HK1120722B - The use of immunogenic composition in preparing a response and enhance the use of the reagent kit for immune responses - Google Patents

Description

Use of immunogenic compositions for the preparation of kits for eliciting and enhancing immune responses

CROSS-REFERENCE TO RELATED APPLICATIONS

According TO 35U.S. C. [ section ]119(e), the present application claims priority from U.S. provisional application No.60/640,402, filed on 29.12.2004, entitled "METHODS TO ELICIT, ENGAGE AND SUSTAIN IMMUNE RESPONSES AGAINST MHC CLASS I-RESTRICTED EPITOPES, FOR PROPHYLACTIC OR THERAPEUTIC PURPOSES"; the disclosure of which is incorporated herein by reference in its entirety.

Background

Technical Field

Embodiments of the invention disclosed herein relate to methods and compositions for inducing class I MHC-restricted immune responses and modulating the nature and intensity of the responses during pathogenic processes, enhancing effective immune intervention. In more detail, embodiments relate to immunogenic compositions, their nature and the order, timing and route of administration with which they are effectively utilized.

Description of the Related Art

Major histocompatibility complex and T cell targeting

T lymphocytes (T cells) are antigen-specific immune cells that function in response to specific antigen signals. B lymphocytes and the antibodies they produce are also antigen-specific entities. However, unlike B lymphocytes, T cells do not respond to antigen in free or solubilized form. For T cell responses to antigens, it requires binding of the antigen to a presentation complex known as the Major Histocompatibility Complex (MHC).

MHC proteins provide a means for T cells to distinguish native or "autologous" cells from foreign cells. MHC molecules are a class of immunoreceptors that present potential peptide epitopes that are subsequently monitored by T cells. There are two types of MHC, MHC class I and MHC class II. CD4+ T cells interact with MHC class II proteins and have primarily a helper phenotype while CD8+ T cells interact with MHC class I proteins and have primarily a cytolytic phenotype, but each may also exhibit a regulatory, especially inhibitory, effect. MHC is a transmembrane protein, with most of its structure on the outer surface of the cell. In addition, both MHC classes have peptide-binding slits on their outsides. Small fragments of endogenous or exogenous proteins are bound in this slit and presented in the extracellular environment.

Cells called Antigen Presenting Cells (APCs) display antigens to T cells using MHC. If the antigen is presented on the MHC, the T cells can recognize the antigen. This requirement is called MHC restriction. If the antigen is not presented by a recognized MHC, the T cell will not recognize and act on the antigen signal. T cells specific for MHC-recognizable peptides bind to these MHC-peptide complexes and enter the next stage of the immune response.

Peptides corresponding to the named MHC class I or class II restricted epitopes are in the simplest form of antigens that can be delivered for inducing, enhancing or manipulating T cell responses. Although peptide epitopes have been shown to be effective in vitro in restimulation of major T cell lines, clones or T cell hybridomas in vivo, their efficacy in vivo is very limited. This is due to two main factors:

(1) poor peptide Pharmacokinetic (PK) patterns, caused by rapid renal clearance and/or in vivo degradation, leading to restricted access to APC;

(2) antigen-induced T Cell Receptor (TCR) -dependent signaling alone (signal 1) is not sufficient to induce or amplify a strong and sustained immune response, and in particular a response consisting of Tc1 or Th1 cells (producing IFN- γ and TNF- α). Furthermore, unless certain immune boosting or modulating adjuvants are used in combination, varying degrees of unresponsiveness or "immune deviation" can be elicited by using large doses of peptides or depot adjuvants to circumvent the limited PK associated with the peptides.

Summary of The Invention

Embodiments of the invention include methods and compositions for modulating, and in particular for inducing, eliciting and/or enhancing immune responses to MHC class I restricted epitopes.

Some embodiments relate to methods of immunization. The method can include, for example, delivering to the mammal a first composition comprising an immunogen that can include or encode at least a portion of a first antigen; and administering a second composition, which may include an enhancer peptide, directly to the lymphatic system of the mammal, wherein the peptide corresponds to an epitope of the first antigen, wherein the first composition and the second composition are not identical. The method may further comprise the step of obtaining, analyzing or detecting an effector T cell response.

The first composition may include a nucleic acid encoding an antigen or immunogenic fragment thereof. The first composition can include a nucleic acid capable of expressing an epitope in pAPC. The nucleic acid may be delivered as a component of a protozoan, bacterial, viral or viral vector. The first composition may, for example, comprise an immunogenic polypeptide and an immunopotentiator. The immunopotentiator may be a cytokine, a toll-like receptor ligand, or the like. Adjuvants may include immunostimulatory sequences, RNA, and the like.

The immunogenic polypeptide may be an enhancing peptide. The immunogenic polypeptide can be a first antigen. The immunogenic polypeptides may be delivered as components of protozoa, bacteria, viruses, viral vectors or virus-like particles, and the like. Adjuvants can be delivered as components of protozoa, bacteria, viruses, viral vectors or virus-like particles, and the like. The second composition may be unadjuvanted and without an immunopotentiator. The delivering step may comprise direct administration to the lymphatic system of the mammal. Administration directly to the lymphatic system of a mammal may include administration directly to lymph nodes or to lymphatic vessels. Direct administration may be to two or more lymph nodes or vessels. The lymph nodes may be, for example, inguinal, axillary, cervical and tonsillar lymph nodes. The effector T cell response may be a cytotoxic T cell response. The effector T cell response may include the production of a proinflammatory cytokine, and the cytokine may be, for example, (gamma) gamma-IFN or TNF α (alpha). The effector T cell response may include the production of T cell chemokines, such as RANTES or MIP-1 α and the like.

The epitope may be, for example, a housekeeping epitope or an immune epitope. The delivery step or administration step may, for example, comprise a single bolus injection (bolus injection), repeated bolus injections. The delivering step or administering step may comprise a continuous infusion, which may, for example, have a duration of between about 8 to about 7 days. The method can include a time interval between the termination of the delivering step and the beginning of the administering step, wherein the time interval can be at least about 7 days. And, the time interval may be, for example, between about 7 and about 14 days, about 17 days, about 20 days, about 25 days, about 30 days, about 40 days, about 50 days, or about 60 days. The time interval may exceed about 75 days, about 80 days, about 90 days, about 100 days, or more.

The first antigen may be a disease-associated antigen, and the disease-associated antigen may be a tumor-associated antigen, a pathogen-associated antigen. Embodiments include methods of treating diseases using the immunological methods. The first antigen may be a target-associated antigen. The target may be a tumor cell, a pathogen-infected cell, or the like. For example, any tumor cell may be targeted. For example, pathogen-infected cells can include, for example, cells infected with bacteria, viruses, protozoa, fungi, and the like, or cells infected with, for example, prions.

Effector T cell responses can be detected by at least one indicator, such as cytokine assays, Elispot assays, cytotoxicity assays, tetramer assays, DTH-responses, clinical responses, tumor shrinkage, tumor clearance, inhibition of tumor development, reduced pathogen titer, pathogen clearance, improvement in disease symptoms, and the like. The method may further comprise obtaining, detecting or analyzing an effector T cell response to the first antigen.

Additional embodiments relate to methods of immunization comprising administering to a subject a composition comprising a nucleic acid encoding a first antigen or an immunogenic fragment thereof

Delivering the first composition to the mammal; administering a second composition comprising a peptide directly to the lymphatic system of the mammal, wherein the peptide corresponds to an epitope of the first antigen. The method may further comprise obtaining, detecting or analyzing an effector T cell response to the antigen.

In addition, embodiments relate to methods of enhancing an existing antigen-specific immune response. The method can include administering a composition including a peptide directly to the lymphatic system of the mammal, wherein the peptide corresponds to an epitope of an antigen, and wherein the composition is not used to induce an immune response. The method may further comprise obtaining, detecting or analyzing the enhancement of the antigen-specific immune response. Such enhancement may include maintaining a response over time, revitalizing sleeping T cells, expanding antigen-specific T cell populations, and the like. In some aspects, the composition does not include an immunopotentiator.

Other embodiments relate to methods of immunization that can include delivering to a mammal a first composition that includes an immunogen that can include or encode at least a portion of a first antigen and at least a portion of a second antigen; administering directly to the lymphatic system of the mammal a second composition comprising a first peptide and a third composition comprising a second peptide, wherein the first peptide corresponds to an epitope of a first antigen, and wherein the second peptide corresponds to an epitope of a second antigen, wherein the first composition may not be identical to the second or third composition. The method may further comprise obtaining, detecting or analyzing effector T cell responses to the first and second antigens. The second and third compositions may each include a first and second peptide. The second and third compositions may be part of a single composition.

Still further embodiments relate to methods of generating antigen-specific tolerogenicity or modulating an immune response. The method can include periodically administering a composition including an adjuvant-free peptide directly to the lymphatic system of a mammal, wherein the peptide corresponds to an epitope of an antigen, and wherein the mammal is naive to the epitope. The method may further comprise obtaining, detecting and analyzing tolerogenic or regulatory T cell immune responses. The immune response may, for example, be useful in treating inflammatory disorders. The inflammatory disorder may be derived from, for example, a class II MHC-restricted immune response. The immune response may include the production of immunosuppressive cytokines such as IL-5, IL-10 or TGB-beta, and the like.

Embodiments relate to methods of immunization comprising administering a series of immunogenic doses directly into the lymphatic system of a mammal, wherein the series may comprise at least 1 priming dose and at least 1 boosting dose, and wherein the priming dose may comprise a nucleic acid encoding an immunogen and wherein the boosting dose may be free of any virus, viral vector, or replicative vector. The method may further comprise obtaining an antigen-specific immune response. The method may, for example, comprise 1 to 6 or more initiator doses. The method can comprise administering multiple priming doses, wherein the doses are administered over a time course of 1 to about 7 days. The priming dose, the boosting dose, or the priming and boosting doses can be delivered in multiple pairs of injections, wherein the first member of a pair can be administered within about 4 days of the second member of the pair, and wherein the time interval between the first members of different pairs can be at least about 14 days. For example, the time interval between the last priming dose and the first boosting dose may be between about 7 and about 100 days.

Other embodiments relate to a panel of immunogenic compositions for inducing an immune response in a mammal, comprising 1 to 6 or more priming doses and at least one boosting dose, wherein the priming doses can comprise a nucleic acid encoding an immunogen, and wherein the boosting doses can comprise peptide epitopes, and wherein the epitopes can be presented or presentable by pAPC expressing the nucleic acid. A dose may further comprise an adjuvant, such as RNA. Priming and boosting doses can be in a vehicle suitable for direct administration to the lymphatic system, lymph nodes, and the like. The nucleic acid may be a plasmid. The epitope may be a class I HLA epitope, such as one listed in tables 1-4. The HLA may preferably be HLA-A2. The immunogen may comprise an array of epitopes, which array may comprise a release sequence. The immunogen may consist essentially of the target-associated antigen. The target-associated antigen can be a tumor-associated antigen, a microbial antigen, any other antigen, and the like. The immunogen may comprise a fragment of the target-associated antigen, which may comprise a cluster of epitopes.

Additional embodiments can include a panel of immunogenic compositions for inducing a class I MHC-restricted immune response in a mammal, comprising 1-6 priming doses and at least one boosting dose, wherein the priming doses can include an immunogen or a nucleic acid encoding an immunogen and an immunopotentiator, and wherein the boosting doses can include a peptide epitope, wherein the epitope can be presented by pAPC. The nucleic acid encoding the immunogen may further comprise an immunostimulatory sequence capable of functioning as an immunopotentiator. The immunogen may be a virus or a replicable vector, which may include or may induce an immunopotentiator. The immunogen may be a bacterium, a bacterial lysate or a purified cell wall component. In addition, bacterial cell wall components can function as immunopotentiators. Immunopotentiators may be, for example, TLR ligands, immunostimulatory sequences, CpG-containing DNA, dsRNA, endocytosis-Pattern Recognition Receptor (PRR) ligands, LPS, quillaja saponin, benzoic acid derivatives (tucaresol), pro-inflammatory cytokines, and the like. In some preferred embodiments for promoting a multivalent response, the panel may comprise, for each administration, multiple priming doses and/or multiple boosting doses corresponding to each individual antigen or combination of antigens. Multiple initiator amounts may be administered as part of a single composition or as part of more than one composition. Booster doses may be administered, for example, at different times and/or to more than one site.

Other embodiments relate to methods of generating various cytokine patterns. In some embodiments of the invention, intranodal administration of the peptide may be effective to enhance the response initially induced with the plasmid DNA vaccine. In addition, the cytokine pattern can be different, as plasmid DNA induction/peptide amplification generally produces more chemokines (chemoattractant cytokines) and less immunosuppressive cytokines than DNA/DNA or peptide/peptide protocols.

The enhancing peptides used in various embodiments correspond to epitopes of an immunizing antigen. In some embodiments, correspondence may include faithfully repeating the native epitope sequence. In some embodiments, correspondence may include that the corresponding sequence may be an analog of the native sequence in which one or more amino acids have been changed or replaced, or the length of the epitope has been changed. The analogs can retain the immune function of the epitope (i.e., they are functionally similar). In a preferred embodiment, the analogue has similar or improved binding to one or more MHC class I molecules compared to the native sequence. Other preferred embodiments are analogs having similar or increased immunogenicity as compared to the native sequence. Strategies for preparing analogs are widely known in the art. An exemplary discussion of such strategies may be found in U.S. patent application No. 10/117,937 (publication No. 2003-0220239a1), filed 4.4.2002; and 10/657,022 filed 9/5/2003 (publication No. 20040180354), both entitled epitope sequences; and U.S. provisional patent application No. 60/581,001 filed on 17.6.2004 and U.S. patent application No. 11/156,253 (publication No. _) filed on 17.6.2005, both entitled SSX-2 peptide analogs; and U.S. provisional patent application No. 60/580,962 and U.S. patent application No. 11/155,929 (publication No. _), both entitled NY-ESO peptide analogs, filed on 17.6.2005; each of which is incorporated herein by reference in its entirety.

A further embodiment relates to the use of the peptide in the preparation of an adjuvant-free medicament for use in prime-and-boost immunization protocols. As described herein, the compositions, kits, immunogens and compounds may be used in medicine for the treatment of various diseases, to enhance immune responses, to generate specific cytokine patterns, and the like. Embodiments relate to the use of an adjuvant-free peptide in a method of enhancing an immune response.

Embodiments relate to methods, uses, therapies and compositions related to epitopes having MHC specificity, including, for example, those listed in tables 1-4. Other embodiments include one or more MHC listed in tables 1-4, including combinations of the same, while other embodiments specifically exclude any one or more MHC or combinations thereof. Tables 3-4 include the frequencies of the listed HLA antigens.

Some embodiments relate to methods of generating an immune response. The method can include delivering a first composition (composition 1) to a mammal, which can include an immunogen comprising or encoding at least a portion of a first antigen (antigen a) and at least a portion of a second antigen (antigen B); and administering a second composition (composition 2), which may include a first peptide (peptide a), and a third composition (composition 3), which may include a second peptide (peptide B), directly to the lymphatic system of the mammal, wherein peptide a corresponds to an epitope of antigen a, and wherein peptide B corresponds to an epitope of antigen B, wherein composition 1 is different from composition 2 or composition 3. The method may further comprise obtaining an effector T cell response to one or both antigens.

In some aspects composition 2 and composition 3 may each comprise peptide a and peptide B. Peptides a and B may be administered, for example, to separate sites or to the same site, including at different times. Composition 1 may include nucleic acid molecules encoding antigen a and antigen B or portions thereof. Furthermore, the composition 1 may for example comprise two nucleic acid molecules, one encoding antigen a or a part thereof and one encoding antigen B or a part thereof.

The first and second antigens may be any antigen. Preferably, the first and second antigens are melanoma antigens, CT antigens, cancer-associated antigens, CT antigens and stromal antigens, CT antigens and neovascular antigens, CT antigens and differentiation antigens, cancer-associated antigens and stromal antigens, and the like. Various combinations of antigens are provided in U.S. application No.10/871,708 (publication No. 20050118186), filed 6/17/2004, entitled combinations of tumor-associated antigens in compositions for various types of cancer; and U.S. provisional application No.60/640,598 filed on 29/12/2004 and U.S. application No. _________, (publication No. ____________________________________________________ (docket No. mannk.049a), filed concurrently herewith, both also entitled combination of tumor-associated antigens in compositions for use in various types of cancer, each. The antigen preferably comprising antigen A or B may be SSX-2, Melan-A, tyrosinase, PSMA, PRAME, NY-ESO-1, and the like. Many other antigens are known to those of ordinary skill in the art. It is understood that more than two compositions, immunogens, antigens, epitopes and/or peptides may be used in this and other embodiments. For example, three, four, five or more of any one or more of the above may be utilized.

Other embodiments relate to methods of generating an immune response, which may include, for example, delivering to a mammal a first composition (composition 1) comprising an immunogen (immunogen 1) and a second composition (composition 2) comprising a second immunogen (immunogen 2), the immunogen 1 may comprise or encode at least a portion of a first antigen (antigen a) and the immunogen 2 may comprise or encode at least a portion of a second antigen (antigen B); and administering a third composition (composition 3) comprising a first peptide (peptide a) and a fourth composition (composition 4) comprising a second peptide (peptide B) directly to the lymphatic system of the mammal, wherein peptide a corresponds to an epitope of antigen a, and wherein peptide B corresponds to an epitope of antigen B, wherein composition 1 is different from composition 2 or composition 3.

For example, composition 2 differs from composition 3 in some aspects. Compositions 1 and 3 can be delivered to the same site, which can be, for example, the inguinal lymph node. Likewise, compositions 2 and 4 can be delivered to a different site than compositions 1 and 3, e.g., to the inguinal lymph node of the other.

Still further embodiments relate to methods of generating an immune response that can include, for example, delivering to a mammal a first composition comprising a means to generate an immune response to a first antigen and a second antigen; and administering a second composition comprising a first peptide and a third composition comprising a second peptide directly to the lymphatic system of the mammal, wherein the first peptide corresponds to an epitope of the first antigen, and wherein the second peptide corresponds to an epitope of the second antigen, wherein the first composition is not identical to the second or third composition. Methods of directing an immune response may, for example, include methods for expressing an antigen or portion thereof.

In addition, some embodiments relate to methods of immunization, which can include, for example, delivering a first composition comprising an immunogen, which can include or encode at least a portion of a first antigen and at least a portion of a second antigen; and a step for enhancing the response to the antigen. Preferably, the step for enhancing the response to the antigen may comprise administering a first peptide corresponding to at least a portion of the first antigen to a secondary lymphoid organ and administering a second peptide corresponding to at least a portion of the second antigen to a different secondary lymphoid organ.

Brief Description of Drawings

FIGS. 1A-C: induction of immune response by intralymphatic immunization

Figure 2 depicts an example of a protocol for controlling or manipulating immunity to MHC class I-restricted epitopes by targeted (lymph node) delivery of antigens.

FIG. 3 shows a visual perspective view of a representative well corresponding to the data set forth in FIG. 4.

FIG. 4 depicts the intensity of the immune response generated by the protocol described in FIG. 2 using ELISPOT measurements and expressed as the number (frequency) of IFN- γ (gamma) -producing T cells recognizing the peptide

Figure 5 shows the cytotoxic characteristics of T cells generated by targeted delivery of antigen as described in figure 2.

Figure 6 depicts cross-reactivity of MHC class I-restricted T cells generated by the protocol described in figure 2.

Figure 7A shows the immune profile, expressed as the ability of lymphocytes to produce members of three classes of biological response modifiers (pro-inflammatory cytokines, chemokines or chemoattractants and immunomodulatory or suppressive cytokines) following application of the immunization protocol described in figure 2.

Figure 7B shows the cell surface marker phenotype of flow cytometry of T cells generated by the immunization protocol described in figure 2. Repeated administration of the peptide to lymph nodes induces immune deviation and modulates T cells.

Fig. 8A and B show the frequency of specific T cells measured by tetramer in mice immunized with DNA, peptide, or DNA and peptide generating/enhancing sequences.

Figure 8C shows the specific cytotoxicity present in vivo in various lymphoid and non-lymphoid organs of mice immunized with DNA ("pSEM"), peptide ("ELA" ═ ELAGIGILTV (SEQ ID NO: 1)), or DNA and peptide generating/enhancing sequences.

Figure 9A shows the persistence/attenuation of circulating tetramer-stained T cells in animals immunized with the peptide and boosted with the peptide as the recall response following peptide boost (boost).

Figure 9B shows persistence/attenuation of circulating tetramer-stained T cells in animals primed with DNA and enhanced with peptide, with recall response after peptide boost.

Figure 9C shows persistence/attenuation of circulating tetramer-stained T cells in animals immunized with DNA and enhanced with DNA, with recall response after peptide boosting.

Figure 10A shows the expansion of antigen-specific CD8+ T cells using different two-cycle immunization protocols.

Figure 10B shows the expansion of antigen-specific CD8+ T cells using different three-cycle immunization protocols.

Figure 10C shows the expansion of circulating antigen-specific T cells detected by tetramer staining in animals primed with different protocols and enhanced with peptides.

Figure 10D shows the expansion of antigen-specific T cells in lymphoid and non-lymphoid organs after different immunization protocols and detected by tetramer staining.

FIG. 11A shows an example of a schedule for immunizing mice with plasmid DNA and peptides

Figure 11B shows the immune response elicited by different immunization protocols (alternating DNA and peptide in either order or in reverse) as determined by ELISPOT analysis.

FIG. 12A shows the in vivo depletion of antigen target cells in blood and lymph nodes of mice immunized with plasmids and peptides.

Figure 12B shows the in vivo depletion of antigen target cells in the spleen and lung of mice immunized with plasmids and peptides.

Fig. 12C shows a summary of the results presented in fig. 12A, B.

Figure 12D shows the correlation between the frequency of specific T cells and the in vivo clearance of antigen target cells in mice immunized by different protocols.

Figure 13A shows a schedule for immunization of mice with plasmid DNA and peptides, and the nature of the measurements made in those mice.

Figure 13B depicts a schedule associated with protocols for determining in vivo clearance of human tumor cells in immunized mice.

Figure 13C shows the in vivo depletion of antigen target cells (human tumor cells) in the lungs of mice immunized with plasmids and peptides.

FIG. 14A shows an immunization protocol for generating the anti-SSX-2 response shown in FIG. 14B.

Figure 14B shows the expansion of circulating SSX-2 specific T cells detected by tetramer staining after application of a DNA priming/peptide boosting protocol.

FIG. 15A shows in vivo clearance of antigen target cells in the spleen of mice subjected to different prime-and-boost regimens to immunize simultaneously against epitopes of Melan A (ELAGIGILTV (SEQ ID NO: 1)) and SSX2(KASEKIFYV (SEQ ID NO: 2)).

FIG. 15B shows in vivo clearance of antigen target cells in blood of mice subjected to different prime-and-boost regimens to immunize simultaneously against epitopes of Melan A (ELAGIGILTV (SEQ ID NO: 1)) and SSX2(KASEKIFYV (SEQ ID NO: 2)).

Fig. 15C summarizes the results shown in detail in fig. 15A, B.

Figure 16 shows the expansion of circulating antigen-specific CD8+ T cells as determined by tetramer staining in mice subjected to two different prime-and-boost protocols.

Fig. 17A and B show circulating antigen-specific T cell persistence in animals subjected to two rounds of prime-and-boost regimens consisting of DNA/peptide (a) or DNA/peptide (B).

Figure 18 shows long term memory in animals subjected to a two-round prime-and-boost regimen consisting of DNA/DNA.

Figure 19 shows a schematic of clinical practice of patient participation and treatment with DNA/peptide prime-and-boost regimens.

FIG. 20 depicts the use of two plasmids: immunization schedule of pCBP expressing SSX241-49 and pSEM expressing Melan A26-35 (A27L).

Figure 21 shows specific cytotoxicity induced by two plasmids administered as a mixture versus separately administered to individual sites.

Figure 22 depicts the addition of a peptide boosting step to the immunization protocol described in figure 20.

Figure 23 presents data demonstrating that peptide boosting rescues sub-optimal epitope immunogenicity even when the carrier and peptide were used separately as a mixture.

Fig. 24A and B depict alternative immunization protocols that induce strong, multivalent responses in clinical practice.

FIG. 25 depicts plasmids capable of eliciting multivalent responses.

Figure 26 presents a scheme for initiating an immune response with a multivalent plasmid and rescuing the response to a suboptimal epitope by intranodal administration of the peptide.

Figure 27A shows the frequency of specific T cells obtained by enhancing the response to the dominant (Melan-a) epitope by initiation with a multivalent plasmid and by intra-nodal administration of the peptide.

FIG. 27B shows the frequency of specific T cells obtained by enhancing the response to a suboptimal epitope (tyrosinase 369-377) by initiation with a multivalent plasmid and by intranodal administration of peptides.

Figure 28A shows specific cytotoxicity obtained by enhancing response to dominant (Melan-a) epitopes by initiation with multivalent plasmids and by intranodal administration of peptides.

FIG. 28B shows the specific cytotoxicity obtained by enhancing the response to a suboptimal epitope (tyrosinase 369-377) by initiation with a multivalent plasmid and by intranodal administration of the peptide.

Figure 29 depicts an immunization protocol that starts with multivalent plasmids and enhances both the response to dominant and subdominant epitopes.

Figure 30A shows the frequency of Melan-a specific T cells obtained by enhancing the response to dominant (Melan-a) and subdominant (tyrosinase) epitopes by initiation with multivalent plasmids and by intranodal administration of peptides.

Figure 30B shows the frequency of tyrosinase-specific T cells obtained by enhancing the response to the dominant (Melan-a) epitope and the subdominant (tyrosinase) epitope by initiation with a multivalent plasmid and by intranodal administration of the peptide.

FIG. 30C shows the frequency of Melan-A and tyrosinase specific T cells in mice initiated with pSEM and enhanced with both Melan-A and tyrosinase peptide. Results from two individual mice are shown.

FIG. 31 shows in vivo cytotoxicity data for co-initiated and enhanced T cells by intranodal administration of peptides corresponding to the dominant (Melan A26-35) and subdominant (tyrosinase 369-377) epitopes as a mixture after multivalent plasmid administration.

FIG. 32: bimulticolor tetramer analysis of pSEM/pBPL immunized animals prior to boost.

FIG. 33: double multicolor tetramer analysis of mouse immune responses induced with a mixture of plasmids pSEM and pBPL and enhanced with SSX2 and a tyrosinase peptide epitope analog.

FIG. 34: double multicolor tetramer analysis of 3 mouse immune responses induced with a mixture of plasmids pSEM and pBPL and enhanced with SSX2 and a tyrosinase peptide epitope analog.

FIG. 35A: first round of enhanced IFN- γ ELISpot assay

FIG. 35B: second round of enhanced IFN- γ ELISpot assay

FIG. 36: in vivo challenge with CFSE of human melanoma tumor cells expressing all four tumor associated antigens. Versions a-D each show a tetramer assay, IFN- γ ELISpot assay, and in vivo tumor cell killing of individual mice after completion of the protocol. Version a shows data from pure control mice, versions B-C show data from two mice of groups 3 and 2, respectively, that have acquired substantially tetravalent immunization, and version D shows data from group 3 mice, whose immunization is essentially monovalent.

Figure 37 describes a general method of inducing multivalent immunity.

Detailed description of the preferred embodiments

Embodiments of the invention provide methods and compositions, for example, for generating immune cells specific for target cells, for directing an effective immune response to target cells, or for affecting/treating inflammatory disorders. The methods and compositions may include, for example, immunogenic compositions such as vaccines and therapeutic agents (therapeutics) as well as methods of prevention and treatment. Disclosed herein is the novel and unexpected discovery that by selecting the form of the antigen, the sequence and timing of its administration, and the delivery of the antigen directly into the secondary lymphoid organs, not only can the intensity of the immune response be controlled, but also its qualitative nature.

Some preferred embodiments relate to compositions and methods for eliciting and enhancing T cell responses. For example, the method can include a priming step in which a composition comprising a nucleic acid encoding an immunogen is delivered to the animal. The compositions may be delivered to different sites in the animal, but are preferably delivered to the lymphatic system, such as lymph nodes. The initiating step may include one or more deliveries of the composition, for example, dispersal over a period of time or dispersal in a continuous manner over a period of time. Preferably, the method may further comprise an enhancement step comprising administering a composition comprising a peptide immunogen. The enhancing step may be performed one or more times, for example in a bolus or continuously over a period of time, at intervals over a period of time. Although not required in all embodiments, some embodiments may include the use of a composition that includes an immunopotentiator or adjuvant.

The disclosure of each of the following applications, including all methods, figures and compositions, is hereby incorporated by reference in its entirety: U.S. provisional application No. 60/479,393, filed on 17.6.2003, entitled method of controlling MHC class I-restricted immune responses; U.S. application No.10/871,707 (publication No. 20050079152), filed 6/17/2004, U.S. provisional application No.60/640,402, filed 12/29/2004 and U.S. application No. ____/, _, (publication No) filed concurrently with the present application, all three of which are entitled "methods of eliciting, enhancing and maintaining an immune response to a class I M HC-restricted epitope for prophylactic or therapeutic purposes"; U.S. application No.10/871,708 (publication No. 20050118186), entitled "combination of tumor-associated antigens in compositions for various types of cancer", filed 6/17/2004; and provisional application No.60/640,598 filed on 29/12/2004, and U.S. patent application No. _____________, (publication No.), (attorney docket No. mannk.049a), filed concurrently with the present application, both entitled "combination of tumor-associated antigens in compositions for use in various types of cancer," each of which is incorporated by reference in its entirety. In addition, the following applications include methods and compositions that may be used with the methods and compositions of the present invention. Plasmids and the principles of plasmid design are disclosed in U.S. patent application No. 10/292,413 (publication No. 20030228634a1), entitled "expression vectors encoding target-associated epitopes and methods for designing the same," which is incorporated by reference herein in its entirety; additional methods, compositions, peptides and peptide analogs are disclosed in the following references: U.S. provisional application No. 60/581,001, U.S. application No. 11/156,253 (publication No. _), entitled "SSX-2 peptide analogs," filed 6/17/2004; each incorporated herein by reference in its entirety; U.S. provisional application No. 60/580,962 filed on 17.6.2004, U.S. application No. 11/155,929 (publication No. _) filed on 17.6.2005, entitled "NY-ESO peptide analogs"; each incorporated herein by reference in its entirety; and U.S. application No. 10/117,937 (publication No. 20030220239) filed 4/2002 and 10/657,022 (publication No. 20040180354) filed 5/9/2003, both entitled "epitope sequences," each of which is incorporated herein by reference in its entirety.

In some embodiments, the specific activity and composition of the immune response elicited may vary depending on the nature of the immunogen and the environment it is exposed to. In particular, although immunization with peptides can produce a cytotoxic/cytolytic T Cell (CTL) response, attempts to further enhance this response with additional injections may instead result in expansion of the regulatory T cell population and a reduction in visible CTL activity. Thus, administration of compositions with high MHC/peptide concentrations on the cell surface within lymph nodes, without additional immune enhancing activity, can be used to purposefully enhance regulatory or tolerogenic responses. Immunogenic compositions that instead provide an abundant immunopotentiating signal (e.g., toll-like receptor ligands [ or cytokines/autocrine factors they will induce), even if only a limited antigen is provided, not only induce a response, but also trigger it as such, so that subsequent encounters with an abundant antigen (e.g., injected peptide) enhance the response without altering the observed activity profile. Thus, some embodiments relate to controlling the immune response pattern, e.g., the type of response obtained and the type of cytokine produced. For example, some embodiments relate to methods and compositions for promoting expansion or further expansion of CTLs, and there are embodiments relating to methods and compositions for promoting expansion of regulatory cells in preference to CTLs.

The disclosed methods are advantageous over many protocols that use only peptides or do not follow the prime-and-boost approach. As noted above, many peptide-based immunization protocols and vector-based protocols have drawbacks. However, if successful, peptide-based immunization or immunopotentiation strategies offer advantages over other approaches, particularly, for example, certain microbial vectors. This is due to the fact that more complex vectors, such as live attenuated viral or bacterial vectors, for example, may induce deleterious side effects when replicated or recombined in vivo; or become ineffective upon repeated administration due to the production of neutralizing antibodies against the vector itself. In addition, when such methods are used to make strong immunogens, the peptides may bypass the need for proteasome-mediated processes (e.g., for proteins or more complex antigens, in a "cross-processing" environment or following cellular infection). That is because processing of a cell antigen presented in a restricted manner to MHC class I is inherently a selection of dominant (favorable) epitopes rather than subdominant epitopes, potentially interfering with the phenomenon of immunogenicity corresponding to potent target epitopes. Finally, effective peptide-based immunization simplifies and shortens the development process of immunotherapy.

Thus, effective peptide-based immune enhancement methods, including in particular those described herein, are highly advantageous for immunotherapy (such as for cancer and chronic infections) or vaccination (against certain infectious diseases). Additional benefits may be obtained by avoiding the simultaneous use of cumbersome, unsafe or complex adjuvant techniques, although such techniques may be used in the various embodiments described herein.

Defining:

unless otherwise clear from the context in which the terms herein are used, the terms listed below should generally have the indicated meanings for the purposes of this specification.

Professional antigen presenting cells (papcs) -cells that have T cell costimulatory molecules and are capable of inducing a T cell response. Fully characterized pAPCs include dendritic cells, B cells and macrophages.

Surrounding cells-cells that are not pAPC.

Housekeeping proteasomes-proteasomes that are normally active in surrounding cells, are usually absent or not strongly active in pAPCs.

Immunoproteasome-the proteasome normally active in pAPCs; immunoproteasome is also active in some surrounding cells of infected tissue or after exposure to interferon.

Epitope-a molecule or substance capable of stimulating an immune response. In a preferred embodiment, epitopes according to this definition include, but are not necessarily limited to, polypeptides and nucleic acids encoding polypeptides, wherein the polypeptides are capable of stimulating an immune response. In other preferred embodiments, epitopes according to this definition include, but are not necessarily limited to, peptides presented on the surface of cells that bind non-covalently to the MHC class I binding slit so that they can interact with the T Cell Receptor (TCR). Epitopes presented by MHC class I can be in immature or mature form. "mature" refers to an MHC epitope distinct from any precursor ("immature"), which may include or consist essentially of a housekeeping epitope, and also includes other sequences in the initial translation product that are removed by processing, including but not limited to proteasomal digestion, N-terminal trimming, or the action of exogenous enzymatic activity, alone or in any combination. Thus, mature epitopes may be provided embedded in somewhat longer polypeptides, the immunological potential of which is at least partially dependent on the embedded epitope; likewise, the mature epitope can be provided in its final form, which can bind to the MHC binding slit recognized by the TCR.

MHC epitope-a polypeptide having known or expected binding affinity for a mammalian class I or class II Major Histocompatibility Complex (MHC) molecule. Some particularly well characterized class I MHC molecules are present in tables 1-4.

Housekeeping epitopes-in a preferred embodiment, a housekeeping epitope is defined as a fragment of a polypeptide that is an MHC epitope and is displayed on a cell in which the activity of the housekeeping proteasome is prominent. In another preferred embodiment, a housekeeping epitope is defined as a polypeptide comprising a housekeeping epitope according to the preceding definition, flanked by one to several additional amino acids. In another preferred embodiment, a housekeeping epitope is defined as a nucleic acid encoding a housekeeping epitope according to the preceding definition. Exemplary housekeeping epitopes are provided in U.S. patent application No. 10/117,937 (publication No. 20030220239a1) filed 4/2002, 11/067,159 (publication No. 2005-0221440a1) filed 25/2/2005, 11/067,064 (publication No. 2005-0142144a1) filed 25/2/2005 and 10/657,022 (publication No. 2004-0180354a1) filed 5/9/2003, and PCT application No. PCT/US2003/027706 (publication No. WO 2004/022709a2) filed 5/9/2003; and U.S. provisional application No. 60/282,211 filed on 6/4/2001; 60/337,017 filed on 11/7/2001; 60/363,210 filed on 3/7/2002 and 60/409,123 filed on 9/6/2002. Each of the listed applications is entitled epitope sequence. Each of the applications mentioned in this paragraph is incorporated herein by reference in its entirety.

Immune epitopes-in a preferred embodiment, immune epitopes are defined as polypeptide fragments, which are MHC epitopes and are displayed on cells in which immunoproteasome activity is prominent. In another preferred embodiment, an immune epitope is defined as a polypeptide comprising an immune epitope according to the previous definition flanked by one to several additional amino acids. In another preferred embodiment, an immune epitope is defined as a polypeptide comprising an epitope cluster sequence comprising at least two polypeptide sequences having known or expected affinity for MHC class I. In a further preferred embodiment, an immune epitope is defined as a nucleic acid encoding an immune epitope according to any one of the above definitions.

Target cells-in a preferred embodiment, the target cells are cells associated with pathogenic conditions that can be acted upon by components of the immune system, such as cells infected with a virus or other intracellular parasite or tumor cells. In another embodiment, the target cell is a cell targeted by the vaccines and methods of the invention. Examples of target cells according to this definition include, but are not necessarily limited to: tumor cells and cells with intracellular parasites such as, for example, viruses, bacteria or protozoa. Target cells may also include cells that are targeted by CTLs as part of an assay to determine or confirm proper epitope release, and that determine T cell specificity or immunogenicity for the desired epitope through cellular processing that expresses immunoproteasome. The cell may be transformed to express the release sequence or the cell may simply be pulsed with the peptide/epitope.

Target Associated Antigen (TAA) -a protein or polypeptide present in a target cell.

Tumor associated antigen (TuAA) -TAA, wherein the target cell is a tumor cell.

HLA epitope-a polypeptide having known or expected binding affinity to human HLA class I or class II complex molecules. Particularly well characterized class I HLAs are presented in tables 1-4.

Antibody-polyclonal or monoclonal native immunoglobulin (Ig), or any molecule consisting wholly or partially of an Ig-binding domain, whether biochemically derived or obtained by using recombinant DNA or by any other method. Examples include, in particular, F (ab), single chain Fv and Ig variable region-phage coat protein fusions.

Substantial similarity-this term is used to refer to sequences that differ from a reference sequence in a disjointed manner, as judged by examination of the sequences. Nucleic acid sequences encoding the same amino acid sequence are substantially similar, although there may be differences at degenerate positions or minor differences in length or any non-coding region composition. Except that amino acid sequences that differ by conservative substitutions or small length changes are substantially similar. In addition, the amino acid sequences comprising housekeeping epitopes that differ in the number of flanking residues at the N-terminus, or immunological epitopes and epitope clusters that differ in the number of flanking residues at either terminus, are substantially similar. Nucleic acids that encode substantially similar amino acid sequences are themselves substantially similar.

Functional similarity-this term is used to refer to sequences that differ from a reference sequence in a disjointed manner, as determined by measuring biological or biochemical properties, although the sequences may not be substantially similar. For example, two nucleic acids may be used as hybridization probes for the same sequence but encoding different amino acid sequences. Even if they differ by non-conservative amino acid substitutions (and therefore do not meet the definition of substantial similarity), the two peptides that induce a cross-reactive CTL response are functionally similar. Paired antibodies or TCRs recognizing the same epitope may be functionally similar to each other, although there are any structural differences. Testing for functional similarity of immunogenicity can be performed by immunizing with an "altered" antigen and testing for the ability of the priming response to recognize the target antigen, including but not limited to antibody responses, CTL responses, cytokine production, and the like. Thus, two sequences can be designed that differ in some way while retaining the same function. Sequence variants of the disclosed or claimed sequences so designed are within the embodiments of the present invention.

Expression cassette-polynucleotide sequence encoding a polypeptide, operably linked to a promoter and other transcriptional and translational regulatory elements including, but not limited to, enhancers, stop codons, internal ribosome entry sites, and polyadenylation sites. The cassette may also include sequences that facilitate its migration from one host molecule to another.

Embedded epitopes-in some embodiments, embedded epitopes are epitopes that are all contained in longer polypeptides; in other embodiments, the term may also include epitopes wherein only the N-terminus or C-terminus is embedded such that the epitope is not all in a position within a longer polypeptide.

Mature epitope-a peptide with no additional sequence other than the one present when the epitope is bound in the MHC peptide-binding slit.

Epitope cluster-polypeptides or nucleic acid sequences encoding the same, which are fragments of a protein sequence, including native protein sequences, comprising two or more known or predicted epitopes having binding affinity for a common MHC restriction element. In a preferred embodiment, the density of epitopes within a cluster is greater than the density of all known or expected epitopes within the full-length protein sequence that have binding affinity for a common MHC restriction element. Epitope clusters are disclosed and more fully defined in U.S. patent application No. 09/561,571 entitled epitope cluster, filed on 28/4/2000, which is incorporated herein by reference in its entirety.

Release sequence-a designed or engineered sequence that contains or encodes a housekeeping epitope embedded in a larger sequence that provides an environment that allows the housekeeping epitope to be released by processing activity, including, for example, immunoproteasome activity, N-terminal tailoring, and/or other processing or activity, alone or in any combination.

CTLp-CTL precursors are T cells that can be induced to exhibit cytolytic activity. By which minor in vitro lytic activity of CTLp is commonly observed, effector functions may be generated by any combination of naive, effector and memory CTLs in vivo.

Memory T cells-regardless of their location in the body, T cells that have been previously activated by an antigen, but are in a dormant physiological state requiring re-exposure to the antigen to gain effector function. Phenotypically they are CD62L-CD44hi CD107 α -IGN- γ -LT β -TNF- α and are in the G0 phase of the cell cycle.

Effector T cells-T cells that readily exhibit effector function when encountering an antigen. Effector T cells are generally able to exit the lymphatic system and enter the immune periphery. It is typically phenotypic as CD62L-CD44HICD107 α + IGN- γ + LT β + TNF- α + and actively circulates.

Effector function-typically T cell activation, typically involves acquisition of cytolytic activity and/or cytokine secretion.

Inducing a T cell response-in many embodiments including generating a T cell response from naive cells or, in some circumstances, from resting cells; the process of activating T cells.

Enhancing T cell responses-in many embodiments include processes that increase cell number, activated cell number, activity level, proliferation rate, or T cell-like parameters involved in a specific response.

Priming-in many embodiments includes administration of an induced T cell lineage immune pattern to induce with a particular stability. In various embodiments, the term "priming" may correspond to "inducing" and/or "initiating".

TOLL-like receptors (TLRs) -TOLL-like receptors (TLRs) are a family of pattern recognition receptors that are activated by specific components of microorganisms and certain host molecules. As part of the innate immune system, it contributes to the first line of defense against many pathogens, and plays a role in adaptive immunity.

TOLL-like receptor (TLR) ligand-any molecule capable of binding to and activating a TOLL-like receptor. Examples include, but are not limited to: double-stranded RNA is known to induce synthesis of poly IC A of interferon. The polymer is composed of polyinosinic acid and one strand of polycytidylic acid, double-stranded RNA, unmethylated CpG oligodeoxyribonucleotides or other immunostimulatory sequences (ISSs), Lipopolysaccharide (LPS), beta-glucan and imidazoquine, and derivatives and analogs thereof.

Immune enhancing adjuvants-adjuvants that activate pAPC or T cells include, for example: TLR ligands, endocytosis-Pattern Recognition Receptor (PRR) ligands, quillaja saponins, benzoic acid derivatives, cytokines, and the like. Some preferred adjuvants are described in Marciani, d.j. drug Discovery Today 8: 934-943, 2003, which is incorporated herein by reference in its entirety.

Immunostimulatory sequences (ISS) -typically oligodeoxyribonucleotides containing unmethylated CpG sequences. CpG can also be embedded in DNA produced by bacteria, in particular in plasmids. Additional embodiments include various analogs; in preferred embodiments, molecules having one or more phosphorothioate linkages or non-physiological bases.

Vaccines-in a preferred embodiment, the vaccine may be an immunogenic composition that provides or helps prevent disease. In other embodiments, the vaccine is a composition that can provide or assist in the cure of a disease. In other embodiments, the vaccine composition may provide or aid in the amelioration of disease. Additional embodiments of vaccine immunogenic compositions are useful as therapeutic and/or prophylactic agents.

Immunization-a process that induces partial or complete protection against disease. Alternatively, a process that induces or enhances an immune system response to an antigen. In the second definition, it may comprise a protective immune response, in particular pro-inflammatory or auto-immune, but may also include a modulating response. Thus in some embodiments, immunity is distinct from tolerance (the process by which the immune system avoids producing pro-inflammatory or auto-immunity), while in other embodiments the term includes tolerance.

TABLE 1

Class I MHC molecules

Class I

Human being

HLA-A1

HLA-A*0101

HLA-A*0201

HLA-A*0202

HLA-A*0203

HLA-A*0204

HLA-A*0205

HLA-A*0206

HLA-A*0207

HLA-A*0209

HLA-A*0214

HLA-A3

HLA-A*0301

HLA-A*1101

HLA-A23

HLA-A24

HLA-A25

HLA-A*2902

HLA-A*3101

HLA-A*3302

HLA-A*6801

HLA-A*6901

HLA-B7

HLA-B*0702

HLA-B*0703

HLA-B*0704

HLA-B*0705

HLA-B8

HLA-B13

HLA-B14

HLA-B*1501(B62)

HLA-B17

HLA-B18

HLA-B22

HLA-B27

HLA-B*2702

HLA-B*2704

HLA-B*2705

HLA-B*2709

HLA-B35

HLA-B*3501

HLA-B*3502

HLA-B*3701

HLA-B*3801

HLA-B*39011

HLA-B*3902

HLA-B40

HLA-B*40012(B50)

HLA-B*4006(B61)

HLA-B44

HLA-B*4402

HLA-B*4403

HLA-B*4501

HLA-B*4601

HLA-B51

HLA-B*5101

HLA-B*5102

HLA-B*5103

HLA-B*5201

HLA-B*5301

HLA-B*5401

HLA-B*5501

HLA-B*5502

HLA-B*5601

HLA-B*5801

HLA-B*6701

HLA-B*7301

HLA-B*7801

HLA-Cw*0102

HLA-Cw*0301

HLA-Cw*0304

HLA-Cw*0401

HLA-Cw*0601

HLA-Cw*0602

HLA-Cw*0702

HLA-Cw8

HLA-Cw*1601M

HLA-G

Mouse (mouse)

H2-K^d

H2-D^d

H2-L^d

H2-K^b

H2-D^b

H2-K^k

H2-K^km1

Qa-1ⁿ

Qa-2

H2-M3

Rat

RT1.A^a

RT1.A¹

Cattle (cow)

Bota-A11

Bota-A20

Chicken with egg yolk

B-F4

B-F12

B-F15

B-F19

Chimpanzee

Patr-A*04

Patr-A*11

Patr-B*01

Patr-B*13

Patr-B*16

Baboon

Papa-A*06

Kiwi fruit

Mamu-A*01

Pig (pig)

SLA (haplotype d/d)

Viral homologues

hCMV class I homolog UL18

TABLE 2

Class I MHC molecules

Class I

Human being

HLA-A1

HLA-A*0101

HLA-A*0201

HLA-A*0202

HLA-A*0204

HLA-A*0205

HLA-A*0206

HLA-A*0207

HLA-A*0214

HLA-A3

HLA-A*1101

HLA-A24

HLA-A*2902

HLA-A*3101

HLA-A*3302

HLA-A*6801

HLA-A*6901

HLA-B7

HLA-B*0702

HLA-B*0703

HLA-B*0704

HLA-B*0705

HLA-B8

HLA-B14

HLA-B*1501(B62)

HLA-B27

HLA-B*2702

HLA-B*2705

HLA-B35

HLA-B*3501

HLA-B*3502

HLA-B*3701

HLA-B*3801

HLA-B*39011

HLA-B*3902

HLA-B40

HLA-B*40012(B60)

HLA-B*4006(B61)

HLA-B44

HLA-B*4402

HLA-B*4403

HLA-B*4601

HLA-B51

HLA-B*5101

HLA-B*5102

HLA-B*5103

HLA-B*5201

HLA-B*5301

HLA-B*5401

HLA-B*5501

HLA-B*5502

HLA-B*5601

HLA-B*5801

HLA-B*6701

HLA-B*7301

HLA-B*7801

HLA-Cw*0102

HLA-Cw*0301

HLA-Cw*0304

HLA-Cw*0401

HLA-Cw*0601

HLA-Cw*0602

HLA-Cw*0702

HLA-G

Mouse

H2-K^d

H2-D^d

H2-L^d

H2-K^b

H2-D^b

H2-K^k

H2-K^km1

Qa-2

Rat

RT1.A^a

RT1.A¹

Cattle

Bota-A11

Bota-A20

Chicken with egg yolk

B-F4

B-F12

B-F15

B-F19

Viral homologues

hCMVI homologue UL18

TABLE 3

Estimated gene frequency of HLA-A antigen

^aFrequency of genes

^bStandard deviation of

TABLE 4

Estimated gene frequency of HLA-B antigen

^aFrequency of genes

^bStandard deviation of

^cThe observed gene count was zero.

TABLE 5

List of CT genes ^* ：

^*See Scanlan et al, "The cancer/testies genes: review, standardization, and commensuration, "Cancer Immunity, Vol.4, p.1 (1/23/2004), which is incorporated herein by reference in its entirety.

The following discussion sets forth an understanding or appreciation for the operation of aspects of the invention. However, the discussion is not intended to limit the patent to any particular theory of operation not presented in the claims below.

Effective immune-mediated neoplastic processes or microbial infections generally involve the induction and expansion of antigen-specific T cells with a variety of abilities such as migration, effector function and differentiation into memory cells. Induction of an immune response can be attempted by various methods and includes administration of different forms of antigen, which have a tunable effect on the strength and nature of the immune response. One limiting factor in achieving immune response regulation is the targeting of papcs that can process and efficiently present the resulting epitopes to specific T cells.

The solution to this problem is to direct antigen delivery to the secondary lymphatic uterus, which is a pAPC and T cell rich microenvironment. The antigen can be delivered, for example, as a polypeptide or as an antigen expressed by any of a variety of vectors. The outcome, depending on the strength and nature of the immunization, can be regulated by factors including, for example, dosage, formulation, nature of the carrier, and molecular environment. Embodiments of the invention may enhance the control of immune responses. Control of the immune response includes the ability to induce different types of immune responses as desired, for example from modulation to pro-inflammatory responses. Preferred embodiments provide enhanced control of the intensity and nature of MHC class I-restricted epitope responses, which is a primary objective of active immunotherapy.

Previous immunization methods present some important limitations: first, conclusions often related to vaccine efficacy are inferred from immunogenicity data generated from one or a set of very limited, hypersensitive read assays. Often, the clinical response is not significant or modest regardless of the inferred efficacy of the vaccination regimen. Second, after immunization, T regulatory cells can be generated and/or expanded along with more conventional T effector cells, and these cells can interfere with the function of the desired immune response. The importance of these mechanisms in active immunotherapy has only recently been recognized.

Intra-nodal administration of the immunogen provides the basis for the intensity and pattern control of the immune response. The efficient in vivo loading of pAPC achieved as a result of said administration enables a high intensity of immunity even by using the simplest form of antigen-peptide epitopes-or in general in connection with poor pharmacokinetics. The nature of the response may be further controlled by the immunogen, the nature of the carrier and the immunization protocol. These protocols can be used to enhance/modify the response in chronic infections or tumor progression.

Immunization generally relies on repeated antigen administration to enhance the intensity of the immune response. The use of DNA vaccines produces high quality responses, but it is difficult to obtain high intensity responses with such vaccines, even if booster doses are repeated. Two characteristics of the response, high quality and low intensity, may be due to the relatively low level of epitope loading on MHC obtained with these vectors. Rather, it is more common to boost the vaccine with antigens encoded in live viral vectors to obtain the high intensity response required for clinical efficacy. However, the use of live vectors entails several disadvantages, including potential safety issues, reduced effectiveness of subsequent booster immunizations due to humoral responses to the vector induced by previous administrations, and costs of production and production. Thus, the use of only live vectors or DNA, while stimulating a high quality response, may result in a limited response intensity or persistence.

Disclosed herein are embodiments relating to schemes and methods that, when applied to peptides, make them effective immunotherapeutic tools. These approaches bypass the PK of poor peptides and produce enhancement and/or control of strong immune responses if applied in a specific, often more complex, context. In a preferred embodiment, administration of the peptide directly to the lymphoid organs following induction of a strong, moderate or even mild (at or below levels detected by conventional methods) initiator of the immune response consisting of Tcl cells results in an unexpectedly strong enhancement of the immune response. While the preferred embodiment of the invention may employ intralymphatic (intralymphatic) administration of the antigen throughout the stages of immunization, intralymphatic administration is the most preferred mode of administration of the peptide without adjuvant. The enhancement with peptides administered intralymphatically can be applied to existing immune responses, which have been previously induced. The prior induction may occur by natural exposure to the antigen or by commonly used routes of administration including, but not limited to, subcutaneous, intradermal, intraperitoneal, intramuscular, and mucosal.

As also shown herein, optimal priming leading to subsequent expansion of specific T cells can be better achieved by exposing naive T cells to a limited amount of antigen (e.g., can give rise to often limited expression of the antigen encoded by the free plasmid) in an abundant co-stimulatory environment (e.g., in lymph nodes). It can cause activation of T cells carrying T cell receptors, it recognizes MHC-peptide complexes on antigen presenting cells with high affinity, and it can lead to the production of memory cells that are more responsive to subsequent stimulation. A beneficial co-stimulatory environment can be enhanced or ensured by the use of an immunopotentiator, and thus intralymphatic administration, while advantageous, is not in all embodiments required to initiate an immune response. In embodiments involving the use of epitope peptides for induction/priming, it is preferred to use relatively low doses of the peptide (compared to the booster dose or MHC-saturating concentrations) to limit presentation, especially if direct intralymphatic administration is utilized. These embodiments generally relate to the inclusion of an immunopotentiator to achieve priming.

Although the poor pharmacokinetics of free peptides prevents their use in most routes of administration, direct administration to secondary lymphoid organs, especially lymph nodes, has proven effective in maintaining more or less continuous antigen levels by continuous infusion or frequent (e.g., daily) injection. Such intratumoral administration for generating CTLs is taught in U.S. patent application nos. 09/380,534, 09/776,232 (publication No. 20020007173a1), current U.S. patent No.6,977,074 and __/__, __ (publication No. __) (attorney docket No. mannk.001cp2c1), and PCT application No. PCTUS98/14289 (publication No. WO9902183a2), each entitled method of inducing CTL responses, each of which is incorporated herein by reference in its entirety. In some embodiments of the invention, the intranodal administration of the peptide is effective to enhance the response initially induced with the plasmid DNA vaccine. Furthermore, the cytokine pattern is unique in that plasmid DNA induction/peptide enhancement generally results in more chemokines (chemoattractant cytokines) and less immunosuppressive cytokine production than either DNA/DNA or peptide/peptide protocols.

Thus, such DNA induction/peptide enhancement regimens may improve the effectiveness of compositions, including therapeutic vaccines for cancer and chronic infections. Advantageous epitope selection principles for these immunotherapies are disclosed in U.S. patent application Nos. 09/560,465, 10/026,066 (publication No. 20030215425A1), 10/005,905, published on 7/20/2004 as 10/895,523 (publication No. 2005-0130920A1), and published on 20/7/2004 as 10/896,325 (publication No. _), all entitled epitope synchronization in antigen presenting cells; 09/561,074 filed on 10/1/2004, currently U.S. Pat. Nos. 6,861,234 and 10/956,401 (publication No. 2005-0069982A1), both entitled methods for epitope discovery; 09/561,571 filed on 28/4/2000 entitled epitope cluster; 10/094,699 filed 3/7/2002 (publication No. 20030046714a1), 11/073,347 filed 6/30/2005 (publication No. _), each entitled anti-neovascular agent for cancer; and 10/117,937 filed 4/2002 (publication No. 20030220239a1), 11/067,159 filed 25/2/25/2005 (publication No. 2005-0221440a1), 10/067,064 filed 25/2/2005 (publication No. 2005-0142114a1), and 10/657,022 (publication No. 2004-0180354a1) and PCT application No. PCT/US2003/027706 (publication No. WO 04/022709a2), each entitled epitope sequences, and each incorporated herein by reference in its entirety. Aspects of the overall vaccine plasmid design are disclosed in U.S. patent application No. 09/561,572 filed on 28/4/2000, and 10/225,568 filed on 20/8/2002 (publication No. 2003-0138808A1), both entitled expression vectors encoding target-related epitopes, and U.S. patent application Nos. 10/292,413 (publication No. 20030228634A1), 10/777,053 (publication No. 2004-0132088A1) filed on 10/2/2004 and 10/837,217 (publication No. _ filed on 30/4/2004), all entitled expression vectors encoding target-related epitopes and methods for their design; 10/225,568 (publication No. 2003-0138808A1), PCT application No. PCT/US2003/026231 (publication No. WO2004/018666), and U.S. patent No.6,709,844 and U.S. patent application No. 10/437,830 (publication No. 2003-0180949A1), each entitled avoidance of unwanted replicative intermediates in plasmid propagation, are filed on 13/5 of 2003 and are each incorporated herein by reference in their entirety. Specific benefits of specific antigen combinations in directing an immune response against a particular cancer are disclosed in U.S. provisional application No. 60/479,554, filed on 17.6.2003, U.S. patent application No.10/871,708 (publication No. 2005-0118186a1), filed on 29.6.2005, PCT patent application No. PCT/US2004/019571 (publication No. WO 2004/112825), U.S. provisional application No.60/640,598, and U.S. patent application nos. _____________________, (publication No. _______________________________________________________________________. The use and advantages of intralymphatic administration of BRMs are disclosed in U.S. provisional patent application No.60/640,727 filed on 29.12.2005 and U.S. patent application No. ___/, (publication No.) (attorney docket No. mannk.046a), filed concurrently with this application, both entitled methods of triggering, maintaining and manipulating immune responses by targeted administration of biological response modifying substances to lymphoid organs, each of which is incorporated herein by reference in its entirety. Additional methods, compositions, peptides and peptide analogs are disclosed in U.S. patent application No. 09/999,186, filed 11/7/2001, entitled methods for commercializing antigens; and U.S. provisional patent application No.60/640,821 filed on 29/12/2005 and application nos. __/__, __ (publication No. _____) filed concurrently herewith (attorney docket No. mannk.048a), both entitled method for bypassing CD4+ cells in the induction of an immune response, each of which is incorporated herein by reference in its entirety.

Additional related disclosures are presented in U.S. patent application No. 11/156,369 (publication No. _) and U.S. provisional patent application No. 60/691,889, both entitled epitope analogs, filed 6/17/2005 and each incorporated herein by reference in its entirety. Also of relevance are U.S. provisional patent application No. 60/691,579, filed on 17.6.2005, entitled methods and compositions for eliciting multivalent immune responses to dominant and subdominant epitopes expressed on cancer cells and tumor stroma, and 60/691,581, filed on 17.6.2005, entitled multivalent prime-and-boost immunotherapy for cancer, each of which is incorporated herein by reference in its entirety.

Surprisingly, repeated intra-nodal injections of peptides according to the conventional activation-potentiation schedule resulted in a reduction in the number of cytolytic responses compared to the response observed following administration of only the initial dose. Examination of the immune response pattern shows that this is a result of immune modulation induction (suppression) rather than no response. This is in contrast to induction-and-enhancement protocols which comprise DNA, usually plasmids, encoding the immunogen. Direct loading of pAPC by intraknot injection of antigens typically reduces or avoids the need for adjuvants that are typically used to correct the pharmacokinetics of antigens delivered via other parenteral routes. The absence of such adjuvants, which are often inflammatory, can therefore facilitate the induction of a different (i.e., regulatory or tolerogenic) immune response pattern than previously observed with peptide immunization. As shown in the examples below, the results support methods of use and compositions according to embodiments of the invention for altering (inhibiting) an ongoing inflammatory response, as the response is measured in the secondary lymphatic uterus away from the initial injection site. This approach is useful even for inflammatory disorders with MHC class II-restricted etiology by targeting the same antigen or any suitable antigen associated with the site of inflammation and relying on bystander effects mediated by immunosuppressive cytokines.

Despite the fact that repeated peptide administration results in a stepwise reduction of the cytolytic immune response, induction with agents such as non-replicating recombinant DNA (plasmids) has a considerable impact on subsequent doses, enabling strong immune enhancement to the epitopes and peptides expressed by the recombinant DNA and eliciting their cytolytic properties. In fact, when a single or multiple administration of the recombinant DNA vector or peptide, respectively, does not result in or results in a modest immune response, induction with DNA and enhancement with peptide results in substantially higher responses, whether in response rate or in response intensity. In the examples shown, the response rate is at least doubled and the response intensity (mean and median) is at least tripled by using a recombinant DNA induction/peptide-enhancement protocol. Thus, a preferred protocol results in immune induction (Tcl immunization) which is capable of treating antigen cells in vivo, in lymphoid and non-lymphoid organs. One limiting factor in most cancer immunotherapy is the limited susceptibility of tumor cells to immune-mediated attack, possibly due to reduced MHC/peptide presentation. In a preferred embodiment, a strong immune expansion is obtained by DNA induction/peptide augmentation, which is generally equal to or greater than the immune response typically observed following infection with virulent microorganisms. This increased intensity contributes to compensating poor MHC/peptide presentation and does lead to the clearance of human tumor cells as shown in specialized preclinical models, such as, for example, HLA transgenic mice.

Such a specific sequence of induction-and-boost regimen, comprising a recombinant DNA priming dose followed by peptide boosting administered to lymphoid organs, can therefore be used for the purpose of induction, enhancement and maintenance of a strong T cell response, e.g. for the prevention or treatment of infectious or neoplastic diseases. These diseases may be cancer (e.g., kidney, ovary, breast, lung, colorectal, prostate, head-and-neck, bladder, uterus, skin), melanoma, tumors of different origin and tumors that typically express established or determinable tumor-associated antigens, such as carcinoembryonic antigens (e.g., CEA, CA 19-9, CA 125, CRD-BP, Das-1, 5T4, TAG-72, etc.), tissue differentiation antigens (e.g., Melan-A, tyrosinase, gp100, PSA, PSMA, etc.) or cancer-testis antigens (e.g., PRAME, MAGE, LAGE, SSX2, NY-ESO-I, etc.; see Table 5). Cancer-testis genes and their relevance for Cancer treatment are described in Scanlon et al, Cancer Immunity 4: 1-15, 2004, which are incorporated herein by reference in their entirety. Tumor neovasculature associated antigens (e.g., PSMA, VEGFR2, Tie-2) may also be used in association with cancer diseases, as disclosed in U.S. patent application Nos. 10/094,699 (publication No. 20030046714A1) and 11/073,347 (publication No. _) filed on 30.6.2005, entitled anti-neovascular formulations for cancer, each of which is incorporated herein by reference in its entirety. The methods and compositions are useful for targeting different organisms and disease conditions. For example, the target organism may include bacteria, viruses, protozoa, fungi, and the like. The target disease may include a disease caused by, for example, a prion. Exemplary diseases, organisms and antigens and epitopes associated with target organisms, cells and diseases are described in U.S. application No. 09/776,232 (publication No. 20020007173a1), currently U.S. patent No.6,977,074, which is incorporated herein by reference in its entirety. Infectious diseases that can be elucidated are those caused by pathogens that tend to produce chronic infections (HIV, herpes simplex virus, CMV, hepatitis b and c viruses, papilloma virus, etc.) and/or are associated with acute infections (e.g. influenza virus, measles, RSV, Ebola virus). Of interest from a prophylactic or therapeutic perspective are viruses with oncogenic potential-such as papilloma virus, Epstein Barr virus and HTLV-I. All of these infectious pathogens have defined or determinable antigens that can be used as a basis for designing compositions such as peptide epitopes.

A preferred application of the method (see, e.g., FIG. 19) involves injection or infusion into one or more lymph nodes, starting with multiple (e.g., 1 to 10 or more, 2 to 8, 3 to 6, preferably about 4 or 5) administrations of recombinant DNA (in a dose range of 0.001-10mg/kg, preferably 0.005-5mg/kg), followed by one or more (preferably about 2) administrations of the peptide, preferably in an immunologically inert carrier or formulation (in a dose range of 1ng/kg-10mg/kg, preferably 0.005-5 mg/kg). Because the dose does not necessarily scale linearly with the subject's body weight, in each of these ranges, the dose for humans may tend to be lower and the dose for mice may tend to be higher. The preferred concentration of plasmids and peptides for injection is generally about 0.1. mu.g/ml to 10mg/ml, and the most preferred concentration is about 1mg/ml, generally regardless of the weight or species of the subject. However, particularly effective peptides may have optimal concentrations towards the lower end of the range, for example between 1 and 100. mu.g/ml. Dosages towards the higher end of these ranges (e.g., 0.5-10mg/ml) are generally preferred when peptide-only regimens are used to improve tolerability. This sequence can be repeated as long as necessary to maintain a strong immune response in vivo. In addition, the time between the last priming dose of DNA and the first boosting dose of peptide is not critical. Preferably it is about 7 days or longer and may exceed several months. The number of repetitions of DNA and/or peptide injections can be reduced by substituting infusions over consecutive days (preferably 2-7 days). It may be advantageous to start an infusion with bolus material similar to that administered by injection, followed by a slow infusion (24-12000. mu.l/day to deliver about 25-2500. mu.g/day of DNA, 0.1-10,000. mu.g/day for peptides). This can be done manually or through the use of a programmable pump, such as an insulin pump. Such pumps are known in the art and are capable of producing periodic spikes and other dosage patterns, which may be advantageous in some embodiments.

The present invention generally describes a single cycle immunization comprising administration of one or an initial dose followed by administration of one or more booster doses. Other embodiments of the invention require repeated cycles of immunization. Such repeated cycling may be used to further enhance the intensity of the response. In addition, when seeking multivalent responses, not all individuals necessarily achieve a significant response to each target antigen as a result of a single-cycle immunization. The immune cycle can be repeated until a particular individual achieves an adequate response to each target antigen. It is also possible to modify a single immune cycle to achieve a more balanced response by adjusting the order, timing or number of doses given for each individual component. Multiple rounds of immune cycles may also be used to maintain a response over time, for example to maintain an effective response effector period to be substantially constant over time, and may be advantageous for the treatment of a disease or other medical condition.

It should be noted that although this method successfully uses peptides in the enhancement step without conjugation to proteins, addition of adjuvants, etc., it is not necessary to lack these components. Thus, conjugated peptides, adjuvants, immunopotentiators, and the like may be used in embodiments. More complex peptide compositions administered to lymph nodes, or having the ability to reach the lymphatic system, including peptide-pulsed dendritic cells composed of or containing different forms of peptide epitopes or antigens, suspensions such as liposomal formulations, aggregates, emulsions, microparticles, nanocrystals, can be replaced with free peptide in this method. Conversely, peptide boosting by intranodal administration may follow any method/pathway activation by achieving induction of T memory cells even at low levels.

In order to reduce the chance of resistance arising from antigen expression mosaicism, or antigen mutation or loss, it is advantageous to immunize against multiple, preferably about 2-4, antigens simultaneously. Any combination of antigens may be used. The pattern of expression of a particular tumor antigen can be used to determine the antigen or combination of antigens used. Exemplary methods are found in U.S. provisional application No. 60/580,969 filed on 17.6.2004, U.S. patent application No. 11/155,288 filed on 17.6.2005, and U.S. patent application No. (publication No.) __________, (publication No.) (attorney docket No. mannk.050cp1), all entitled "combinations of tumor-associated antigens for use in methods of diagnosing various types of cancer"; and each is incorporated by reference herein in its entirety. Specific antigen combinations particularly suited for selected cancer treatments are disclosed in U.S. provisional patent application No. 60/479,554 and U.S. patent application No.10/871,708 (publication No. 2005-0118186A1) and PCT application No. PCT/US2004/019571, which are incorporated by reference above. In order to trigger an immune response to multiple antigens or multiple epitopes from a single antigen, these methods can be used to deliver multiple immunogenic entities, either separately or as a mixture. When the immunogens are delivered separately, it is preferred that different entities are administered to different lymph nodes or to the same lymph node at different times, or to the same lymph node at the same time. This can be particularly relevant for peptide delivery, for which it is difficult to design a single formulation that provides solubility and stability for all component peptides. A single nucleic acid molecule may encode multiple immunogens. Alternatively, for multiple antigens, multiple nucleic acid molecules encoding one or a subset of all component immunogens can be mixed together so long as the required dosage can be provided without the need for high concentrations of nucleic acid, such high concentrations causing their viscosity to become problematic.

In a preferred embodiment, the method requires direct administration into the lymphatic system. In a preferred embodiment this is administration to lymph nodes. Afferent lymphatic vessels are similarly preferred. The choice of lymph nodes is not critical. The inguinal lymph node is preferred due to its size and accessibility, but axillary and cervical nodules and tonsils are similarly favored. Administration to a single lymph node is sufficient to induce or enhance an immune response. Application to multiple nodules can improve the reliability and intensity of the response. For embodiments that promote multivalent responses and thus use multiple potentiating peptides, it is preferred that only a single peptide be administered to any particular lymph node on any particular occasion. Thus, for example, one peptide may be administered to the right inguinal lymph node and a second peptide to the left inguinal lymph node at the same time. Additional peptides may be administered to other lymph nodes even if they are not the site of induction, since the initial and booster doses do not have to be administered to the same site due to migration of T lymphocytes. Or, for example, any additional peptide may be administered to the same lymph node several days later for administration of the previously enhanced peptide, since the time interval between induction and enhancement is generally not a critical parameter, although in preferred embodiments the time interval may be greater than about one week. If their MHC-binding affinities are similar, separate administration of the enhancing peptide is generally of less importance, but its importance increases as the affinities become more different. Incompatible formulations of the various peptides may also be preferred for separate administration.

Patients who may benefit from these immunization methods may be recruited using methods to determine their MHC protein expression patterns and general levels of immune responses. In addition, the level of immunity can be monitored using standard techniques in conjunction with entry into the peripheral blood. Finally, the treatment regimen can be adjusted based on changes in response and antigen expression during the induction or enhancement phase. For example, it may be preferable to administer repeated priming doses until a detectable response is obtained, and then administer a booster peptide dose, rather than a booster dose administered after a set of priming doses. Similarly, if its effectiveness declines, the number of antigen-specific regulatory T cells increases or some other evidence of tolerance is observed, the booster or maintenance dose of the predetermined peptide may be discontinued and a further priming dose administered prior to restoring the booster with the peptide. The integration of diagnostic techniques for assessing and monitoring immune responses using immunological methods is discussed more fully in provisional U.S. patent application No. 60/580,964 filed on 17.6.2004 and U.S. patent application No. 11/155,928 (publication No. _) filed on 17.6.2005, both entitled "improving the efficacy of active immunotherapy by integrating diagnostic and therapeutic methods", each of which is incorporated herein by reference in its entirety.

The practice of many of the method embodiments of the present invention involves the use of at least two different compositions and, particularly where more than one target antigen is present, it may include several compositions that are administered together and/or at different times. Embodiments of the invention thus include groups and subsets of immunogenic compositions and their respective dosages. Multivalency can be achieved using synergistic use of compositions comprising multivalent immunogens, combinations of monovalent immunogens, compositions comprising one or more monovalent immunogens or different combinations thereof. The manufacture of multiple compositions for a particular treatment regimen or method according to these methods identifies immunotherapeutic products. In some embodiments all or a subset of the product components are packaged together in a kit. In some cases induction and enhancement compositions targeting a single epitope or group of epitopes can be packaged together. In other cases multiple inducing compositions may be fitted in one kit and the corresponding enhancing compositions in another. Or the compositions may be packaged and sold separately with instructions, in printed form or on a machine-readable medium, describing how they can be used in conjunction with each other to achieve the advantageous results of the method of the invention. Further modifications will be apparent to persons skilled in the art. The use of various packaging regimens that do not include the use of the entire composition in a particular regimen or manner facilitates personalization of the treatment, e.g., based on tumor antigen expression or observed response to immunotherapy or various components thereof, such as U.S. provisional application No. 60/580,969 filed on day 6/17 2004, U.S. patent application No. 11/155,288 (publication No.) filed on day 6/17 2005, and U.S. patent application No. __/__, __ (attorney docket No. nomank.050cp1) filed on day 29/12/2005, all entitled "tumor-associated antigen combinations for diagnosis of various types of cancer"; and provisional U.S. patent application No. 60/580,964 and U.S. patent application No. 11/155,928 (publication No. _), both entitled "improving the efficacy of active immunotherapy by integrating diagnostic and therapeutic methods", each of which is incorporated by reference in its entirety.

In some embodiments, numbers expressing quantities of components, properties such as molecular weight, reaction conditions, and so forth, 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 set forth in the written description and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, numerical parameters should be analyzed in light of the number of reported significant digits 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 possible. In some embodiments of the invention, a given value may contain certain errors necessarily resulting from the standard deviation found in their respective testing 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 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 separate 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 unless 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 groups of substitute elements or embodiments of the invention disclosed herein are not to be considered as limiting. Each member may be referred to and claimed individually or in any combination with other members of the groups or other elements found herein. It is contemplated that one or more group members may be included in or deleted from the group for convenience and/or patentability. When any such inclusion or deletion is present, the specification is considered herein to encompass a group modified to thereby conform to all markush group written descriptions 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 contemplated that skilled artisans may employ such variations as appropriate, and the invention may 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 employed are within the scope of the invention. Accordingly, by way of example, and not limitation, alternative forms of the invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to only those shown and described.

The following examples are for illustrative purposes only and are not intended to limit the scope of the invention or its various embodiments in any way.

Example 1 high efficiency induction of immune response by intralymphatic immunization.

Mice loaded with a chimeric single-stranded transgene expressing human MHC class I (A x 0201, designated "HHD"; see Pascolo et al J.Exp.Med.185 (12): 2043-51, 1997, which is incorporated herein by reference in its entirety) were immunized by intra-nodal administration as follows. By applying different injection routes: five groups of mice (n ═ 3) were immunized to induction with a plasmid expressing Melan-a26-35 a27L analog (pSEM) and boosted after one week, subcutaneously (sc), intramuscularly (im) and intralymphatically (in, using direct inoculation of inguinal lymph nodes). The immunization and dose schedule is shown in fig. 1A. After one week of enhancement, mice were sacrificed; splenocytes were prepared and stained with labeled anti-CD 8mAbs and tetramers recognizing Melan-a 26-35-specific T cell receptors. Representative data are shown in FIG. 1B: while subcutaneous and intramuscular administration achieved a frequency of about or less than 1% tetramer + CD8+ T cells, intralymphatic administration of the plasmid achieved a frequency of more than 6%. In addition, splenocytes were stimulated with Melan-A peptide in vitro and 51 Cr-labeled target cells (T2 cells) were detected at various E: T ratios (FIG. 1C). Using this standard cytotoxicity assay, splenocytes from animals immunized with intralymph node injection showed the highest levels of in vitro lysis at various E: T ratios.

Example 2. Effect of the sequence of administration of the different forms of immunogen.

HHD mice were immunized by intracorporeal administration of plasmids (pSEM) or peptides (Mel A; ELAGIGILTV; SEQ ID NO: 1) in different sequences. Immunogenic polypeptides encoded by pSEM are disclosed in U.S. patent application 10/292,413 (publication No. 20030228634A1), entitled expression vectors encoding target-associated epitopes and methods for designing the same, which are incorporated herein by reference in their entirety.

The immunization protocol (fig. 2) included:

i) induction phase/induction dose: on days 0 and 4, 25 μ l (microliters) of sterile saline was injected bilaterally into the inguinal lymph node, containing 25 μ g (micrograms) of plasmid or 50 μ g (micrograms) of peptide.

ii) booster dose: as described above in example 1 and initiated 2 weeks after completion of the induction phase.

Upon isolation of splenocytes and in the presence of pAPC, immune responses were measured by standard techniques after in vitro stimulation with homologous peptides. The pattern of the preferred immune response is described by considering the results from multiple assays, facilitating the assessment of various effector and regulatory functions and providing a more comprehensive observation of the response. The type of assay used may be considered rather than its number; for example, one assay for a different proinflammatory cytokine plus an assay for a chemokine or for an immunosuppressive cytokine would be more beneficial than two assays for a different proinflammatory cytokine.

Example 3 ELISPOT assay of mice immunized as described in example 2

ELISPOT assay measures the frequency of cytokine-producing, peptide-specific T cells. FIG. 3 shows a repeated representative embodiment; and figure 4 presents a summary of the data expressed as number of cytokine producing cells/number of 106 effector cells, respectively. The results indicate that in contrast to mice immunized with peptides, plasmid-immunized or plasmid-primed/peptide-enhanced mice develop an increased frequency of IFN- γ (gamma) -producing T cells recognizing Melan-a peptide. Four of the four mice primed with plasmid and boosted with peptide exhibited frequencies in excess of 1/2000. In contrast, two of the four mice immunized with plasmid throughout the protocol exhibited a frequency of over 1/2000. Mice using only the peptide as an immunogen did not receive an elevated response consisting of IFN- γ -producing T cells. In fact, repeated administration of the peptide reduces the frequency of these cells, in stark contrast to administration of the peptide following plasmid priming.

Example 4 cytolytic Activity assay of mice immunized as described in example 2

Pooled (pooled) spleen cells (spleens collected, minced, red blood cells lysed) were prepared from each group and incubated for 7 days with LPS-stimulated, Melan-a peptide-coated cognate pAPC in the presence of rIL-2. Cells were washed and incubated for 4 hours at various rates with 51 Cr-labeled T2 target cells pulsed with Melan-A peptide (ELA). The radioactivity released in the supernatant was measured using a gamma counter. The response was quantified as% lysis ═ (sample signal-background)/(maximum signal-background) x 100, where background represents the radioactivity released by only target cells when incubated in assay medium, and maximum signal is the radioactivity released by target cells lysed with detergent. Figure 5 illustrates the results of the cytotoxicity assays described above. The level of cytolytic activity obtained after in vitro stimulation with the peptide was higher for those groups that had received DNA as an induction dose in vivo than for those groups that had received the peptide as an induction dose. Consistent with the ELISPOT data above, the immune response induced with the DNA composition produced a stable, scalable effector function, whereas immunization with only the peptide produced a lesser response, the values of which were further reduced upon repeated administration.

Example 5 Cross-reactivity

Splenocytes were prepared and used as in example 4 above, for target cells coated with three different peptides: melan-a analogue immunogens and those representing human and murine epitopes corresponding thereto. As shown in figure 6, similar cytolytic activity was observed on all three targets, confirming cross-reactivity of the response to the native sequence.

Example 6 repeated administration of the peptide to lymph nodes induces immune deviation and modulates T cells.

The cytokine pattern of specific T cells generated by the above immunization method was assessed by ELISA or luminex (r) (and in fig. 2). (Luminex)The assay is a method of measuring cytokine production by T cells in culture in a multiplex format. ) Supernatants from mixed lymphocyte cultures generated as described above for seven days were used to measure the following biological response modifiers: MIP-1 alpha, RANTES and TGF-beta (capture ELISA, using antibodies coated with anti-cytokines and specific reagents such as biotin-labeled antibodies, streptavidin-horseradish peroxidase anda plate of colorimetric substrate; r&D Systems). Luminex using T1/T2 and T inflammation kit supplied by professional manufacturer (BD Pharmingen)Other cytokines were measured.

The data in fig. 7A compares three different immunization protocols and shows the unexpected effect of this protocol on the immune response pattern: although the plasmid elicits T cells capable of inducing secretion of pro-inflammatory cytokines, repeated peptide administration results in the production of regulatory or immunosuppressive cytokines such as IL-10, TGF- β, and IL-5. It will be appreciated that the immunization schedule for the peptide-only protocol provides for the presence of epitopes within the lymphatic system periodically rather than continuously, which instead extends the effector phase of the response. Finally, peptide enhancement after plasmid priming resulted in increased yields of the T cell chemokines MIP-1 alpha and RANTES. T cell chemokines such as MIP-I α and RANTES may play an important role in regulating trafficking to tumors or sites of infection. During immune surveillance, T cells specific for target-associated antigens may encounter cognate ligands, proliferate and produce mediators including chemokines. These may enhance T cell recruitment at sites that recognize the antigen, resulting in a more efficient response. Data were obtained from supernatants obtained from bulk cultures (mean ± SE of replicates, two independent measurements).

Cells were recovered from lung interstitial tissue and spleen by standard methods and stained with antibodies against CD8, CD62L, and CD45RB along with tetramer reagents that recognize Melan-a-specific T cells. The data in fig. 7B represent gated populations of CD8+ tetramer + T cells (Y-axis CD45RB and X-axis CD 62L).

Together, these results demonstrate the immune bias (strong induction of reduced IFN-. gamma., TNF-. alpha.production, increased IL-10, TGF-. beta.and IL-5, CD62L-CD45 Rblob CD8+ tetramer + regulatory cells) in animals injected with peptide alone.

Example 7 by combining non-replicating plasmids (priming) with peptides(enhancement) alternative administration to lymph nodes to induce immune response with high efficiency

Three groups of HHD mice transgenic for human MHC class I hla. a2 gene were immunized by intralymphatic administration of Melan-a tumor-associated antigen. Animals were activated (induced) by direct inoculation into the inguinal lymph node with either pSEM plasmid (25. mu.g/lymph node) or ELA peptide (ELAGIGILTV (SEQ ID NO: 1), MelanA26-35A27L analog) (25. mu.g/lymph node), and a second injection three days later. Ten days later, mice were boosted with pSEM or ELA in the same manner, and three days later with a final boost to boost the response (see fig. 11A for a similar immunization program), resulting in the following induction & boost combinations: pSEM + pSEM, pSEM + ELA and ELA + ELA (12 mice per group). Ten days later, the immune response was monitored using Melan-a specific tetrameric reagent (HLA-a x 0201MARTl (ELAGIGILTV (SEQ ID NO: 1)) -PE, Beckman Coulter). One mouse was bled via the posterior orbital sinus vein and PBMCs were isolated using density centrifugation (Lympholyte Mammal, Cedarlane Labs) at 2000rpm for 25 minutes. PBMCs were co-stained with mouse-specific CD8 antibody (BD Biosciences) and Melan-a tetrameric reagent and percent specificity was determined by flow cytometry using a FACS calibre flow cytometer (BD). The percentage of Melan-a specific CD8+ cells generated by different activation/boost combinations is shown in fig. 8A and 8B. The plasmid-activated/peptide-boosted group (pSEM + ELA) elicited a strong immune response with an average tetramer percentage of 4.6 among all animals. Responding mice were defined as having a tetramer percentage of 2 or greater, which represents a number equal to the mean plus 3 standard deviations (SE) of the non-immunized control group. These values are considered in the art to be very strong responses and can generally be obtained only by using replicating vectors. The pSEM + ELA immunized group of 12 contained 10 mice as effectors and compared to the control group (p (fisher) ═ 0.036), which represents a statistically significant difference. Two other immunization series, pSEM + pSEM and ELA + ELA, resulted in 6 mice as effectors from 12 mice, but had p-values greater than 0.05, making them statistically insignificant. To measure the immunity of these mice, the animals were challenged in vivo with peptide-coated target cells. Splenocytes were isolated from littermate control HHD mice and incubated with 20. mu.g/mL ELA peptide for 2 hours. These cells were then fluorescently stained with CFSEhi (4.0 μ M for 15 min) and co-injected intravenously into immunized mice with an equal ratio of control splenocytes not incubated with peptide, fluorescently stained with CFSEIo (0.4 μ M). Specific depletion of target cells was measured 18 hours later by removing spleen, lymph nodes, PBMCs and lungs from the challenged animals (5 mice per group) and measuring CFSE fluorescence by flow cytometry. The results are shown in fig. 8C. In the pSEM + ELA activation/boost group, 4 of 5 mice showed strong immune responses and successfully cleared approximately 50% of the target in each tissue examined. Representative histograms (PBMCs) for each experimental group are also shown.

Example 8 peptide boosting effectively restores immune memory cells in animals induced by DNA and dormant to tetramer levels close to baseline.

Melan-a tetramer levels were measured in mice (5 mice per group) after immunization as described in fig. 9A. Tetramer levels returned close to baseline 5 weeks after completion of the immunization program. Animals were boosted with ELA peptides at week 6 to determine whether an immune response could be restored. Animals immunized with the previous pSEM plasmid (DNA/DNA, FIG. 9C) after ELA enhancement showed an unprecedented expansion of Melan-A specific CD8+ T cells, with levels in the range of greater than 10%. On the other hand, animals receiving prior ELA peptide (fig. 9A) injections received little benefit from ELA boosting, as shown by the lower frequency of tetramer staining cells. Mice receiving DNA, and subsequently peptide as a first immunization exhibited a significant but moderate expansion when receiving peptide boost compared to the other group. (FIG. 9B). These results clearly demonstrate the strong theoretical basis of DNA/DNA-priming and peptide-boosting immunization strategies.

Example 9 immunization was optimized to obtain a high frequency of specific T cells in lymphoid and non-lymphoid organs.

Mice subjected to priming with a series of two sets of plasmid injections, followed by peptide-boosting, developed potent immune responses, as described in fig. 9A-C. Further evidence for this is shown in fig. 10A-C, which illustrates tetramer levels before (fig. 10A) and after (fig. 10B) peptide administration. Tetramer levels in a single mouse were clearly observed and represent up to 30% of the T cell population of total CD8+ in mice receiving the DNA/peptide immunization protocol. These results are summarized in the graph of fig. 10C. In addition, high tetramer levels were evident in blood, lymph nodes, spleen and lungs of animals receiving this selected immunization protocol (fig. 10D).

A number of additional experiments have been performed to identify the phenotype of CTLs produced by this protocol. The immune pattern initiated with the conditions was labeled as peptide boosting resulted in significant expansion of CD43+, CD44+, CD69+, CD62L-, CD45RBdim, peptide-class I MHC-specific T cell populations. These specific T cells reside in non-lymphoid organs and when additionally specifically stimulated, rapidly achieve expression of CD107 a and IFN- γ in a manner dependent on the density of the stimulatory peptide complex.

Example 10 the precise order of administration of the plasmid and peptide immunogens determines the magnitude of the immune response.

Six groups of mice (n-4) were immunized with plasmids expressing Melan-a26-35 a27L analog (pSEM) or Melan-a peptide using activation and enhancement by direct inoculation into the inguinal lymph node. The immunization schedule is shown in FIG. 11A (50 μ g plasmid or peptide dose/lymph node, two-sided). Two groups of mice were activated with plasmids and boosted with plasmids or peptides. In contrast, two groups of mice were started with peptides and boosted with peptides or plasmids. Finally, two groups of control mice were initiated with peptide or plasmid but not boosted. Four weeks after the last inoculation, spleens were harvested and spleen cell suspensions were prepared, pooled and stimulated with Melan-a peptide in anti-IFN- γ antibody coated ELISPOT plates. At 48 hours after incubation, analysis was performed and the frequency of cytokine-producing T cells that recognized Melan-A was automatically counted. Data are presented in fig. 5B as the frequency of specific T cells/1 million responding cells (mean of triplicates + SD). The data indicate that reversing the sequence of plasmid and peptide initiation and enhancer amounts has a considerable effect on the overall response intensity: priming with plasmid followed by peptide potentiation produced the strongest response, whereas starting with peptide dose followed by plasmid potentiation produced significantly weaker responses, similar to repeated administration of peptide.

Example 11 correlation of immune responses to immunization protocols and in vivo efficacy as demonstrated by the clearance of target cells in lymphoid and non-lymphoid organs.

To evaluate the immune response obtained by the prime-and-boost protocol, 4 groups of animals (n-7) were challenged in vivo with Melan-a coated target cells. Splenocytes were isolated from littermate control HHD mice and incubated with 20. mu.g/mL ELA peptide for 2 hours. These cells were then fluorescent stained with CFSEhi (4.0 μ M for 15 min) and co-injected intravenously into immunized mice with an equal ratio of control splenocytes stained with CFSElo fluorescence (0.4 μ M). Specific depletion of target cells was measured 18 hours later by removing spleen, lymph nodes, PBMCs and lungs from the challenged animals and measuring CFSE fluorescence by flow cytometry. Figures 12A and 12B show CFSE histograms from tissues of non-immunized control animals or animals receiving peptide/peptide, DNA/peptide or DNA/DNA immunization protocols (two representative mice are shown per group). The DNA-prime/peptide-boost group showed high levels of specific killing of target cells in lymphoid as well as non-lymphoid organs (fig. 12C) and showed a unique immunization protocol that showed a specific correlation with tetramer levels (fig. 12D, r2 ═ 0.81 or higher for all tissues tested).

Example 12 Elimination of human tumor cells in animals immunized by a selected prime-and-boost regimen

Immunization with Melan-a antigen was further tested by challenge of mice with human melanoma tumor cells, followed by immunization with a selection protocol. Figure 13A shows the selection immunization strategy for the 3 groups tested. As illustrated in fig. 13B, immunized mice received two intravenous injections of human target cells, mixed with CFSEhi fluorescently labeled 624.38hla.a2+ and equal ratios of 624.28hla.a 2-control cells labeled with CFSElo. After 14 hours, mice were sacrificed and lungs (organs in which human target was accumulated) were analyzed by flow cytometry for specific target cell lysis. Fig. 13C shows representative CFSE histograms from each group of mice. DNA-priming followed by peptide-boosting clearly immunizes mice against human tumor cells as demonstrated by the almost 80% specific killing of the target in the lung. Longer series of DNA-primed injections also resulted in a further increased frequency of CD8+ cells reacting with Melan-a tetramer.

Example 13 DNA-priming, peptide-boosting strategy elicits a positive response to the SSX 2-derived epitope, KASEKIFYV (SSX 2) _41-49 ) The strong immunity of (2).

Immunization of animals against the SSX2 tumor associated antigen using the immunization procedure defined in fig. 14A showed a strong immune response. Figure 14B shows representative tetramer staining of mice activated (primed) with pCBP plasmid and boosted (enhanced) with SSX241-49K41F or K41Y peptide analogs. These analogs cross-react with T cells specific for SSX241-49 epitopes. These examples illustrate that the prime-and-boost regimen can result in SSX2 antigen specificity that is 80% of that of efficient CD8T cells. The pCBP plasmid and the principles of its design are disclosed in U.S. patent application No. 10/292,413 (publication No. 20030228634a1), entitled expression vector encoding a target-associated epitope and methods for its design, which are incorporated herein by reference in their entirety. Additional methods, compositions, peptides and peptide analogs are disclosed in U.S. provisional application No. 60/581,001 filed on 17.6.2004 and U.S. application No. 11/156,253 filed on 17.6.2005, both entitled SSX-2 peptide analogs; each of which is incorporated herein by reference in its entirety. Additional methods, compositions, peptides and peptide analogs are disclosed in U.S. provisional application No. 60/580,962 filed on 17.6.2004 and U.S. application No. 11/155,929 filed on 17.6.2005, each entitled NY-ESO peptide analogs; and each is incorporated by reference herein in its entirety.

Example 14 prime-and-boost strategies can be used to simultaneously elicit immune responses to epitopes located on different antigens.

Four groups of HHD mice (n ═ 6) were treated with pSEM only; only pCBP; a mixture of pSEM and pCBP; or left LNs were immunized with pSEM and right LNs were injected intranodal with pCBP. These injections were performed 10 days after the ELA or SSX2 peptides were boosted in the same manner. All immunized mice were compared to the unimmunized controls. Mice were challenged with splenocytes from HHD littermates coated with ELA or SSX2 peptides, using a trimodal CFSE in vivo cytotoxicity assay, which allows simultaneous assessment of specific lysis of both antigen targets. Equivalent control-CFSE^lo、SSX2-CFSE^medAnd ELA-CFSE^hiCells were injected intravenously into immunized mice, and 18 hours later mice were sacrificed, and the elimination of target cells was measured in spleen (fig. 15A) and blood (fig. 15B) by CFSE fluorescence using flow cytometry. Fig. 15A and 15B show the specific lysis percentage of SSX2 and Melan-a antigen targets from individual mice and fig. 15C summarizes the results in a histogram format. Immunization of animals with the mixture of the two vaccines resulted in immunity to both antigens and elicited the highest immune response, showing specific lysis for mean SSX2 percent in the spleens of 30+/-11 and 97+/-1 for Melan-A.

The induction of changes in multivalent responses, including responses to subdominant epitopes, is further exemplified in examples 24-34.

Example 15 repeated cycles of DNA priming and peptide enhancement achieve and maintain strong immunity.

Three groups of animals (n-12) received two cycles of the following immunization protocol: DNA/DNA/DNA; DNA/peptide or DNA/peptide. After each round of immunization, Melan-a tetramer levels were measured in mice and presented in fig. 16. The initial DNA/DNA/peptide immunization cycle produced 21.1+/-3.8 percent of the mean tetramer + CD8+ T cells-almost 2-fold higher than the other two groups. After the second round of prime-and-boost immunization, the mean tetramer percentage of the DNA/peptide group increased 54.5% to 32.6 +/-5.9-2.5 times higher than the DNA/peptide level and 8.25 times higher than the DNA/DNA group level. In addition, little increase in frequency of tetramer-positive T cells was obtained by other immunization programs under these conditions.

Example 16 Long-term memory B T cells were triggered by means of immune induction and enhancement presented by alternating plasmid and peptide vectors.

Four HHD transgenic animals (3563, 3553, 3561, and 3577) received the following two cycles of prime-and-boost regimen: DNA/peptide. The first cycle included immunizations at days-31, -28, -17, -14, -3, 0; the second cycle included immunizations on days 14, 17, 28, 31, 42 and 45. Mice were boosted with peptide on day 120. Melan-a tetramer levels were measured in mice periodically 7-10 days after each round of immunization and before 90 days after the second round of immunization. The arrows in the graph correspond to each cycle completion. (FIG. 17A). All four animals mount a response after the last boost, confirming the persistence of the immune memory rather than the induction of tolerance.

Five HHD transgenic animals (3555, 3558, 3566, 3598 and 3570) received the following two cycles of prime-and-boost regimen: DNA/peptide. As previously described, the first cycle includes immunization at days-31, -28, -17, -14, -3, 0; the second cycle included immunizations on days 14, 17, 28, 31, 42 and 45. Mice were boosted with peptide on day 120. Melan-a tetramer levels were measured periodically 7-10 days after each round of immunization and before 90 days after the second round of immunization (fig. 17B). In this experiment, peptides were injected in each round instead of later DNA to elicit a diminished immunological memory or reduced response by comparing the prime-and-boost regimen.

Example 17 Long-term memory T-cells with considerable expansion capacity were generated by intranodal DNA administration.

Seven HHD transgenic animals received two cycles of the following immunization protocol: DNA/DNA/DNA. The first cycle included immunizations at days-31, -28, -17, -14, -3, 0; the second cycle included immunizations on days 14, 17, 28, 31, 42 and 45. Mice were boosted with peptide on day 120. Melan-a tetramer levels were measured in mice periodically 7-10 days after each round of immunization and before 90 days after the second round of immunization. (FIG. 18). All seven animals showed a critical% frequency of tetramer + cells during and after two rounds of immunization, but mount a strong response after peptide boosting, confirming significant immunological memory.

Example 18 various combinations of antigen plus immunopotentiating adjuvant are effective for priming of CTL responses.

Intranodal administration of peptides is a very effective method of enhancing the immune response elicited by intralymphatic administration of agents (replicative or non-replicative) that include or are conjugated to adjuvants such as TLRs.

A subject (e.g., a mouse, human, or other mammal) is primed by intranodal infusion or injection of a vector such as a plasmid, virus, peptide plus adjuvant (CpG, dsRNA, TLR ligands), recombinant protein plus adjuvant (CpG, dsRNA, TLR ligands), inactivated microorganism or purified antigen (e.g., a cell wall component with immunopotentiating activity) and enhanced by intranodal injection of the peptide without adjuvant. Immune responses before and after boosting, measured by tetramer staining and other methods, showed a significant increase in intensity. In contrast, boosting by other routes with adjuvant-free peptides did not achieve the same increase in immune response.

Example 19. Intra-nodal administration of peptides is a very effective method to enhance the immune response triggered by antigen plus immunopotentiating adjuvant by any route of administration.

A subject (e.g., a mouse, human, or other mammal) is immunized by parenteral or mucosal administration of a vector such as a plasmid, virus, peptide plus adjuvant (CpG, dsRNA, TLR ligands), recombinant protein plus adjuvant (CpG, dsRNA, TLR ligands), inactivated microorganism, or purified antigen (e.g., a cell wall component with immunopotentiating activity) and enhanced by peptide intranodal injection without adjuvant. Immune responses before and after boosting, measured by tetramer staining and other methods, showed a significant increase in intensity. In contrast, boosting with adjuvant-free peptides by other non-internodal routes did not achieve the same increase in immune response.

Example 20 disruption of tolerance using prime-and-boost immunization protocols.

To break tolerance to self-antigens (e.g., tumor-associated antigens) or restore an immune response, a subject (e.g., mouse, human, or other mammal) is immunized with a vector such as a plasmid, virus, peptide plus adjuvant (CpG, dsRNA, TLR mimetics), recombinant protein plus adjuvant (CpG, dsRNA, TLR mimetics), inactivated microorganism, or purified antigen and boosted by intracorporeal injection of the adjuvant-free peptide (corresponding to the self epitope). Immune responses before and after boosting, measured by tetramer staining and other methods, showed a significant increase in the intensity of the immune response ("resistance destruction").

Example 21. clinical practice for prime-and-boost immunization.

Diagnosing a patient as in need of treatment for a tumor or infectious disease using clinical and laboratory criteria; the patient is treated or not treated with first line (first line); and to evaluation for active immunotherapy. Depending on the nature of the disease and the characteristics of the therapeutic product, participation is based on additional criteria (antigenic characteristics, MHC haplotype, immune response). Treatment was performed by intralymphatic injection or infusion (bolus, programmable pump or other method) in the precise order by the vector (plasmid) and protein antigen (peptide) (fig. 19). The most preferred protocol involves repeated cycles involving plasmid priming followed by booster doses of peptide. The frequency and duration of these cycles can be modulated according to responses measured by immunological, clinical and other methods. The composition administered may be monovalent or multivalent, comprising multiple carriers, antigens, or epitopes. Administration may be to one or more lymph nodes simultaneously or in a staggered manner. Patients receiving this treatment exhibit an improvement in symptoms.

Example 22 clinical practice of induction of immune bias or inactivation of pathogenic T cells.

Patients with autoimmune or inflammatory disorders are diagnosed with clinical and laboratory criteria, treated or not treated with first line therapy, and involve evaluation of active immunotherapy. Depending on the nature of the disease and the characteristics of the therapeutic product, participation is based on additional criteria (antigenic characteristics, MHC haplotype, immune response). Treatment is by intralymphatic injection or infusion (bolus, programmable pump or other method) in the absence of T1-adjuvant-promoting peptide and/or with immune modulators that enhance immune bias. However, regular bolus injections are the preferred means for creating immune bias by this method. Treatment with the peptide may be performed weekly, bi-weekly, or less frequently (e.g., monthly) until the desired effect on the immune or clinical state is achieved. As in group 2 of fig. 2, the treatment may comprise a single administration or multiple intensive administrations. Maintenance therapy may be performed after initiation, using a conditioning regimen that includes less frequent injections. The composition administered may be monovalent or multivalent, comprising multiple epitopes. Preferably the composition is free of any components which prolong the retention of the peptide in the lymphatic system. In addition to the appropriate clinical method, administration can be simultaneous or in a staggered fashion to one or more lymph nodes and the response monitored by measuring T cells specific for the immune peptide or for unrelated epitopes ("epitope spreading").

EXAMPLE 23 immunogenic compositions (e.g., viral vaccines)

According to the following schedule: on days 0, 3, 14 and 17, six (n ═ 6) groups of HLA-a2 transgenic mice were injected bilaterally into the inguinal lymph node with 25 μ g of plasmid vector. The vector encodes three A2 restriction epitopes from HIV gag (SLYNTVATL (SEQ ID NO: 3), VLAEAMSQV (SEQ ID NO: 4), MTNNPPIPV (SEQ ID NO: 5)), two from pol (KLVGKLNWA (SEQ ID NO: 6), ILKEPVHGV (SEQ ID NO: 7)) and one from env (KLTPLCVTL (SEQ ID NO: 8)). Two weeks after the last round of priming, mice were injected with a cocktail containing all these five peptides (5 μ g/peptide/knob three days interval on both sides). As a parallel, five groups of mice were injected with a single peptide (two-sided 5 μ g/peptide/knot three days apart). Seven days later, mice were bled and responses were assessed by tetramer staining for each peptide. Then, half of the mice were challenged with recombinant vaccinia virus expressing env, gag or pol (103TCID 50/mouse) and at day 7, viral titers were measured in the ovaries by using a conventional plaque assay. The other half was sacrificed, spleen cells were stimulated with peptide for 5 days and cytotoxic activity was measured on target cells coated with peptide. As a control, mice were injected with plasmid or peptide only. Mice primed with plasmid and boosted with peptide showed stronger immunity to all five peptides by tetramer staining and cytotoxicity.

More generally, to break tolerance to non-self antigens such as viruses, bacteria, parasites or microorganisms, restore an immune response or induce immunity, a subject (e.g., a mouse, human or other mammal) is immunized with a vector such as a plasmid, virus, peptide plus adjuvant (CpG, dsRNA, TLR mimetics), recombinant protein plus adjuvant (CpG, dsRNA, TLR mimetics), inactivated microorganism or purified antigen (e.g., a cell wall component) and boosted by intracompartmental injection of the peptide without adjuvant (corresponding to the target epitope). Immune responses before and after boosting, measured by tetramer staining and other methods, showed a significant increase in the intensity of the immune response. The strategies can be used to protect against infection or to treat chronic infections caused by pathogens such as HBV, HCV, HPV, CMV, influenza virus, HIV, HTLV, RSV, and the like.

Example 24. with two plasmids: immunization schedule of pCBP expressing SSX241-49 and pSEM expressing Melan-A26-35 (A27L).

As shown in fig. 20, two groups of HHD mice (n ═ 4) used pSEM and pCBP as a mixture; or the left inguinal lymph node was immunized twice on days 0 and 4 with intraparenchymal injection of pSEM and the right inguinal lymph node with pCBP. The amount of plasmid was 25. mu.g/plasmid/dose. After two weeks, animals were sacrificed and cytotoxicity was measured against T2 cells, with or without peptide delivered into the T2 cells in a pulsed manner.

Example 25 vector isolation rescues the immunogenicity of suboptimal epitopes.

Animals immunized as described in example 24 were sacrificed, spleen cells pooled and grouped and stimulated in parallel with one of the two peptides, Melan-A26-35(A27L) or SSX 241-49. Cytotoxicity was measured by incubation with 51 Cr-loaded, peptide-pulsed-in T2 target cells. The data in figure 21 shows the average value of specific cytotoxicity (n-4/panel) against various target cells.

The results indicate that the use of the plasmid mixture interferes with the response elicited by the pCBP plasmid; however, isolation of the two plasmids at the site of administration rescues the activity of pCBP. Co-administration of different vectors carrying different antigens leads to the establishment of a hierarchy involving immunogenicity. Vector isolation rescues the immunogenicity of suboptimal components, producing multivalent responses.

Example 26 adding a peptide boosting step to the immunization protocol.

As shown in fig. 22, pSEM and pCBP were used as a mixture; or the left inguinal lymph node was injected intranodal with pSEM and the right inguinal lymph node with pCBP to immunize four groups of HHD mice (n ═ 6) twice on day 0 and day 4. As a control, mice were immunized with either pSEM or pCBP plasmid only. The amount of plasmid was 25. mu.g/plasmid/dose. Two weeks later on day 14 and day 17, animals were boosted with Melan-a and/or SSX2 peptides, in response to plasmid immunization with dose and combination. Two weeks later on day 28, animals were stained with CFSE and challenged with splenocytes fired (pulse) with or without Melan-a (ela) or SSX2 peptide pulses for assessment of cytotoxicity in vivo.

Example 27. peptides enhance the immunogenicity to rescue sub-optimal epitopes even when the carrier and peptide are used separately as a mixture.

Animals were immunized as described in example 26 and challenged with splenocytes from HHD littermates coated with ELA or SSX2 peptide, using a trimodal CFSE in vivo cytotoxicity assay, which allowed simultaneous assessment of specific lysis of both antigen targets. Equivalent control-CFSE^lo、SSX2-CFSE^medAnd ELA-CFSE^hiCells were infused intravenously into immunized mice, and after 18 hours mice were sacrificed, and target cell depletion in the spleen (fig. 23) was measured by CFSE fluorescence using flow cytometry. The figure shows the percentage of SSX2 and Melan-a antigen target specific lysis from a single mouse, mean and SEM for each group.

Interestingly, immunization of animals with a mixture comprising two vaccines, plasmid first and peptide second, resulted in immunization against both antigens and the highest immune response, showing the average SSX2 specific lysis percentages in the spleen of 30+ -11 and 97+ -1 for Melan-A. Thus, as illustrated in fig. 23, peptides enhance immunogenicity that can rescue sub-dominant epitopes even when the carrier and peptide are used separately as a mixture.

Example 28. clinical practice for prime-and-boost immunization.

Figure 24 shows two protocols for inducing a strong multivalent response: in the first protocol (a), even if plasmids and peptides are used as a mixture, multivalent immune responses are restored using the peptides for enhancement. In the second protocol (B), plasmid and peptide component separation provides induction of multivalent immune responses, respectively. The peptide is preferably administered to the same lymph node to which the priming plasmid for the common epitope is administered. This is not absolutely required, however, because T memory cells lose CD62L expression and therefore reside in other lymphoid organs. The time interval between initiation and enhancement shown in fig. 24 is suitable but not considered critical. Substantially less preferred shorter time intervals and longer time intervals are fully acceptable.

Example 29. Single plasmid elicits multivalent response.

Plasmid pSEM, described in FIG. 25 and the tables below, contains multiple peptides from two different antigens (Melan-A and tyrosinase) in close proximity together in the open reading frame ("synchronized polypeptide coding sequence"). It therefore has the potential to express and induce immunity against more than a single epitope. The encoded peptide sequence is as follows: 1-9 parts of tyrosinase; Melan-A/MART-126-35 (A27L); tyrosinase 369-; and Melan-A/MART-131-96.

The cDNA sequence of the polypeptide in the plasmid is under the control of a promoter/enhancer sequence from cytomegalovirus (CMVp), which allows for efficient transcription of the polypeptide message when absorbed by antigen presenting cells. The bovine growth hormone polyadenylation signal (BGH polyA) at the 3 'end of the coding sequence provides a messenger's polyadenylation signal to enhance its stability and transfer from the nucleus into the cytoplasm. To facilitate plasmid transport into the nucleus, a Nuclear Import Sequence (NIS) from simian virus 40 is inserted into the plasmid backbone. One copy of the CpG immunostimulatory motif was designed into the plasmid to further boost the immune response. Finally, two prokaryotic genetic elements in the plasmid are responsible for the amplification of the kanamycin resistance gene (Kan R) and the pMB bacterial origin of replication in e. Further description of pSEM can be found in U.S. patent application No. 10/292,413, in which pSEM is variously named pMA2M and pVAXM3, incorporated by reference above.

Example 30 protocol for "rescuing" or enhancing immune responses to suboptimal epitopes after challenge by using multivalent carriers.

A well-known limitation of vectors co-expressing therapeutically relevant epitopes is that within the newly designed environment, one epitope will play a major role with respect to immune induction, while the rest will be minor (especially when the epitope is bound to the same MHC restriction element).

In fig. 26, such a scheme is depicted as: eight groups of HHD mice (n ═ 4) were immunized with pSEM by intralymph node injection on days 0, 3, 14, and 17. The amount of plasmid was 25. mu.g of plasmid per dose. On days 28 and 31, mice were administered intranodal enhancement peptides corresponding to Melan-A26-35 (FIG. 27A) or tyrosinase 369-377 (FIG. 27B), also at 25 μ g peptide/dose. Immune responses were measured by tetramer staining of CD8+ T cells in peripheral blood using Melan-a or tyrosinase specific reagents two weeks after immunization was completed.

The results in FIG. 27 show that activation with pSEM caused a significant response to Melan-A, but not tyrosinase. In parallel, animals immunized with peptide alone did not show a detectable tetrameric response to either epitope. Together, these data suggest that the Melan-A epitope plays a major role in immunity associated with tyrosinase epitopes. However, after boosting with tyrosinase ("native peptide"), the immune response to tyrosinase (fig. 27B, first group) was similar in intensity compared to the levels obtained for Melan-a (fig. 27A, second and fourth groups) in animals immunized with Melan-a peptide after activation with pSEM.

In summary, intralymphatic administration of the tyrosinase peptide rescued the immune response to this epitope initiated by pSEM, overcoming the secondary nature associated with Melan-a epitopes in the context of the vector used to initiate the response (pSEM).

Example 31. protocol for "rescuing" or enhancing immune responses to suboptimal epitopes by using multivalent vectors after priming: evaluation of cytotoxic immunity.

Immunization was performed as described in example 30: eight groups of HHD mice (n ═ 4) were immunized with pSEM by lymph node injection on days 0, 3, 14, and 17. The amount of plasmid was 25. mu.g/dose. On days 28 and 31, mice were immunized with peptides administered to the lymph nodes (25 μ g peptide/dose) corresponding to Melan-A26-35 (FIG. 28A) or tyrosinase 369-377 (FIG. 28B). Following ex vivo re-stimulation of spleen cells with Melan-a or tyrosinase epitope peptides, immunity was assessed by cytotoxicity analysis 14 days after completion of immunization. In summary, splenocytes were prepared (spleens collected, minced, red blood cells lysed) and incubated for 7 days with LPS-stimulated, Melan-a (fig. 28A) or tyrosinase (fig. 28B) peptide-coated homologous pAPC in the presence of rIL-2. Cells were washed and incubated with 51 Cr-labeled Melan-A +, tyrosinase +624.38 target cells at various ratios for 4 hours. Radioactivity released into the supernatant was measured using a gamma counter. Responses were quantified as% lysis ═ (sample signal-background)/(maximum signal-background) x 100, where background represents the radioactivity released by only target cells when incubated in assay medium, and maximum signal is the radioactivity released by target cells lysed with detergent.

As in example 30, the results of fig. 28 demonstrate that boosting rescue/boost immunity to an epitope (tyrosinase) by an internodal peptide, which is secondary in the context of the immune initiation vector (pSEM).

Example 32. protocol for co-inducing and enhancing immune responses to two epitopes-a dominant and a subdominant epitope simultaneously within the starting vector environment.

In the previous two examples, rescue of suboptimal epitope responses in the absence of enhancement of dominant epitope responses was demonstrated. Next, an attempt was made to enhance both responses simultaneously.

In fig. 29, the scheme is described as: four groups of HHD mice (n ═ 6) were immunized with pSEM by intralymph node injection on days 0, 3, 14, and 17. The amount of plasmid was 25. mu.g/dose. On days 28 and 31, mice were simultaneously immunized with peptides corresponding to the epitopes Melan A26-35 (left inguinal lymph node) and tyrosinase 369-377 (right inguinal lymph node) at 25 μ g of peptide per dose. Immune responses were measured by tetramer staining of CD8+ T cells in peripheral blood using Melan-a (fig. 30A) or tyrosinase (11B) specific reagents two weeks after immunization was completed. Data are expressed as mean% tetramer + cells within the CD8+ subgroup. Activated with the pSEM plasmid and with the peptide analog Melan A26-35A27 Nva { E (Nva) AGIGILTV as determined by multicolor tetramer staining; SEQ ID NO: 9 (left lymph node) and tyrosinase 369-377V377Nva { YMDGTMSQ (Nva); SEQ ID NO: 10} (right lymph node) enhanced animals showed multivalent immune responses specific for each epitope (fig. 30C). A dot plot was plotted for total CD8 positive cells from peripheral blood and represents the competing immune response in a single mouse. Tetramer levels were calculated as the percentage of CD8 positive T cells.

The results in figure 30 show that by co-administration of Melan a and tyrosinase peptide, one can co-enhance the immune response to both Melan a and tyrosinase epitopes, both in a dominant/suboptimal relationship in the context of the immune initiation vector (pSEM).

Example 33 utilization of peptide mixtures in the starting vector environment co-induces and enhances cytolytic responses to two epitopes-one dominant and one subdominant epitope.

To further investigate the simplified product formulation, an alternative approach was tested, combining the use of bivalent plasmids expressing dominant and subdominant epitopes, followed by enhancement of the response to each epitope by administration of a mixture of dominant and subdominant peptides rather than administration of the peptides individually-as described in the previous examples.

Six groups of HHD mice (n ═ 6) were immunized with pSEM plasmid (or not immunized, respectively) in the lymph nodes as described in the previous examples, and boosted with peptides (as a mixture of Melan-a + various tyrosinase peptides) at a dose of 12.5 μ g/peptide/dose, as per the schedule: plasmid was used on days 0 and 3; the cycle was repeated two weeks later with peptide on days 14 and 17. The tyrosinase peptides used were: tyr 369-; tyr 1-9, which is encoded by a plasmid but not presented by transformed cells; and Tyr 207-215, which is not encoded by a plasmid.

The immune response was measured by CFSE assay two weeks after completion of the immunization protocol as described above. Briefly: splenocytes were isolated from littermate control HHD mice and incubated with 20. mu.g/mL ELA or 20. mu.g/mL tyrosinase peptide for 2 hours. These cells were subsequently treated with CFSE^hiAnd CFSE^medFluorescent staining and ratiometric staining with CFSE^loFluorescently stained control splenocytes were co-injected intravenously into immunized mice. Spleens were removed after 18 hours and specific depletion of target cells was measured using flow cytometry and% in vivo specific lysis was calculated by the following equation:

{[1-(％CFSE^{hi or med}/％CFSE^lo)]-[1-(％CFSE^{hi or med}Control/% CFSE^loControl)]}x100

Where each% term in the equation represents the proportion of the total sample represented by each peak.

Overall, the results presented in figure 31 (specific lysis of Melan-a epitope-coated or tyrosinase epitope-coated splenocytes in vivo;% X-axis depicts the peptides used for boosting) demonstrate that the use of peptide mixtures in the boosting phase of the plasmid priming/peptide boosting regime results in co-boosting of immunity to the dominant (Melan-a) and subdominant (tyrosinase 369-377) epitopes. Furthermore, the use of peptides alone does not produce an effective response. For this combination of peptides, a significant response to both epitopes was obtained. However, it should be noted that the expectation for success from a mixture of peptides is greater when the MHC-binding affinities of the various peptides are similar, while the expectation for success decreases as the affinities become more disparate.

Example 34 Induction of a response with higher multivalence

In this study, immunization was induced with two bivalent plasmids and enhanced with four peptide epitope analogs. As before, plasmid pSEM was used to induce immunity to Melan-A and tyrosinase epitopes and to enhance the response using the analogs Melan-A (A27Nva) and tyrosinase (V377 Nva). Plasmid pBPL was also used to induce immunity to sites SSX241-49, NY-ESO-1157-165. Immunogenic polypeptides encoded by pBPL are disclosed in U.S. patent application 10/292,413 (publication No. 20030228634A1), entitled expression vectors encoding target-associated epitopes and methods for their design, which are incorporated herein by reference in their entirety. Enhanced utilization of peptide epitope analogs SSX241-49(A42V) and NY-ESO-1157-165 (L158Nva, C165V). Further discussion of epitope analogs is provided in the epitope analog applications cited and set forth above by reference. These analogs typically have superior affinity and stability for binding to MHC compared to native sequences, but cross-react with TCRs that recognize native sequences.

Three groups of female HHD-A2 mice were treated with a mixture of pSEM/pBPLImmunisation was performed bilaterally to the inguinal lymph node (100. mu.g of each plasmid per day; 25. mu.l per injected node). Throughout the protocol, group 1(n ═ 10) received only plasmids, and injections were performed on days 1, 4, 15, 18, 28, 32, 49, and 53. Groups 2 and 3 (n-25 per group) received plasmid injections on days 1, 4, 15 and 18 and peptide injections on the following day. On day 25, blood was collected from the immunized animals and CD8 was analyzed by flow cytometry using MHC-tetramer assay⁺T cells. Responses were compared to naive littermate control mice (n ═ 5).

Mice in group 2 were boosted by administration of the peptide tyrosinase V377Nva (25 μ g/day) to the right lymph node and SSX2a42V (25 μ g/day) to the left lymph node on days 28, 32, 49 and 53. Group 3 animals were boosted by administering the peptide tyrosinase V377Nva (25. mu.g/day) to the right lymph node and SSX2A42V (25. mu.g/day) to the left lymph node on days 28 and 32, followed by NY-ESO-1L158Nva, C165V (12.5. mu.g/day) to the right lymph node and Melan-A A27Nva (25. mu.g/day) to the left lymph node on days 49 and 53. All injections were 25. mu.l/knot injected. On days 39 and 60, blood was collected from each group and analyzed for CD8+ T cells using the tetramer assay. Responses were compared to naive littermates (n-5).

At days 41 and 63, selected animals from each group were sacrificed and spleens were removed for IFN γ ELISPOT analysis of spleen cell suspensions.

On day 62, selected animals from each group received an intravenous injection of CFSE-labeled 624.38 human melanoma cells, which expressed all four tumor-associated antigens, and were used as targets for SSX2, NY-ESO-1, tyrosinase, and Melan a-specific CTLs in immunized mice.

Plasmids were formulated in clinical buffer (in H)₂127mM NaCl, 2.5mM Na in O₂HPO₄，0.88mM KH₂PO₄，0.25mM Na₂EDTA, 0.5% ETOH; 2mg/ml of each plasmid, 4mg/ml total). Melan-A26-35(A27Nva), tyrosinase 369-377(V377Nva) and SSX241-49(A42V) analogs were formulated at 1.0mg/mlIn PBS. NY-ESO 157-165(L158Nva, C165V) peptide analogs were prepared for immunization in PBS containing 5% DMSO at a concentration of 0.5 mg/ml. Cytometric data were collected using a BD FACS Calibur flow cytometer and analyzed by gating lymphocyte populations using CellQuest software. PBMCs were purified using FITC-conjugated rat anti-mouse CD8a (Ly-2) monoclonal antibody (BD Biosciences, 553031) and MHC tetramers: HLA-A0201 SSX2(KASEKIFY (SEQ ID NO: 11)) -PE MHC tetramer (Beckman Coulter, T02001), HLA-A0201 NY-ESO (SLLMWITQC) (SEQ ID NO: 12) -APC MHC tetramer (Beckman Coulter, T02001), HLA-A0201 Melan-A (ELAGIGILTV (SEQ ID NO: 1)) -PE MHC tetramer (Beckman Coulter, T02001) or HLA-A0201 tyrosinase (YMTMSQV (SEQ ID NO: 13)) -APCMHC tetramer (Beckman Coulter, T02001) co-stain.

IFN-. gamma.ELISpot analysis was performed as follows. Spleens were removed from the euthanized animals on days 27 and 62, and monocytes were isolated by density centrifugation (Lympholyte Mammal, Cedarlane Labs) and resuspended in HL-1 medium. Spleen cells (5 or 3X10 per well)⁵Cells) were incubated with 10. mu.g of Melan-A26-35A 27L, tyrosinase 369-377, SSX241-49 or NY-ESO-1157-165 peptide in triplicate in 96-well filter plates (Multiscreen IP membrane 96-well plates, Millipore). Samples were incubated at 37 ℃ with 5% CO prior to development₂And incubation at 100% humidity for 42 hours. Mouse IFN- γ coated antibody was used to coat the filter prior to incubation with splenocytes and biotinylated detection antibody was added to develop the signal after lysing the cells and washing the cells with water from the filter (IFN- γ antibody pair, Ucytech). GABA conjugates from Ucytech and proprietary substrates were used for IFN- γ spot visualization. CTL responses in immunized animals were measured 24 hours after development on an AID International plate reader using ELISpot reading software version 3.2.3, which calibrates the IFN- γ spot assay.

In vivo cytotoxicity assays were performed on day 61 as follows. Human 624.38(HLA A0201)^pos) CFSE for cultured melanoma tumor cells^hi(Vybrant CFDA SE cell tracer kit, molecular probes) fluorescent staining (1.0. mu.l)M15 min), 624.28HLA-a2(HLAA 0201)^neg) Using CFSE^loFluorescent staining (0.1. mu.M for 15 min). Two mice from each group (groups 1,2 and 3) selected on the basis of high tetramer levels and received intravenous injection with equivalent amounts of CFSE^loLabeled 624.28(HLAA 0201)^neg) Mixed 20x10⁶CFSE^hiLabeled 62438(HLAA 0201)^pos) 2 naive control mice of human melanoma cells were divided into two aliquots delivered at 2 hour intervals. HLAA 0201 is determined after about 14 hours by killing mice, removing lung tissue, preparing a single cell suspension and measuring CFSE fluorescence by flow cytometry^posSpecific elimination of human target cells. The percentage of specific lysis was calculated as indicated above.

The immune response obtained was evaluated at various points in the protocol. Figure 32 shows the response obtained 7 days after the fourth plasmid injection as judged by tetramer analysis, which is common to all three groups. Significant responses were observed for all but the tyrosinase epitope. Melan-A26-35 and NY-ESO-1157-165 were revealed as dominant epitopes and the response to suboptimal epitopes was enhanced by applying tyrosinase V377Nva and SSX2A42V peptide epitope analogs to groups 2 and 3 in order to generate a more balanced tetravalent immune response. Group 1 received another round of immunization with the plasmid mixture. Further immunization with plasmids (group 1) as shown in fig. 33 boosted the response to the dominant epitope only, whereas administration of peptides corresponding to two subdominant epitopes produced a significant and more balanced response to all four epitopes. Figure 34 shows the response of selected individual animals, indicating that a true tetravalent response can be generated. IFN- γ ELISpot analysis of a subset of mice sacrificed at day 27 confirmed the conventional pattern observed from tetramer data (fig. 35A). Another group of mice were sacrificed at day 62 after further rounds of boosting on day 59 and subjected to IFN- γ ELISpot analysis (FIG. 35 b). The plasmid mixture was reused for the last round of immunization of group 1 and the pattern of response remained similar to that observed after the earlier round. Only those peptides corresponding to sub-optimal epitopes (group 2) were used to maintain a relatively balanced response to the four epitopes. Peptides corresponding to all four epitopes were administered in group 3. Although that is still observedThe significant response of these epitopes, the degree of dominance of Melan-A epitopes, is again manifested at a significant loss of response to tyrosinase epitopes. It should be noted that the absolute intensity of the responses described in fig. 35A and B are not directly comparable because of the conventional responses of the groups of sacrificed animals at two different time points. Cytolytic activity in vivo was also assessed by challenge with CFSE labeled human tumor cells expressing all four target antigens. These tumor cells are derivatives of cell line 624.38, which naturally express SSX2, PRAME, tyrosinase, and Melan-A, which have been transformed with plasmid vectors to similarly stably express NY-EOS-1. As would be expected in naive mice analyzed by ELISpot for only background levels of tetramer or IFN- γ responses, with HLA-a2^-In contrast to control, HLA-A2 was absent⁺Specific depletion of tumor cells (fig. 36A). However specific depletion was observed in mice with significant tetravalent responses and the more balanced the responses obtained the better the results. Epitope-specific responses of FIGS. 36B (71% specific lysis) and 36C (95% specific lysis) seen by tetramer and ELISpot analysis were compared. No specific lysis was observed in mice with a significant monovalent response. In vivo cytotoxicity due to a monovalent response is seen above (in example 7), but the target cells in this experiment have significantly stronger epitope expression. Thus, it is seen here that multivalent responses overcome the protective effect of low target antigen expression levels.

Example 35 general procedure for the induction of multivalent immunity.

The method may include the following steps (depicted in fig. 37):

epitopes from different antigens or the same antigen are identified. The epitopes may have an advantage/disadvantage relationship relative to each other (e.g., due to widely varying degrees of expression or presentation, TCR skill bias, etc.), or be co-dominant in their natural environment.

The sequences associated with the epitopes are recovered and expression vectors containing these epitopes in the same reading frame or in the same vector are designed. The new environments may establish or alter the relationship of immune superiority/inferiority with each other compared to their natural environment.

Immunization with a vector that results in an initial response may predominate one specificity (dominant epitope) over another.

The response to a subdominant epitope is enhanced by administration of the corresponding peptide. The peptide may be a native sequence or an analogue thereof. The peptide may be administered to the same site, or more preferably to a different site, alone or simultaneously with other peptides corresponding to dominant and/or subdominant epitopes.

Any of the methods described in the examples and elsewhere herein can be and are modified to include different compositions, antigens, epitopes, analogs, and the like. For example, any other cancer antigen may be utilized. In addition, many epitopes are interchangeable, and epitope analogs including those disclosed, described or introduced herein can be utilized. The methods are useful for generating immune responses, including multivalent immune responses to various diseases and disorders.

Many modifications and alternative elements of the invention have been disclosed. Further modifications and alternative elements will be apparent to those skilled in the art. Various embodiments of the invention may explicitly include or exclude any such variations or elements.

Each reference cited herein is incorporated by reference in its entirety.

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Claims (49)

1. Use of a first immunogen, a second immunogen and a first peptide in the manufacture of a kit for immunizing a mammal,

wherein a first immunogen is comprised in a first composition and a second immunogen is comprised in a second composition, the first immunogen encoding at least a portion of the immunogenicity of a first antigen and the second immunogen encoding at least a portion of the immunogenicity of a second antigen; and wherein

The first peptide is contained in a third composition for direct administration to the lymphatic system of said mammal, wherein said first peptide corresponds to an epitope of said first antigen, wherein said third composition is different from said first or second composition.

2. The use of claim 1, wherein the first and second compositions are the same.

3. The use of claim 2, wherein the first and second immunogens are comprised in a single macromolecule.

4. The use of claim 1, wherein the first and second compositions are delivered as a mixture.

5. The use of claim 1, said kit further comprising a fourth composition comprising a second peptide for subsequent direct administration to the lymphatic system of said mammal, wherein said second peptide corresponds to an epitope of said second antigen, wherein said fourth composition is different from said first or second composition.

6. The use of claim 5, wherein said third and fourth compositions each comprise said first and second peptides.

7. The use of claim 1, wherein the first and second compositions are for delivery to separate sites.

8. The use of claim 5, wherein the first and second peptides are for administration to separate sites.

9. The use of claim 1, wherein the first composition and the third composition are for delivery to the same site.

10. The use of claim 5, wherein the second composition and the fourth composition are for delivery to the same site.

11. The use of claim 5, wherein the first and second peptides are for simultaneous administration.

12. The use of claim 5, wherein the first and second peptides are for administration on different days.

13. The use of claim 1, wherein said first antigen is selected from the group consisting of tyrosinase, Melan-A, SSX-2, NY-ESO-1, PRAME, PSMA, VEGFR2, VEGF-a, and PLKl, and said second antigen is selected from the group consisting of tyrosinase, Melan-A, SSX-2, NY-ESO-1, PRAME, PSMA, and VEGFR 2.

14. The use of claim 1, wherein direct administration to the lymphatic system comprises administration to the inguinal lymph node.

15. Use according to claim 1, wherein the immunization comprises induction of a CTL response.

16. Use according to claim 1, wherein the first or second composition comprises the same epitope peptide as the first peptide, and wherein the third composition differs from the first or second composition at least by comprising a larger dose of the epitope peptide.

17. The use of claim 1, wherein at least one of the first or second compositions is for delivery with an immunopotentiator.

18. The use of claim 17, wherein at least one of the first composition or the second composition comprises the immunopotentiator.

19. The use of claim 1, wherein the first and second compositions and the third composition are used in repeated cycles of immunization.

20. The use of claim 19, wherein the cycling is repeated for a sufficient time to maintain an immune response effective to fulfill a medical need.

21. The use of claim 20, wherein the cycling repeats increase the multivalency of the immune response.

22. A panel of immunogenic compositions for inducing an immune response in a mammal, comprising 1 or more priming doses and at least 1 boosting dose for each of 2 or more antigens, wherein the priming doses for each antigen comprise a nucleic acid encoding an immunogen and further comprise an immunopotentiator, wherein the immunogen comprises at least a portion of the immunogenicity of the antigen, and wherein the boosting doses comprise peptides for subsequent direct delivery to the lymphatic system of the mammal, wherein the one or more priming and boosting doses are different, wherein the peptides correspond to epitopes of the immunogen.

23. The panel of claim 22, where at least one of the compositions is multivalent.

24. The panel of claim 22, where the nucleic acid encoding the immunogen further comprises an immunostimulatory sequence that acts as an immunopotentiator.

25. The panel of claim 22, where the immunopotentiator is selected from the group consisting of TLR ligands, immunostimulatory sequences, CpG-containing DNA, dsRNA, endocytosis-Pattern Recognition Receptor (PRR) ligands, LPS, quillaja saponin, tocarol, and pro-inflammatory cytokines.

26. The panel of claim 22, where the amount of the priming agent is suitable for intranodal delivery.

27. The panel of claim 26, where at least one of the priming doses comprises a nucleic acid.

28. The panel of claim 27, where the daily dosage of nucleic acids is 25-2500 μ g.

29. The panel of claim 27, where the booster dose is 5 to 5000 μ g peptide per kilogram of indicated receptor.

30. Use of a first immunogen, a second immunogen, and a first peptide in the manufacture of a medicament comprising a set of immunogenic compositions for eliciting and enhancing an immune response, wherein the medicament comprises:

a first composition comprising the first immunogen, wherein the first immunogen encodes at least a portion of the immunogenicity of a first antigen;

a second composition comprising the second immunogen, wherein the second immunogen encodes at least a portion of the immunogenicity of a second antigen; and

a third composition comprising the first peptide for administration directly to the lymphatic system after the first and second compositions, wherein the first peptide corresponds to an epitope of the first antigen, wherein the third composition is not identical to the first or second composition.

31. The use of claim 30, wherein the first and second compositions are the same.

32. The use of claim 31, wherein the first and second immunogens are comprised in a single macromolecule.

33. The use of claim 30, wherein the medicament further comprises a fourth composition for administration directly to the lymphatic system after the first and second compositions, wherein the fourth composition comprises a second peptide corresponding to an epitope of the second antigen, and wherein the fourth composition is not identical to the first or second composition.

34. The use of claim 33, wherein the third and fourth compositions each comprise the first and second peptides.

35. The use of claim 30, wherein the first or second composition further comprises an immunopotentiator.

36. The use of claim 33, wherein the first and second compositions are for delivery to separate sites.

37. The use of claim 33, wherein the third and fourth compositions are for administration to separate sites.

38. The use of claim 33, wherein the first immunogen is for delivery to the same site as the first peptide is delivered to.

39. The use of claim 33, said first and second peptides for administration at the same time.

40. The use of claim 33, said first and second peptides for administration on different days.

41. The use of claim 30, wherein said first antigen is selected from the group consisting of tyrosinase, Melan-A, SSX-2, NY-ESO-1, PRAME, PSMA, VEGFR2, VEGF-a, and PLKl, and said second antigen is selected from the group consisting of tyrosinase, Melan-A, SSX-2, NY-ESO-1, PRAME, PSMA, and VEGFR 2.

42. The use of claim 30, wherein the immune response comprises a CTL response.

43. Use of a set of immunogenic compositions in the manufacture of a kit for eliciting and enhancing an immune response in a mammal,

wherein the first composition comprises a first immunogen encoding at least a portion of the immunogenicity of the first antigen and the second composition comprises a second immunogen encoding at least a portion of the immunogenicity of the second antigen,

and wherein a third composition comprises a first peptide for subsequent direct administration to the lymphatic system of said mammal, wherein said first peptide corresponds to an epitope of said first antigen, wherein said third composition is different from said first or second composition.

44. Use of a panel of immunogenic compositions comprising

One or more priming doses for each of the two or more antigens; and

at least one of the booster doses is present,

wherein the priming dose for each antigen comprises a nucleic acid encoding the immunogen and further comprises an immunopotentiator, wherein the immunogen comprises at least a portion of the immunogenicity of the antigen; and wherein the booster dose comprises a first peptide administered directly to the lymphatic system of the mammal, wherein the peptide corresponds to an epitope of a first antigen of the two or more antigens.

45. Use of a first immunogen, a second immunogen and a first peptide in the manufacture of a kit for immunizing a mammal,

wherein the first immunogen is comprised in a first composition and the second immunogen is comprised in a second composition, the first immunogen comprising at least a portion of the immunogenicity of a first antigen, the second immunogen comprising at least a portion of the immunogenicity of a second antigen; and wherein

The first peptide is contained in a third composition that is then used for direct administration to the lymphatic system of the mammal, wherein the first peptide corresponds to an epitope of the first antigen, wherein the third composition is different from the first or second composition, wherein at least one of the first or second composition is used for delivery with an immunopotentiator.

46. A panel of immunogenic compositions for inducing an immune response in a mammal comprising 1 or more priming doses and at least 1 boosting dose for each of 2 or more antigens, wherein the priming doses for each antigen comprise an immunogen and further comprise an immunopotentiator, wherein said immunogen comprises at least a portion of the immunogenicity of said antigen; and wherein the booster dose comprises a peptide for subsequent direct administration to the lymphatic system of said mammal, wherein said 1 or more priming and booster doses are different, wherein said peptide corresponds to an epitope of said immunogen.

47. Use of a first immunogen, a second immunogen and a first peptide in the manufacture of a medicament comprising a set of immunogenic compositions for inducing and enhancing an immune response, wherein the medicament comprises:

a first composition comprising the first immunogen, wherein the first immunogen comprises at least a portion of the immunogenicity of a first antigen;

a second composition comprising the second immunogen, wherein the second immunogen comprises at least a portion of the immunogenicity of a second antigen; and

a third composition comprising a first peptide for administration directly to the lymphatic system after the first and second compositions, wherein the first peptide corresponds to an epitope of the first antigen, wherein the third composition is different from the first or second composition;

wherein at least one of the first composition or the second composition is for delivery with an immunopotentiator.

48. Use of a set of immunogenic compositions in the manufacture of a kit for inducing and enhancing an immune response in a mammal, wherein the first composition comprises a first immunogen comprising at least a portion of a first antigen that is immunogenic and the second composition comprises a second immunogen comprising at least a portion of a second antigen that is immunogenic; and wherein a third composition comprises a first peptide for subsequent direct administration to the lymphatic system of the mammal, wherein the third composition is different from the first or second composition, and wherein at least one of the first or second composition is for delivery with an immunopotentiator.

49. Use of a panel of immunogenic compositions in the manufacture of a medicament for inducing an immune response in a mammal, comprising:

1 or more priming doses for each of 2 or more antigens; and

at least 1 part of the enhancer amount,

wherein the priming dose for each antigen comprises an immunogen and additionally an immunopotentiator, wherein the immunogen comprises at least a portion of the immunogenicity of the antigen; and wherein the booster dose comprises a first peptide administered directly to the lymphatic system of the mammal, wherein the peptide corresponds to an epitope of a first antigen of the 2 or more antigens.