively on days 0, 1,2 and 3, 10 is used^-12，10^-11，10^-10And 10^-9Exponentially increasing gp33 dose for M (° c); the same 0.25X 10 is used every day^-9Gp33 dose of M was carried out for 4 days (. tangle-solidup.); or 10 days at 0, 1,2 and 3 days^-9，10^-10，10^-11And 10^-12Exponential decrease of M gp33 dose (. cndot.). Control cells without gp33 stimulation were described as (.). IL-2, IL-10 and IFN- γ in the supernatants were determined daily (FIG. 8B) and after 6 days, at 5 hours⁵¹CTL activity was determined in Cr release assay (fig. 8A). The values represent the average of two replicate (fig. 8A) and three replicate (fig. 8B) cultures.

Similar to the in vivo findings, exponentially increasing doses of immunogen induced the strongest CTL response, followed by daily administration of the same dose of gp33, although administration of the immunogen in bolus or with a reduced dose pattern resulted in weaker CTL responses. The difference is even greater when cells are stimulated with one-tenth of the peptide dose. CTL activity was associated with IL-2 production (FIG. 8B, upper panel). Exponentially increasing immunogen doses induced the highest amount of IL-2, while constant daily immunogen doses induced much less IL-2. Constant antigen stimulation induced high amounts of IL-10 with an earlier onset compared to exponentially increasing antigen stimulation (fig. 8B, middle panel). IFN- γ is transiently produced at an early stage in cells stimulated with a bolus or exponentially decreasing amount of immunogen (FIG. 8B, bottom panel). In contrast, daily stimulation by a constant or exponentially increasing dose induces specific T cells to secrete higher amounts of IFN- γ.

Thus, as observed, sustained and adequate antigenic stimulation is necessary to maintain IFN- γ production. The fact that exponentially increasing antigen stimulation appears to produce less IFN- γ than a constant daily dose can be explained by the high in vitro stability of IFN- γ and the continuous accumulation due to earlier production of IFN- γ.

In summary, in vitro stimulation of clonal T cells with exponentially increasing doses of immunogen produced higher amounts of IL-2 and IFN- γ and stimulated IL-10 at later time points compared to all other antigenic patterns. These observations are consistent with the conclusion that the enhanced immune response resulting from increased antigen stimulation operates at the clonotype level. These phenomena have also been shown to be accompanied by a higher avidity of the T cell antibodies, which is important for efficient interaction between T cells and dendritic cells (Bousso and Robey.2003.nat Immunol (natural immunology) 4: 579-585, which is incorporated herein by reference in its entirety).

IL-2 production is a hallmark of CD4+ and CD8+ T cell activation and plays an important role in regulating several phases of the T cell response. The combined use of TCR (signal 1) and costimulatory molecule (signal 2) induced only limited clonal expansion of T cells. The extensive expansion of T cells and differentiation into effector cells to exhibit a prolific T cell response requires signaling by IL-2R (Signal 3; Malek, T.R. and Bayer, A.L., Nat.Rev.Immunol. (review in Natural immunology), 4, 665-74, 2004, the entire contents of which are incorporated herein by reference), and by CD8⁺T cell production of autocrine IL-2 is CD8 in vivo⁺Key driver for T cell expansion (Malek, t.r.&Bayer, a.l., nat.rev.immunol. (natural immunological review), 4, 665-74, 2004, supra; d' Souza, w.n., et al, J Immunol, 168, 5566-72, 2002, which are incorporated herein by reference in their entirety). On the other hand, IL-10 is a major inhibitor of T cell proliferation, primarily through regulation of dendritic cells (Moore, k.w., et al, annu.rev.immunol. (immunization)Yearly review), 19, 683-765, 2001, the entire contents of which are incorporated herein by reference). Thus, the in vivo data of the present invention demonstrate that at the clonal level, T cells are able to decode the kinetics of antigen exposure.

Example 10: linearly increasing antigen stimulation enhances CD8⁺T cell response

Studies were conducted to determine whether T cell responses could be enhanced by increasing antigen stimulation. In the experiment, 1X 10⁶Transgenic gp 33-specific T cells were transfected into C57BL/6 wild-type recipient mice to increase precursor T cell frequency and facilitate assessment of immune responses.

All mice were immunized with the same cumulative dose of gp33 peptide of mixed CpG ODN (125 μ g gp33 and 12.5nmol CpG total) using different vaccination protocols as follows: s1) one single dose at day 0 in a bolus injection; s2) 4 equivalent doses over 4 days; s3) dose linearly decreasing within 4 days; and s4) dose increasing linearly over 4 days. In addition, several groups of mice were immunized with a single dose of CpG followed by a linearly increasing dose of gp33 peptide (s5), or with a single dose of gp33 followed by a linearly increasing dose of CpG (s 6). Mice infected intravenously with 250pfu of LCMV virus on day 0 were used as positive controls. At day 6, day 12 and day 8, CD8 was quantified by intracellular IFN- γ staining of blood lymphocytes re-stimulated in vitro with gp33 peptide⁺T cell response.

CpG ODN were selected as adjuvants because they strongly enhance CD8⁺T cell responses (Krieg, A.M., Annu Rev Immunol (annual review in immunology) 20, 709-60, 2002; Schwarz, K. et al, Eur J Immunol (J. Eur. Immunol) 33, 1465-70, 2003, supra). Phosphorothioate stabilised ODN was cleared from plasma with a half-life of 30-60 minutes (Farman, c.a).&Kornbrust, D.J., Toxicol Pathol 31 supplement, 119-22, 2003, supra). However, CpG ODN is relatively stable in tissue with a half-life of 48 hours (Mutwiri, G.K., et al, J Control Release 97, 1-17, 2004, supra). Furthermore, it is noted in the literature that plasma proteases degrade free peptides at detectable levels within 60 minutes (Falo, L.D., Jr., et al, Proc Natl Acad Sci USA 89, 8347-50, 1992; Widmann, C., et al, J Immunol (J. Immunol) 147, 3745-51, 1991, supra).

It was observed that immunity leading to CD8+ T cell responses of comparable magnitude to infection with LCMV wild type was provided by administration of gp33 and CpG in a linearly increasing fashion. It was also observed that despite induction of a strong CD8+ T cell response, immunization with a uniform daily dose of gp33 and CpG was significantly weaker compared to stimulation with elevated doses. Furthermore, it was observed that the efficacy of the immunization was significantly reduced when one of the vaccine components was delivered as a single dose, but still significant compared to the control used for the first time in the experiment.

Similar observations were made in naive wild type mice that did not receive TCR transgenic cells. C57BL/6 mice immunized with linearly increasing vaccine (gp33 and CpG) doses showed significantly enhanced induction of CD8+ T cells compared to other vaccination protocols that induced barely detectable specific CD8+ T cell frequencies. These results demonstrate that, independent of the overall dose, the kinetics of vaccination is a key parameter for immunogenicity.

Example 11: linearly increasing antigenic stimulation enhances protective antiviral responses

Studies were conducted to determine whether the protective antiviral response could be enhanced by increasing antigen stimulation. In experiments, female wild-type C57BL/6 mice were immunized according to different protocols (s1-s 4 as described in example 10) with fixed cumulative doses of gp33 peptide and CpG (125 μ g gp33 and 12.5nmol CpG total), followed by stimulation with LCMV or recombinant vaccinia virus expressing LCMV glycoprotein (vacc-gp) at the time point when the T cell response was already in the contractile or memory phase (Kaech, s.m., et al, Nat Rev Immunol (natural immunological review) 2, 251-62, 2002, supra). Protection against both viruses depends exclusively on CD8+ T cells (Binder, D. and Kundig, T.M., J Immunol 146, 4301-7, 1991; Kundig, T.M., et al, Proc. Natl. Acad. Sci., USA (Proc. Natl. Acad. USA): 93, 9716-23, 1996, supra).

Mice were immunized with linearly increasing amounts (s4) or injected (s1) with gp33 peptide and CpG using boluses as described in example 10 (n-4). Negative control mice were untreated (naive), and positive control mice were infected with LCMV (250 pfu). Mice were bled at day 10 and day 30 for analysis of gp33 specific effector or memory CTLs using gp 33-MHC-tetramers and flow cytometry, or IFN- γ producing CD8 following in vitro re-stimulation with gp33 at day 30⁺T cells. It was observed that linearly increasing doses of peptide (gp33) and CpG induced significantly more frequent IFN-. gamma.producing effects and memory cells and gp 33-tetramer positive memory (CD 44) than single immunizations^hi) A cell. On day 30, all mice were stimulated by intraperitoneal injection of 250pfu LCMV. Four days later, virus titers were measured in the spleen. On day 30, mice were stimulated intraperitoneally with 250pfu LCMV. Four or five days later, the spleen or ovaries were collected for LCMV determination. It was observed that although the bolus (s1) -immunized mice did not receive significant protection against viral replication, the linearly increasing vaccination induced significant protection against LCMV titers (p < 0.01) when compared to mice used for the first time in the experiment or bolus immunization.

In another set of experiments, C57BL/6 mice were immunized using a different protocol, followed by 1.5X 10 on day 8 or 24⁶pfu of recombinant vaccinia virus (vacc-gp) was stimulated intravenously. Five days later, vacc-gp replication was measured in the ovaries. It was observed that only mice immunized in a dose-increasing manner were able to present significantly protective CD8⁺T cell responses, by orders of magnitude better than other peptide immunization protocols, inhibit viral replication.

Example 12: linearly increasing antigen stimulation favors prolonged T cell stimulation

To test how the immuno-kinetics affected CD8⁺Proliferation of T cells, as described in example 10, with a single dose (s1)) Mice were injected with gp33 peptide and CpG at the same daily dose (s2) or at a linearly increasing dose (s 4). One group of mice was not treated as a negative control. To monitor proliferation, all mice received 10 intravenously from transgenic TCR318 mice one day before the first immunization⁷CFSE-labeled splenocytes. At different time points, lymphocytes were isolated by tail bleeds and analyzed by flow cytometry for CD8 expression and CFSE staining. It was observed that linearly increasing stimulation significantly prolonged T cell proliferation relative to a single bolus injection stimulation protocol.

Example 13: linearly increasing numbers of peptide pulsed DCs enhanced CD8⁺T cell response

To investigate the contribution of different numbers of APCs, C57BL/6 mice were immunized with the same total number of peptide-pulsed DCs, but using different kinetics. Bone marrow derived DCs were loaded with HPV E7(aa49-57, RAHYNIVTF, SEQ ID NO: 2) peptide and a total of 1.2X 10 on day 1⁵Individual cells were injected as a bolus into the groin node (s1), or on day 1 (2 × 10)⁴Cells), day 3 (4X 10)⁴Cells), and day 6 (6 × 10)⁴Cells) were administered the same total number of cells in a linear increase (s4) pattern. In addition, the vaccine was administered intralymphatically to ensure a constant total number of DCs available for T cell priming. Mice used for the first time in the experiment were used as negative controls. On days 17 and 22, peripheral blood was analyzed by flow cytometry for E7-tetramer positive CD8⁺T cell frequency and IFN-. gamma.ELISPOTs were analyzed in spleen. On day 21, three vaccinated and ten naive mice were stimulated with the HPV-transformed tumor cell line C3.43. Tumor progression was monitored by calibre measurements (mm) to calculate tumor volume. Survival after stimulation was studied in C57BL/6 mice immunized by s.c injection with DCs loaded with VSV np52 peptide.

It was observed that linearly increasing doses (s4) induced a higher number of antigen-specific CD8 than the bolus injected vaccine (s1)⁺T cells, which are positive for MHC-E7-tetramer and produce IFN- γ CD8 as measured on days 17 and 22, respectively⁺Frequency of T cells. Also observeMice vaccinated with the dose escalation protocol were resistant to stimulation with the HPV-transformed tumor cell line C3.43, whereas mice vaccinated with only a single bolus injection were unprotected.

C57BL/6 mice were immunized by s.c injections of DCs loaded with the VSV np52 peptide. 1.2X 10 doses were administered as boluses on day one (s1), or as equivalent doses on days 1, 3, 6 (s2) or as escalating doses (s4)⁵DCs. Mice used for the first time in the experiment were used as controls. On day 14, dose 10 was administered⁶EL-4N.1 cells i.p stimulated all mice (Kundig et al, J.Immunol. 150, 4450-4456, 1993, supra). It was observed that the survival of mice immunized with the DCs (s4) regimen loaded with the VSVnp52 peptide was significantly better than that of mice immunized according to the (s1) or (s2) regimen given DCs in equal amounts on the three days.

The various methods and techniques described above provide many ways to implement the present invention. Of course, it is to be understood that not necessarily all such objects or advantages may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods may be practiced in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. Various advantageous or disadvantageous alternatives are mentioned herein. It should be understood that some preferred embodiments specifically include one, another, or several advantageous features, while other embodiments specifically include one, another, or several disadvantageous features, while others specifically mitigate an existing disadvantageous feature by including one, another, or several advantageous features.

In addition, those skilled in the art will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, may be mixed and matched by one of ordinary skill in this art to implement methods in accordance with principles described herein. Among the various elements, features and steps, some may be specifically included in different embodiments, while others are specifically excluded.

Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments, and/or uses, modifications and equivalents thereof.

Many variations and alternative elements of the present invention have been disclosed. Still other variations and alternative elements will be apparent to those skilled in the art. Among these variations, include, but are not limited to, a specific number of antigens in the screening set or targeted by the therapeutic product, the type of antigen, the type of cancer, and the specific antigen specified. Various embodiments of the invention may specifically include or exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like, used to describe and claim certain embodiments of the present invention, are to be understood as being modified in some instances by the term "about". Accordingly, in some embodiments, the numerical parameters of the written representations and the appended claims are approximations that may vary depending upon the advantageous properties sought to be obtained by a particular embodiment. In some embodiments, numerical parameters should be analyzed in terms of the number of significant digits reported and by applying conventional rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practical. The numerical values set forth in some embodiments of the invention may include certain errors necessarily resulting from the standard error found in their respective measurements.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing particular embodiments of the invention (especially in the context of certain of the following claims) are to be construed to cover both the singular and the plural. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided with respect to certain embodiments herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The groupings of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in combination with other members of other groups or other elements found herein. It is contemplated that one or more members of a group may be included in, or deleted from, the group for convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein considered to comprise the modified group and thus satisfies the written description of all markush claim groups used in the appended claims.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. It is intended that the skilled person can use such variations as are appropriate, and that the invention can be practiced otherwise than as specifically described herein. Accordingly, many embodiments of the invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

In addition, throughout this specification, reference is made to a number of patents and printed publications. Each of the above-cited references and printed publications is hereby incorporated by reference in their entirety.

Finally, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be used are also within the scope of the invention. Thus, by way of example, but not limitation, alternative configurations of the present invention may be used in accordance with the teachings herein. Accordingly, the invention is not limited to that as specifically shown and described.

Claims (40)

1. Use of a plurality of immunogenic compositions for stimulating a class I MHC-restricted T cell response in the manufacture of a kit for the treatment or prevention of an infectious or neoplastic disease, wherein the plurality of immunogenic compositions for stimulating a class I MHC-restricted T cell response are consecutive doses, wherein each dose administered after an initial dose is greater than the immediately preceding dose, and wherein the consecutive doses increase as an exponential function of the initial dose, wherein an enhanced response is obtained compared to immunization with the same cumulative dose without consecutive increasing doses, wherein the immunogenic composition comprises an immunogen provided as a protein or peptide.

2. The use of claim 1, wherein the immunogenic composition comprises an immunogen, plus an immunopotentiator or biological response modifier.

3. The use of claim 2, wherein the immunopotentiator or biological response modifier is selected from the group consisting of: cytokines, pathogen-associated molecular patterns (PAMPs), TLR-ligands, immunostimulatory sequences, recognition receptor (PRR) ligands for endocytic patterns, Lipopolysaccharide (LPS), quillaja saponin, and tucaresol.

4. The use of claim 3, wherein the cytokine is a chemokine.

5. The use of claim 3, wherein the immunostimulatory sequence is a CpG-containing DNA or dsRNA.

6. The use of claim 1, wherein the exponential function is defined by ≧ 2^n-1Is defined by the exponential factor of (c).

7. The use of claim 6, wherein the exponential factor is 5^n-1。

8. The use of claim 1, wherein the consecutive doses comprise more than 2 doses.

9. The use of claim 8, wherein the consecutive doses comprise more than 6 doses.

10. Use according to claim 1, wherein the last dose is administered within 6 days of the first dose.

11. The use of claim 1, wherein the enhanced response comprises an increase in the number of reactive T cells.

12. The use of claim 1, wherein the enhanced response comprises increased cytokine production.

13. The use of claim 12, wherein the cytokine is IL-2 or IFN- γ.

14. The use of claim 1, wherein the enhanced response comprises a delay in peak production of immunosuppressive cytokines.

15. The use of claim 14, wherein the immunosuppressive cytokine is IL-10.

16. The use of claim 1, wherein the enhanced response comprises an increase in cytolytic activity.

17. The use of claim 1, wherein the immunogenic composition is suitable for direct delivery to the lymphatic system of a mammal.

18. The use of claim 17, wherein direct delivery to the lymphatic system of a mammal comprises intranodal delivery.

19. The use of claim 1, wherein the immunogenic composition is suitable for subcutaneous, intramuscular, intradermal, transdermal, transmucosal, nasal, bronchial, oral, or rectal administration to a mammal.

20. Use according to claim 1, wherein the immunogen is provided as a polypeptide, synthetic epitope or mimotope.

21. The use of claim 1, wherein the immunogen stimulates a response to an antigen selected from the group consisting of: viral antigens, bacterial antigens, fungal antigens, differentiation antigens, tumor antigens, and embryonic antigens.

22. The use of claim 1, wherein the immunogen stimulates a response to an oncogene antigen.

23. The use of claim 1, wherein the immunogen stimulates a response to an antigen of a mutated tumor suppressor gene.

24. The use of claim 1, wherein the immunogen stimulates a response to a unique tumor antigen caused by a chromosomal translocation.

25. The use of any one of claims 21-24, wherein the antigen is a self-antigen.

26. Use according to claim 3, wherein the immunopotentiator is a TLR-ligand.

27. The use of claim 26, wherein the TLR-ligand is CpG-containing DNA.

28. A kit comprising a panel of immunogenic compositions for stimulating a class I MHC-restricted T cell response, the immunogenic compositions comprising an immunogen provided as a protein or peptide, plus an immunopotentiator or biological response modifier, wherein the doses of each member of the panel are related as an exponential series, further comprising instructions for administering the compositions to a subject in need thereof.

29. The kit of claim 28, wherein the dose is in an exponential series consisting of ≥ 2^n-1Is defined by the exponential factor of (c).

30. The kit of claim 28, wherein the exponential series of doses is comprised of an exponential factor of 5^n-1And (4) limiting.

31. The kit of claim 28, wherein the immunopotentiator or biological response modifier is selected from the group consisting of: cytokines, PAMPs, TLR-ligands, immunostimulatory sequences, endocytic Pattern Recognition Receptor (PRR) ligands, LPS, quillaja saponin, and tocarol.

32. The kit of claim 31, wherein the cytokine is a chemokine.

33. The kit of claim 31, wherein the immunostimulatory sequence is a CpG-containing DNA or dsRNA.

34. The kit of claim 28, wherein the immunogen, and the immunopotentiator or biological response modifier are contained in separate containers.

35. The kit of claim 28, wherein the immunogen and the immunopotentiator or biological response modifier are contained in the same container.

36. The kit of claim 28, comprising two or more doses of the immunogenic composition, each in a separate suitable container.

37. The kit of claim 36, wherein the suitable container is a syringe, ampoule or vial.

38. A set of syringes comprising successively increasing doses of an immunogenic composition for stimulating a MHC class I-restricted T cell response, wherein each dose following an initial dose is greater than the immediately preceding dose in each syringe of the set of syringes, wherein the successively increasing doses increase as an exponential function of the initial dose, and wherein the immunogenic composition comprises an immunogen provided as a protein or peptide, and an immunopotentiator or biological response modifier, thereby enhancing the T-cell response in a subject.

39. A set of vials comprising successively increasing doses of an immunogenic composition for stimulating a MHC class I-restricted T cell response, wherein each dose following an initial dose is greater than the immediately preceding dose in each vial of the set of vials, wherein the successively increasing doses increase as an exponential function of the initial dose, and wherein the immunogenic composition comprises an immunogen provided as a protein or peptide, and an immunopotentiator or biological response modifier, thereby enhancing the T-cell response in the subject.

40. Use of a plurality of immunogenic compositions in the manufacture of a kit for stimulating a class I MHC-restricted T cell response in a mammal, wherein the amount of the plurality of immunogenic compositions is a consecutive dose and each dose after an initial dose is greater than the immediately preceding dose, and wherein the consecutive dose increases as an exponential function of the initial dose, wherein the immunogenic compositions comprise an immunogen provided as a protein or peptide.