WO2008151197A2 - Tumor-derived endogenous toll-like receptor 4 ligands - Google Patents

Abstract

Disclosed herein are proteins, compositions and methods for inducing dendritic cell activation, maturation, polarization, and/or filamentous actin rearrangement in immature dendritic cells, inducing immune responses in subjects, targeting or a dendritic cell for phagocytosis or TLR4 signaling, and reversing, inhibiting, or reducing immune suppression by a cancer in a subject.

Description

A FAMILY OF TUMOR-DERIVED ENDOGENOUS TOLL-LIKE RECEPTOR 4 LIGANDS AND METHODS OF MAKING AND USING THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[01] This application claims the benefit of U.S. Provisional Application Serial No.

60/941,712, filed 4 June 2007, pending, and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION.

[02] The present invention generally relates to a tumor derived molecular adjuvant family and their use in compositions and methods.

2. DESCRIPTION OF THE RELATED ART.

[03] Pattern-recognition receptors such as the Toll-like receptors (TLR), distinguish infectious non-self from noninfectious self and control adaptive immune responses by modulating the innate immune response, such as inducing maturation of dendritic cells (DC). Depending on the pathogen-associated molecular patterns (PAMP), specific TLR of the innate immune system can induce distinct cellular responses via intracellular adaptor proteins. For example, TLR4 recognizes lipopolysaccaride (LPS) on the cell wall of Gram-negative bacteria and induces inflammatory cytokines, including IL-6, TNF, and IL- 12, through intracellular adaptors called MyD 88 and TIRAP. Direct interactions of PAMP and TLR on the surface of the adaptive immune system, such as B cells and CD4⁺CD25⁺ T regulatory cells, also had powerful immune modulatory functions. See Peng, G., et al. (2005) Toll-like receptor 8-mediated reversal of CD4+ regulatory T cell function. Science 309(5739): 1380-4. Recently, the involvement of TLR was indicated in susceptibility to autoimmune conditions, for example, duplication of the TLR7 gene contributed to the susceptibility to lupus and the intrinsic bias to developing in the Y-linked autoimmune accelerator mice. See Pisitkun, P., et al. (2006) Autoreactive B cell responses to RNA-related antigens due to TLR7 gene duplication. Science 312(5780): 1669-72; and Subramanian, S., et al. (2006) From the Cover: A Tlr7 translocation accelerates systemic autoimmunity in murine lupus. PNAS USA 103(26):9970-5.

[04] It is speculated that the initiation of spontaneous immune responses against tumor-associated antigens (TAA) resembles that against bacteria and viral products, in which the innate immune system such as immature DC and macrophages sense "danger signals". See Matzinger, P. (1994) Tolerance, danger, and the extended family. Ann. Rev. Immunol. 12:991-1045. On one hand, no innate immune receptors have been identified to exert similar functions as the TLR for PAMP in anti-tumor immunity. On the other hand, cell death causes cancer to release such endogenous factors as heat shock proteins (Basu, S., et al. (2001) CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 14(3):303-13), apoptotic bodies (Albert, MX., et al. (1998) Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392(6671):86-9), and uric acid (Shi, Y. et al. (2003) Molecular identification of a danger signal that alerts the immune system to dying cells. Nature 425(6957):516-21), rather than PAMP, to alert the innate immune system.

[05] TAA are not considered as "danger signals", but are generally perceived to be associated with the above danger signals, which serve as endogenous adjuvants to initiate anti-tumor immune responses. According to this paradigm, "spontaneous" anti-tumor immune responses may preferentially recognize products resulting from genetic alternations within cancer cells, against which the host keeps less stringent immune tolerance. However, human TAA identified to date are mostly non-mutated self-antigens. See Van Der Bruggen, P., et al. (2002) Tumor-specific shared antigenic peptides recognized by human T cells. Immunol Rev. 188(1):51-64.

SUMMARY OF THE INVENTION

[06] The present invention provides proteins comprising an NY-ESO-I polypeptide having at least one peptide which is not naturally associated with the NY-ESO-I polypeptide fused to the N-terminus or the C-terminus of the NY-ESO-I polypeptide. In some embodiments, the NY-ESO-I polypeptide is selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; amino acid residues 74- 180 of SEQ ID NO:1; SEQ ID NO:9; SEQ ID NO:12; and SEQ ID NO:13, said NY- ESO-I polypeptide may optionally have up to five amino acid residues truncated from the N-terminus, the C-terminus, or both. In some embodiments, the NY-ESO-I polypeptide has a methionine amino acid residue at the N-terminus, in other embodiments, the NY-ESO-I polypeptide does not have a methionine residue at the N-terminus. In some embodiments, the protein is purified or isolated.

[07] In some embodiments, the peptide is tag, a linker, all or part of a leader sequence, all or part of an antigen, all or part of an allergen, all or part of a transmembrane domain of a receptor, or all or part of an androgen-regulated protein. In some embodiments, the leader sequence is an Ig k chain leader sequence or a leader sequence from a human RANTES precursor. In some embodiments, the tag is a hamagglutinin A tag, a Flag tag, a Myc epitope tag, or a histadine tag. In some embodiments, the antigen is a carbonic anhydrase 9 antigen, a human tumor- associated antigen gplOO. In some embodiments, the allergen is Bet via, Art vl, or PMSA antigen. In some embodiments, the androgen-regulated protein is a platelet derived growth factor receptor transmembrane domain. In some embodiments, the linker comprises 1 to about 20, 1 to about 10, or 1 to about 6 amino acid residues.

[08] In some embodiments, the peptide is selected from the group consisting of:

SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:35; SEQ ID NO:36; SEQ ID NO:37; SEQ ID NO:38; and SEQ ID NO:39, wherein said peptide may optionally have up to five amino acid residues truncated from its N-terminus, C-terminus, or both. In some embodiments, the peptide contains a methionine amino acid residue at the N-terminus, in other embodiments, the peptide does not have a methionine residue at the N-terminus.

[09] In some embodiments, the protein comprises the amino acid sequence set forth in SEQ ID NO:6; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:27; or SEQ ID NO:28. In some embodiments, the protein consists essentially of the amino acid sequence set forth in SEQ ID NO:6; SEQ ID NO: 14; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:27; or SEQ ID NO:28. In some embodiments, the protein consists of the amino acid sequence set forth in SEQ ID NO:6; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:27; or SEQ ID NO:28.

[10] In some embodiments the present invention provides a protein comprises the amino acid sequence set forth in SEQ ID NO:9; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO:15; SEQ ID NO:19; SEQ ID NO:21; or SEQ ID NO:25. In some embodiments the present invention provides a protein consists essentially of the amino acid sequence set forth in SEQ ID NO:9; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO:15; SEQ ID NO:19; SEQ ID NO:21; or SEQ ID NO:25. In some embodiments the present invention provides a protein consists of the amino acid sequence set forth in SEQ ID NO:9; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO:19; SEQ ID NO:21; or SEQ ID NO:25.

[11] In some embodiments, the present invention provides a protein comprising the amino acid sequence set forth in SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; or amino acid residues 74-180 of SEQ ID NO:1. In some embodiments, the present invention provides a protein consisting essentially of the amino acid sequence set forth in SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; or amino acid residues 74-180 of SEQ ID NO : 1. In some embodiments, the present invention provides a protein consisting of the amino acid sequence set forth in SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; or amino acid residues 74-180 of SEQ ID NO:1.

[12] In some embodiments, a protein of the present invention is in the form of an oligomeric structure. In some embodiments, the oligomeric structure comprises two chains of the protein. In some embodiments, the oligomeric structure comprises more than two, for example 3, 4, 10, 20, 30, 40, or 50 and up to 100 chains of the protein. In some embodiments, the oligomeric structure is substantially similar to the oligomeric structure of wildtype NY-ESO-I formed under the same conditions. In some embodiments, the protein is covalently linked to a vault protein or a nanoparticle.

[13] In some embodiments, the present invention provides a cell which expresses a protein as disclosed herein. In some embodiments, the cell is a tumor cell and the protein is expressed on the surface of the cell. The cell may be mammalian, preferably human. In these embodiments, the cell is a recombinant cell, i.e. engineered using methods known in the art to express the protein of the present invention.

[14] In some embodiments, the present invention provides a polynucleotide which encodes a protein as disclosed herein. In some embodiments, the present invention provides a vector which contains a polynucleotide as disclosed herein. In some embodiments, the present invention provides an expression vector which expresses a protein as disclosed herein. In some embodiments, the present invention provides a recombinant cell which contains the polynucleotide, vector, or expression vector.

[15] In some embodiments, the present invention provides compositions which comprise a protein, a recombinant cell, a polynucleotide, a vector, an expression vector, as disclosed herein or compositions comprising combinations thereof. The compositions may comprise a pharmaceutically acceptable carrier. [16] A method for activating the NFkB pathway in a cell by binding a TLR4 receptor of a dendritic cell which comprises contacting the TLR4 receptor with as claimed herein or a protein having SEQ ID NO:1; SEQ ID NO:2; or SEQ ID NO:3. In some embodiments, the protein is in the form of an oligomeric structure. In some embodiments, the oligomeric structure is substantially similar to that of wildtype NY- ESO-I formed under the same conditions. In some embodiments, the protein is a recombinant protein, a purified protein or an isolated protein. In some embodiments, the protein consists essentially of SEQ ID NO:1; SEQ ID NO:2; or SEQ ID NO:3. In some embodiments, the protein consists of SEQ ID NO:1; SEQ ID NO:2; or SEQ ID NO:3. In some embodiments the protein may be fused with another polypeptide. In some embodiments, the protein comprises amino acids 75-180 of SEQ ID NO:1 and may optionally have one, some or all of the cysteine residues substituted with serine residues. In some embodiments, the amount of upregulation may be decreased by contacting the TLR4 receptor with a protein having SEQ ID NO: 1 and one or more of its cysteine residues replaced with an amino acid which does not form disulphide bonds, e.g. serine.

[17] In some embodiments, the present invention provides methods of inducing dendritic cell activation, maturation, polarization, and/or filamentous actin rearrangement in immature dendritic cells which comprises contacting a protein, a recombinant cell, a polynucleotide, a vector, an expression vector, and compositions with TLR4 receptors on the immature dendritic cells or binding the TLR4 receptors as disclosed herein.

[18] In some embodiments, the present invention provides methods of inducing an immune response in a subject which comprises administering to the subject an immunogenic amount of a protein, a recombinant cell, a polynucleotide, a vector, an expression vector, and compositions with TLR4 receptors on the immature dendritic cells or binding the TLR4 receptors as disclosed herein.

[19] In some embodiments, the present invention provides methods of modulating, increasing or decreasing the ability of the protein disclosed herein to elicit antibodies, modulate ThI and/or Th2 responses, produce cytokines and/or chemokines, or induce dendritic cell activation, maturation, polarization, filamentous actin rearrangement and/or phagocytosis which comprises increasing or decreasing the number of disulphide bonds in the NY-ESO-I polypeptide. [20] In some embodiments, the present invention provides methods of targeting

(e.g. increasing its susceptibility) a dendritic cell for phagocytosis or TLR4 signaling which comprises administering to the dendritic cell a protein, a recombinant cell, a polynucleotide, a vector, an expression vector, and compositions with TLR4 receptors on the immature dendritic cells or binding the TLR4 receptors as disclosed herein. [21] In some embodiments, the present invention provides methods of reversing, inhibiting, or reducing immune suppression by a cancer in a subject which comprises administering to the subject a TLR4 agonist or a protein, a recombinant cell, a polynucleotide, a vector, an expression vector, and compositions with TLR4 receptors on the immature dendritic cells or binding the TLR4 receptors as disclosed herein. [22] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description serve to explain the principles of the invention.

DESCRIPTION OF THE DRAWINGS

[23] This invention is further understood by reference to the drawings wherein:

[24] Figures 1 A-ID show the physiological and morphological modulation of human immature DC by NY-ESO-I .

[25] Figure IA shows the mean fluorescent intensity of F-actin stained with FITC- labeled phalloidin on immature DC treated with various reagents was compared to DC incubated with medium alone. The ratios were plotted in, which represent 4 independent experiments with immature DC from 4 different donors. Student t-tests indicated significant difference (p<0.05) between samples treated with NY-ESO-I, LPS, LPS+PK vs. untreated samples.

[26] Figure IB shows images of F-actin stained with FITC-labeled phalloidin and nucleus stained with DAPI on Day-6 immature DC pre-treated with medium, clinical GMP grade gplOO protein at 10 μg/ml, clinical GMP grade NY-ESO-I at 3 μg/ml, NY-ESO-I pretreated with proteinase K (PK), LPS at 100 ng/ml, LPS pretreated with proteinase K.

[27] Figure 1C shows expression of CD83 and ICAM (i.e. CD54) on the surface of immature DC after incubation with medium (purple histogram) and the indicated reagents. [28] Figure ID shows chemokine/cytokine secretion in the supernatant of human immature DC co-cultured with the indicated reagents, MIP lα, MIP lβ, TARC, IL6 an TNFα. NY-ESO-I did not exhibit a significant increase in production of other cytokines and chemokines, including ITAC, MDC, MIG, RANTES, IFNα, ILlα, ILlβ, ILlO, IL12p40 and IL12p70.

[29] Figures 2A-2D shows that NY-ESO-I forms oligomers held together by intermolecular disulfide bonds.

[30] Figure 2A shows human 293 cells transfected with a plasmid encoding NY-

ESO-I (lane 1) and NY-ESO-I fused with an N-terminal Flag tag (Flag-ESO, lane 3) reacted with mAB131 and M2 monoclonal antibody specific to NY-ESO-I and the Flag epitope, respectively. Lanes 2 and 4 contained lysates from 293 cells transfected with a plasmid encoding the green fluorescent protein.

[31] Figure 2B shows a Western blot using mAB131 of ESOcsl, ES0cs2, ES0cs3, and NY-ESO-I, from left to right.

[32] Figure 2C shows a Western blot on melanoma cell lines (lanes #1 and #2) and human embryonic stem cells of passage 3, 17, and 70 (lanes #3-5) with recombinant NY-ESO-I (lane #6) as control.

[33] Figure 2D shows that the NY-ESO-I (1 μg/ml) but not ES0cs3 was able to bind to immature DC. ESOR62H was a control mutant with an Arg to His change at amino acid 62. NY-ESO-I treated with proteinase K completely abolished the binding to immature DC.

[34] Figures 3A-3C show that activation of immature DC by NY-ESO-I is dependent on TLR4.

[35] Figure 3 A shows secretion of IL-6 from bone marrow derived immature DC of wild-type, TLR2-/-, and TLR4-/- mice. DC were cultured in medium from the supernatant of 293 cells transfected with plasmids encoding secreted NY-ESO-I (RANTES-ESO), secreted ES0cs3 (RANTES-ES0cs3), or GFP, or in the same medium containing exogenously added recombinant NY-ESO-I 2807 (3 μg/ml), LPS (100 ng/ml), or CPG (l μM).

[36] Figure 3B shows that bone marrow derived DC from the wild-type, TLR2-/-, and TLR4-/- mice were used for binding to NY-ESO-I, which was subsequently detected by staining with a FITC-labeled anti-ESO Ab.

[37] Figure 3C shows co-immunoprecipitation of TLR4 with NY-ESO-I but not gplOO. Membrane fractions of DC2.4 were co-incubated with His-ESO (lane 1) and His-gplOO (lane 2), followed by precipitation with anti-His antibodies. Western blot was conducted with anti-TLR4 Ab. Lane 3 is DC2.4 membrane fraction showing

TLR4 bands.

[38] Figures 4A-4D show that NY-ESO-I is highly immunogenic in mice.

[39] Figure 4A shows the Ab responses in HLA- A2. I/Kb mice in response to 10 μg of NY-ESO-I, 45 μg β-galactosidase, or 10⁷ pfu Vaccinia virus encoding NY-ESO-I

(wESO). [40] Figure 4B shows the Ab responses to ova and NY-ESO-I free of adjuvant at two doses (i.e. 3 and 0.3 μg/mouse). [41] Figures 4C and 4C shows the amount of IgG, IgGl, and IgG2a Ab against

NY-ESO-I and CS variants of NY-ESO-I in serum samples from mice. Three 6-8 week old female C57/BL6 mice were immunized via the intraperitoneal route with the indicated recombinant proteins (50 μg purified protein in the absence of adjuvant for each mouse). Two weeks later, serum samples from each mouse was examined for the presence of IgG, IgGl, and IgG2a Ab against NY-ESO-I using ELISA. The

OD450 is presented in Figures 4C and 4D. [42] Figure 4C shows Th2-dependent IgG antibodies induced by NY-ESO-I and

CS variants of NY-ESO-I as a function of the oligomeric structure. [43] Figure 4D shows Thl-dependent IgG antibodies induced by NY-ESO-I and

CS variants of NY-ESO-I as a function of the oligomeric structure. [44] Figure 5A shows the total IgG antibodies against the allergen Bet vl were measured following the course of gene gun immunization with the indicated plasmids encoding Bet vl alone or Bet vl fused with NY-ESO-I . Arrows indicated the points of gene gun immunization. [45] Figure 5B shows the subclasses of IgG antibodies against Art bl and NY-

ESO-I measured 2 weeks after the 3^rd gene gun immunization. [46] Figure 5 C shows the sera from mice immunized with plasmid encoding the

CA9-ES0 fusion protein and CA9 alone were analyzed against lysates from 293/GFP

(lane 1), 293/CA9 (lanes 2 and 4), and 293/ESO (lanes 3 and 5) in a Western blot.

The arrows in lanes 2 and 3 indicated the full-length CA9 and NY-ESO-I proteins, respectively. [47] Figure 5D shows that gp 1 OO :201 -220-ESO but not the gp 1 OO protein alone was cross-presented to human gpl00:208-217 specific CD8+ T cells in vitro. DETAILED DESCRIPTION OF THE INVENTION

[48] Mammals recognize pathogen-associated molecular patterns present in bacterium, virus, parasite, and fungus through Toll-like receptors (TLR). While searching for intrinsic factors from tumor-associated antigens (TAA), it was unexpectedly found that an endogenous tumor-associated antigen is a toll-like receptor 4 (TLR4) ligand and that TLR4 initiates or modulates anti-tumor immune responses in vivo.

[49] In particular, based on a hypothesis that intrinsic factors from some TAA may contribute to the initiation of anti-tumor immune responses in vivo, a non-mutated cancer/testis antigen, wild-type NY-ESO-I, was selected for further study. As used herein "wild-type NY-ESO-I", which is alternatively referred to herein as "NY-ESO- 1" without the indication of "wild-type" has the following amino acid sequence: MQAEGRGTGGSTGDADGPGGPGI PDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAP RGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRS LAQDAPPLPVPGVL LKE FTVSGNILTIRLTAADHRQLQLS I SSCLQQLSLLMWITQCFLPVFLAQPPSGQRR ( SEQ I D N0 : l ) .

[50] As provided herein, the present invention provides NY-ESO-I and NY-ESO-I polypeptides (including fusion polypeptides comprising NY-ESO-I fused to at least one other polypeptide and derivatives of NY-ESO-I), cells which express the polypeptides and methods of making and using thereof. As used herein, "derivatives of NY-ESO-I" are those which have 1 to 30, 1 to 20, 1 to 10, and 1 to 5 amino acids changes from SEQ ID NO:1. In some embodiments, all or some of the cysteine amino acids of wild type NY-ESO-I (SEQ ID NO:1) are substituted or deleted with an amino acid, e.g. serine, which does not form disulphide bonds.

[51] As used herein, the terms "protein", "polypeptide" and "peptide" are used interchangeably to refer to two or more amino acids linked together.

[52] As used herein, a "fusion" polypeptide refers to the expression product of two or more nucleic acid molecules that are not natively expressed together as one expression product. For example, a native protein X comprising subunit A and subunit B, which are not natively expressed together as one expression product, is not a fusion protein. However, recombinant DNA methods known in the art may be used to express subunits A and B together as one expression product to yield a fusion protein comprising subunit A fused to subunit B. A fusion protein may comprise amino acid sequences that are heterologous, e.g., not of the same origin, not of the same protein family, not functionally similar, and the like.

[53] As used herein, a "receptor" refers to a molecular structure within a cell or on the surface characterized by (1) selective binding of a specific substance and (2) a specific physiologic effect that accompanies the binding, e.g., membrane receptors for peptide hormones, neurotransmitters, antigens, complement fragments, and immunoglobulins and nuclear receptors for steroid hormones and include natural and synthetic biomolecules, such as proteins, polypeptides, peptides, nucleic acid molecules, carbohydrates, sugars, lipids, lipoproteins, small molecules, natural and synthetic organic and inorganic materials, synthetic polymers, and the like.

[54] As used herein, "specifically binds" refers to the character of a receptor which recognizes and interacts with a ligand but does not substantially recognize and interact with other molecules in a sample under given conditions.

[55] As used herein, "nucleic acid" or "nucleic acid molecule" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally- occurring nucleotides (e.g., α-enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term "nucleic acid molecule" also includes so- called "peptide nucleic acids", which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.

[56] An "isolated" nucleic acid molecule or polypeptide refers to a nucleic acid molecule or a polypeptide that is in an environment that is different from its native environment in which the nucleic acid molecule or polypeptide naturally occurs. Isolated nucleic acid molecules or polypeptides include those having nucleotides or amino acids flanking at least one end that is not native to the given nucleic acid molecule or polypeptide. For example, a promoter P for a protein X is inserted at the 5 ' end of a protein Y which does not natively have P at its 5 ' end. Protein Y is thus considered to be "isolated".

[57] As used herein, "such as" and "for example, are used interchangeably to describe a group of elements, which group may comprise other similar elements.

[58] In all amino acid sequences disclosed herein, a methionine amino acid residue need not be present at the amino terminus. Thus, in the claims, references to specific sequence identifiers includes the entire sequence represented by the sequence identifier or the sequence without the methionine amino acid residue at the amino terminus, e.g. a claim to a polypeptide comprising or consisting of SEQ ID NO:9, refers to the sequence with the methionine and the sequence without the methionine. As such, claim limitations wherein the methionine amino acid residue is specifically included or excluded are contemplated herein.

[59] The polypeptides of the present invention may comprise, consist essentially of, or consist of one of the amino acid sequences as set forth herein and may comprise 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acid mutations, substitutions, and/or deletions. In some embodiments, the amino acid mutations, substitutions, or deletions occur in regions which do not result in an oligomeric structure that is substantially different from the oligomeric structure of wild-type NY-ESO-I . In other embodiments, the amino acid mutations, substitutions, or deletions occur in regions, e.g. cysteine residues, which do result in an oligomeric structure that is different from the oligomeric structure of wild-type NY-ESO-I.

[60] In some embodiments, the polypeptides of the present invention are capable providing a therapeutic benefit in a subject. Preferably, the subject is mammalian, more preferably, the subject is human.

[61] As used herein, an "immune response" refers to a humoral or cellular response caused by exposure to an antigenic substance. A "protective immune response" refers to humoral immune responses, cellular immune responses, or both, that are sufficient to result in an observable therapeutic benefit in a subject.

[62] The polypeptides of the present invention need not be identical to those exemplified herein so long as the subject polypeptides exhibit functional and/or structural characteristics that are substantially similar to those exemplified herein when assayed or evaluated under the same conditions. For example, an ESO 1-74 polypeptide may have at least one amino acid mutation, substitution, deletion, or a combination thereof, that is different from ESO 1-74, but when assayed under the same conditions, the ESO 1-74 polypeptide exhibits characteristics which are substantially similar to ESO 1-74. Thus, the polypeptide is considered to be an ESOl- 74 polypeptide.

[63] The polypeptides of the present invention may also be modified to provide a variety of desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of its activity. By using conventional methods in the art, one of ordinary skill will be readily able to make a variety of polypeptides having mutated linker regions and then screen the polypeptides for stability, toxicity, and immunogenicity according to the present invention.

[64] Additionally, single amino acid substitutions, deletions, or insertions can be used to determine which residues are relatively insensitive to modification. Amino acid substitutions are preferably made between relatively neutral moieties, such as alanine, glycine, proline, and the like. Substitutions with different amino acids, of either D or L isomeric forms, or amino acid mimetics can be made. The number and types of substitutions, deletions, and insertions depend on the functional attributes that are sought such as hydrophobicity, immunogenicity, three-dimensional structure, and the like.

[65] An "amino acid mimetic" as used herein refers to a moiety other than a naturally occurring amino acid residue that conformationally and functionally serves as a suitable substitute for an amino acid residue in a polypeptide of the present invention. A moiety is a suitable substitute for an amino acid residue if it does not interfere with the activity of the unmodified polypeptide. Examples of amino acid mimetics include cyclohexylalanine, 3-cyclohexylpropionic acid, L-adamantyl alanine, adamantylacetic acid, and the like. See e.g. Morgan and Gainor, (1989) Ann. Repts. Med. Chem. 24:243-252. [66] Individual amino acid residues may be incorporated in the polypeptides of the present invention with peptide bonds or peptide bond mimetics. A peptide bond mimetic include peptide backbone modifications of the amide nitrogen, the .alpha.- carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. See e.g. Spatola (1983) CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, Vol. VII, Weinstein ed. The polypeptides of the present invention may include an additional methionine as the first amino acid residue on the protein amino terminus or a histadine tag at one of its ends. The polypeptides may be truncated by up to about five (5) amino acid residues from the either terminus or both. Co-translational or post-translational surface modifications, such as the addition of covalently attached sugars or lipids, may be made to the polypeptides of the present invention.

[67] As used herein "sequence identity" means that two sequences are identical over a window of comparison. The percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[68] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

[69] The polypeptides of the present invention may be made by conventional methods known in the art. The polypeptides of the present invention may be manually or synthetically synthesized using conventional methods and devices known in the art. See e.g., Stewart and Young (1984) SOLID PHASE PEPTIDE SYNTHESIS, 2 ed. Pierce, Rockford, IL. The polypeptides of the present invention may be obtained or purified using protein purification techniques such as reverse phase high-performance liquid chromatography (HPLC), ion-exchange or immunoaffinity chromatography, filtration or size exclusion, or electrophoresis known in the art. See e.g., Scopes (1982) PROTEIN PURIFICATION, Springer- Verlag, NY.

[70] In some embodiments, the polypeptides of the present invention are substantially purified. As used herein, a "substantially purified" compound refers to a compound that is removed from its natural environment and is at least about 60% free, preferably about 75% free, and most preferably about 90% free from other macromolecular components with which the compound is naturally associated.

[71] Alternatively, the polypeptides of the present invention may be made by recombinant DNA techniques known in the art. Thus, the present invention provides polynucleotides that encode the polypeptides of the present invention.

[72] A polynucleotide encoding a polypeptide of the present invention is then inserted in to a vector such as a cloning vector or an expression vector. An expression vector allows the polypeptide to be expressed when present in a host. Either the expression vector or the host may comprise the regulatory sequences necessary for expression of the polypeptide. Where the regulatory sequences are within the expression vector, the regulatory sequences are operatively linked to the sequence encoding the polypeptide. As used herein, "operably linked" means that the nucleotide sequence of interest is linked to at least one regulatory sequence in a manner that allows the polypeptide to be expressed in an in vitro transcription/translation system or in a host cell. Regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). See e.g., Goeddel (1990) GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY, Academic Press, San Diego, CA.

[73] It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the desired expression levels of the polypeptide, the compatibility of the host cell and the expressed polypeptide, and the like.

[74] The vectors can be designed for expressing the polypeptides of the present invention in prokaryotic or eukaryotic host cells such as bacterial cells, insect cells, plant cells, yeast cells, or mammalian cells using methods and host cells known in the art. Thus, the present invention also provides host cells comprising polynucleotides that encode the polypeptides of the present invention. Host cells include the progeny or potential progeny of the primary cell in which the polynucleotide was introduced. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope and meaning of host cell.

[75] A polypeptide of the present invention may be used to prepare antibodies by immunizing a suitable subject, e.g., rabbit, goat, mouse or other mammal with the polypeptide using methods known in the art. The antibodies raised against the polypeptides of the present invention may be used in therapeutic methods. Thus, the present invention also provides antibodies that are raised against or derived from the polypeptides of the present invention, and methods of using thereof.

[76] Antibodies of the present invention may be produced by methods known in the art. See e.g., Coligan (1991) CURRENT PROTOCOLS IN IMMUNOLOGY. Wiley/Greene, N.Y.; and Harlow and Lane (1989) ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Press, N. Y.; Stites, et al. (1986) BASIC AND CLINICAL IMMUNOLOGY. 4th ed. Lange Medical Publications, Los Altos, Calif; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE. 2d ed. Academic Press, New York, N.Y.; and Kohler and Milstein (1975) Nature 256:495-497. Therapeutic antibodies may be produced specifically for clinical use in humans by conventional methods known in the art. See Chadd, H. E. and S. M. Chamow (2001) Curr. Opin. Biotechnol. 12:188-194 and references therein.

[77] As used herein, "antibody" refers to immunoglobulin molecules and immunologically active portions that comprise an antigen binding site which specifically binds an antigen. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')₂ fragments which may be generated by treating the antibody with an enzyme such as pepsin. Polyclonal and monoclonal antibodies against the polypeptides of the present invention may be made by conventional methods known in the art.

[78] The polypeptides, polynucleotides, or antibodies of the present invention may be administered, preferably in the form of pharmaceutical compositions, to a subject. Pharmaceutical compositions of the present invention are those comprising, consisting essentially of, or consisting of an amount, such as an immunogenic amount or a therapeutically effective amount, of at least one of the polypeptides of the present invention and a pharmaceutically acceptable vehicle. The present invention provides immunogenic compositions which may comprise, consist essentially of, or consist of an active immunizing agent, such as a polypeptide of the present invention, or a passive immunizing agent, such as an antibody raised against the polypeptide of the present invention. The immunogenic composition may elicit an immune response that need not be protective or the immunogenic composition may provide passive immunity. A vaccine elicits a local or systemic immune response that is protective against subsequent challenge by the immunizing agent such as the polypeptides of the present invention, or an immunologically cross-reactive agent. Methods known in the art may be used to determine the feasibility of using the polypeptides of the present invention as vaccines. A protective immune response may be complete or partial, i.e. a reduction in symptoms as compared with an unvaccinated subject.

[79] As used herein, an "immunogenic amount" is an amount that is sufficient to elicit an immune response in a subject and depends on a variety of factors such as the immunogenicity of the polypeptide, the manner of administration, the general state of health of the subject, and the like. The typical immunogenic amounts for initial and boosting immunization for therapeutic or prophylactic administration ranges from about 0.01 mg to about 0.1 mg per about 65-70 kg body weight of a subject. For example, the typical immunogenic amount for initial and boosting immunization for therapeutic or prophylactic administration for a human subject ranges from about 0.01 mg to about 0.1 mg. Examples of suitable immunization protocols include initial immunization injections at time 0 and 4 or initial immunization injections at 0, 4, and 8 weeks, which initial immunization injections may be followed by further booster injections at 1 or 2 years.

[80] As used herein, a "therapeutically effective amount" refers to an amount of a polypeptide, a polynucleotide, or an antibody that results in an observable therapeutic response in subject as compared to a control. Again, the skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including previous treatments, the general health and age of the subject, and the like. A therapeutically effective amount may be readily determined using methods known in the art. It should be noted that treatment of a subject with a therapeutically effective amount of a polypeptide, a polynucleotide, or an antibody of the present invention can include a single treatment or a series of treatments.

[81] The pharmaceutical compositions may include an adjuvant. As used herein, an "adjuvant" refers to any substance which, when administered with or before the polypeptide, polynucleotide, or antibody of the present invention, aids the polypeptide, polynucleotide, or antibody in its mechanism of action. Thus, an adjuvant in a vaccine is a substance that aids the immunogenic composition in eliciting an immune response. Suitable adjuvants include incomplete Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, nor-MDP), N-acetylmuramyl-Lalany-Disoglutaminyl-L-alanine-2-(r-2'- dipa-lmitoyl-sn-g lycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835 A, MTP- PE), and RIBI, which comprise three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (NPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by conventional methods in the art.

[82] The compositions of the present invention may be administered to a subject by any suitable route including oral, transdermal, intranasal, inhalation, intramuscular, and intravascular administration. It will be appreciated that the preferred route of administration and pharmaceutical formulation will vary with the condition and age of the subject, the nature of the condition to be treated, the therapeutic effect desired, and the particular polypeptide, polynucleotide, or antibody used.

[83] As used herein, a "pharmaceutically acceptable vehicle" or "pharmaceutically acceptable carrier" refers to and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Pharmaceutically acceptable vehicles include those known in the art. See e.g. REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY. 20th ed. (2000) Lippincott Williams & Wilkins. Baltimore, MD.

[84] The pharmaceutical compositions of the present invention may be provided in dosage unit forms. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. [85] Toxicity and therapeutic efficacy of such compounds can be determined by

Standard pharmaceutical procedures in cell cultures or experimental animals. For example, one may determine the lethal dose of a compound, LC50 (the dose expressed as concentration of compound x exposure time that is lethal to 50% of the population) or the LD50 (the dose lethal to 50% of the population), and the ED50 (the dose therapeutically effective in 50% of the population) by conventional methods in the art. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[86] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[87] The present invention also provides polypeptides, polynucleotides, antibodies, or compositions of the present invention may be provided in kits along with instructions for use. A kit comprising a pharmaceutical composition may include the pharmaceutical composition as a single dose or multiple doses. The kit may include a device for delivering the pharmaceutical composition. The device may be a multi- chambered syringe for intramuscular delivery, a microneedle or set of microneedle arrays for transdermal delivery, a small balloon for intranasal delivery, or a small aerosol generating device for delivery by inhalation.

[88] As provided in the Examples below, NY-ESO-I was found to induce rearrangement of filamentous actin, maturation and polarization of dendritic cells (DC) in vitro. Activation of immature DC was found to be dependent on TLR4- dependent and the native oligomeric structure of NY-ESO-I. ESOcsl, ES0cs2 and ES0cs3 (based on NY-ESO-I, but have cysteine-to-serine substitutions) exhibit decreased oligomeric structures and less TLR4 activation capabilities as compared to NY-ESO-I . Thus, the present invention provides methods and compositions for inducing rearrangement of filamentous actin, maturation and polarization of DC. The present invention also provides methods and compositions for inducing rearrangement of filamentous actin, maturation and polarization of DC in an amount less than that provided by NY-ESO-I.

[89] Also as disclosed, immunization of mice with the wild-type NY-ESO-I produced an integrated ThI and Th2-dependent response featuring IgGl and IgG2a antibodies; while ESOcsl, ES0cs2, and ES0cs3, which exhibit less TLR4 activation capacities, each produced a ThI -dependent response of primarily IgG2a antibody. Such induced Ab responses diminished in TLR4-/- mice. Thus, the present invention provides methods and compositions for inducing an integrated ThI and Th2- dependent response featuring both IgGl and IgG2a antibodies or only a ThI- dependent response of primarily IgG2a antibodies in a subject.

[90] As provided in the Examples, NY-ESO-I can be fused with other proteins, such as allergens and tumor-associated antigens, to modulate immune responses against the other proteins. Thus, the present invention provides fusion polypeptides and methods and compositions comprising the fusion polypeptides.

[91] Compositions, including vaccines, and methods for inducing efficient phagocytosis coupled with proper DC maturation. As shown herein, NY-ESO-I binds to both complement CIq receptor (calreticulin or CRT) and TLR4. Therefore, the present invention provides NY-ESO-I and fusion polypeptides as potent molecular adjuvants for modulating T cell and T cell-dependent antibody responses against a variety of antigens in allergy, autoimmune disease, and cancer. The present invention also provides irradiated tumor cells expressing cell-surface anchored NY- ESO-I or secreted NY-ESO-I as well as other polypeptides disclosed herein that are able to enhance both phagocytosis and TLR4 signaling which can be used as cross- primers of cytotoxic T cells and switches of T helper cell dependent antibody responses.

[92] As provided herein, fusion polypeptides comprising NY-ESO-I fused to another antigen mediates, either up-regulates or down-regulates, the immune response to the antigen. Therefore, the present invention provides compositions and methods for mediating an immune response to a given antigen.

[93] Thus, genetically modified cancer cell lines expressing membrane-anchored

NY-ESO-I or secreted NY-ESO-I lead to break of immune tolerance and anti-tumor efficacy in vivo.

Dendritic cell (DC) Targeted Vaccines

[94] In some embodiments, the present invention relates to translational fusion and engineered tumor cells as DC targeted vaccines. Translational fusion vaccines include fusion proteins and genes encoding fusion proteins. CA9-ESO, gplOO-NY- ESO-I, and PSMA-NY-ESO-I are examples of such fusion proteins disclosed herein. The efficacy of CA9-ES0 against kidney cancer, gplOO-NY-ESO-1 against melanomas, and PSMA-NY-ESO-I against prostate cancer model may be determined using methods known in the art..

[95] Engineered tumor cells that express cell-surface anchored or secreted NY-

ESO-I may be used as DC targeted vaccines. Irradiate tumor cells expressing PSMA- NY-ESO-I and irradiated Myc-CaP cell lines expressing cell-surface anchored ESO are examples of such engineered tumor cells which may be used as vaccines against prostate cancer.

[96] Cancer stem cells expressing a cell-surface anchored version of NY-ESO-I or

LAGE-I may be engineered using methods known in the art. Human cancer stem cells or cancer stem-like cells have been reported in breast cancer, leukemia, glioblastoma multiform, prostate cancer, non-small cell lung cancer, kidney cancer, liver cancer, and the like. Ideally, cancer stem cells can be isolated with antibodies against particular cell surface markers such as CD44+/CD24-/PSMA- from prostate cancer cell lines, and then subject to retroviral or lentiviral mediated gene delivery to express cell-surface NY-ESO-I or LAGE-I . These cells will then be irradiated and injected as whole cell vaccines.

[97] Thus, the present invention provides cells, such as cancer cells, which express recombinant cell surface-anchored NY-ESO-I and NY-ESO-I family members, such as ESOcsl, ES0cs2, ES0cs3, and LAGE-I, to enhance immunogenicity. The present invention also provides combined expression of GM-CSF (as in the GVAX cell vaccine platform) and cell surface-anchored NY-ESO-I and ESO-NY-I family members and methods of using to further enhance irradiated cell vaccines. Immune Modulatory Adjuvant

[98] As provided herein, integrated Thl/Th2 responses are induced by the wild type NY-ESO-I, while ESOcsl, ES0cs2, and ES0cs3 induce a predominant ThI type of response. It is believe that the immune modulating effect of NY-ESO-I is a function of its tertiary structure. Therefore, the present invention provides compositions and methods for inducing different immune responses.

[99] As provided herein ESOcsl, ES0cs2 and ES0cs3 bind DC to a less extent than NY-ESO-I . NY-ESO-I based polypeptides which have increased binding to DC as compared with NY-ESO-I, may be created using methods known in the art. For example, randomized in vitro mutagenesis may be used to create new NY-ESO-I polypeptides with higher binding and DC stimulating activities. These polypeptides with higher binding can be use as molecular adjuvants.

[100] As another example, a polypeptide of the present invention, such as ESO1-74, may be fused with a vault protein to achieve even higher oligomeic structures as compared to NY-ESO-I and thus further enhance the binding with DC surface CRT/TLR4. Vaults are large barrel-shaped particles found in the cytoplasm in all mammalian cells. See Kickhoefer, V.A., et al. (2005) Engineering of vault nanocapsules with enzymatic and fluorescent properties. PNAS USA 102(12): 4348- 52. Polypeptides can be targeted to the surface at both ends of the vault particle by expression as fusion proteins with the C terminus of MVP domain. Fusion of ESOl- 74 with the MVP protein may result in the assembly of 48 copies of the CRT/TLR4 binding domain at each end of a vault nanoparticle. Such fusion proteins can be used as a nano-sized molecular adjuvant that can be delivered directly or combined with specific vaccines against cancer, infectious diseases, and auto-immune diseases.

[101] Thus, the polypeptides of the present invention may be covalently linked to a vault protein and/or a nanoparticle known in the art. As used herein, a "nanoparticle" is a particle that has nano-size dimensions.

[102] The polypeptides and compositions of the present invention may be used in immunotherapies and methods of treating or preventing diseases such as malaria using a fusion protein, such as circumsporozoite protein (CSP) fused to a polypeptide of the present invention. [103] The following examples are intended to illustrate but not to limit the invention.

Example 1

Construction of plasmids and introduction of site-directed mutagenesis [104] Construction ofESOl-74 and ESO1-95. PCR products containing the coding region of truncated NY-ESO-I proteins were amplified using a cDNA as a template. The truncated NY-ESO-I products were ESO 1-74 AND ESO 1-95. ESO 1-74 consists of amino acid residues 1 to 74 of NY-ESO-I and has the following amino acid sequence:

MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAP RGPHGGAASGLNG (SEQ ID N0:2).

[105] ESO1-95 consists of amino acid residues 1 to 95 of NY-ESO-I and has the following amino acid sequence:

MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAP RGPHGGAASGLNGCCRCGARGPESRLLEFYLAMP (SEQ ID N0:3).

[106] The truncated NY-ESO-I proteins were produced using methods known in the art. ESO 1-74 was produced as previously described. See Zeng, G. et al. (2000) J. Immunol. 165:1153-1159. The coding region of ESO 1 -94 was amplified using the following primers:

Forward: 5' GCTCCGGACATATGCAGGCCGAAGGCCGGGG (SEQ ID NO : 4 ) (the introduced Ndel site is underlined) ;

Reverse : 5 ' ATCCTACTCGAGTTAAGGCATGGCGAGGTAGAA ( SEQ I D NO : 5 ) ( the introduced Xhol site i s underl ined) . [107] A eukaryotic version of the NY-ESO-I expression vector was employed as previously described. See Wang, R.F. et al. (1998) J. Immunol. 161 :3596-3606. [108] Construction of Flag-ESO . The full-length nucleotide sequence which encodes NY-ESO-I was introduced to the pFlag-CMV-2 vector (Sigma-Aldrich Co., St. Louis, MO) to result in a fusion between the coding region of NY-ESO-I and the N-terminal Flag epitope to give a polypeptide having the following amino acid sequence (underline indicates Flag tag; bold indicates linker; and lower case indicates the gene product of interest, i.e. NY-ESO-I):

MDYKDDDDKLmqaegrgtggstgdadgpggpgipdgpggnaggpgeagatggrgprgagaa rasgpgggaprgphggaasglngccrcgargpesrllefylampfatpmeaelarrslaqd applpvpgvllkeftvsgniltirltaadhrqlqlsissclqqlsllmwitqcflpvflaq pps gqrr ( SEQ I D NO : 6 ) . [109] Construction of ESOcsl. PCR was used to introduce site-directed mutagenesis to replace cysteines at amino acid positions 75, 76, and 78 of NY-ESO-I with serines using the following primers:

Forward: 5' TCCTCCAGATCCGGGGCCAGGGGGCCGGAGAG (SEQ ID NO : 7 )

(underlined residues are all changed from a guanadine) ;

Reverse: 5' TCCATTCAGCCCTGAAGCCGCGCCGCCAT (SEQ ID NO : 8 )

(phosphorylated at the 5' end) .

[110] The long PCR using methods known in the art was performed on a previously- described expression vector pET-28-NY-ESO-l (formerly Novagene, now EMD Chemicals, Inc., Gibbstown, NJ) using a high fidelity PCR kit from Invitrogen (Carlsbad, CA). The resultant PCR product was religated and sequenced to ensure that no PCR-introduced error was present. The polypeptide with serines at amino acid positions 75, 76, and 78 of NY-ESO-I was named ESOcsl and has the following sequence:

MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAP RGPHGGAASGLNGSSRSGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVL LKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR (SEQ ID NO: 9) .

[Ill] Construction ofESOcs2. The same approach used to construct ESOcsl was used to introduce cysteine to serine substitutions at amino acid 152 and 165 using a known wild-type NY-ESO-I expression vector (See Zeng, G. et al. (2000) J. Immunol. 165:1153-1159) as a PCR template using the following primers:

Forward: 5' TTGATGTGGATCACGCAGTCCTTTCTGCCCGTG (SEQ ID NO:10); Reverse: 5' CAGGGAAAGCTGCTGGAGAGAGGAGCTGATG (SEQ ID NO: 11) (mutated nucleotides are underlined) .

[112] The polypeptide with serines at amino acid positions 152 and 165 of ESO-NY-

1 was named ES0cs2 and has the following sequence:

MQAEGRGTGGSTGDADGPGGPGI PDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAP RGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRS LAQDAPPLPVPGVL LKEFTVSGNILTIRLTAADHRQLQLS I SS SLQQLSLLMWITQS FLPVFLAQPPSGQRR ( SEQ I D N0 : 12 ) . [113] Construction ofESOcs3. To replace all 5 cysteine residues with serine residues at amino acid positions 75, 76, 78, 152, and 165 of NY-ESO-I, another PCR was carried out using the same primers that were used to construct plasmid encoding ESOcs2. The PCR was performed on a plasmid template encoding ESOcsl as disclosed herein. The polypeptide with 5 serine residues at amino acid positions 75, 76, 78, 152, and 165 of ESO-NY-I was named ES0cs3 and has the following sequence:

MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAP RGPHGGAASGLNGSSRSGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVL LKEFTVSGNILTIRLTAADHRQLQLSISSSLQQLSLLMWITQSFLPVFLAQPPSGQRR (SEQ ID N0:13) .

[114] ESOcsl, ESOcs2, and ESOcs3 expression vectors. To construct eukaryotic expression plasmids that encode ESOcsl, ES0cs2, and ES0cs3, PCR was used to amplify the NY-ESO-I coding region with the corresponding mutations. The resultant PCR products were subsequently cloned into pcDNA3. ITOPO vectors (Invitrogen, Carlsbad, CA). All plasmids were sequenced to make sure the absence of extra mutations introduced by PCR. These plasmids were purified using endotoxin- free maxiprep kits purchased from Qiagen Inc. (Santa Clarita, CA).

[115] NY-ESO-I is naturally a cytoplasmic protein. To express NY-ESO-I and its variants on the cell surface, the pDisplay vector (Invitrogen, Carlsbad, CA) was used. The cDNAs encoding full-length NY-ESO-I, ESOcsl, ES0cs2, ES0cs3, LAGE-Ib, High mobility group box-1 (HMGB-I), as well as control GFP was cloned into the Sail and Hindϊll sites of the pDisplay vector using methods known in the art. [116] In the following cell-surface anchored protein sequences, italics indicates an

Ig k chain leader sequence; bold indicates a Hamagglutinin A tag; underline indicates a linker; the gene product of interest is in lower case; double underline indicates a Myc epitope; and REGULAR UPPER CASE indicates a platelet derived growth factor receptor transmembrane domain. [117] Cell-surface anchored NY-ESO-I has the following sequence:

METgrLLLlWLLLlWPGSrGgYPYDVPDYAGAQPARmqaegrgtggstgdadgpggpgipd gpggnaggpgeagatggrgprgagaarasgpgggaprgphggaasglngccrcgargpesr llefylampfatpmeaelarrslaqdapplpvpgvllkeftvsgniltirltaadhrqlql sissclqqlsllmwitqcflpvflaqppsqqrrQVDEQKLISEEDLNGVGQDTQEVIWPH SLPFKWVISAILALWLTIISLIILIMLWQKKPR (SEQ ID N0:14). [118] Cell-surface anchored LAGE-I has the following sequence:

METgrLLLlWLLLlWPGSrGgYPYDVPDYAGAQPARmqaegqgtggstgdadgpggpgipd gpggnaggpgeagatggrgprgagaarasgprggaprgphggaasaqdgrcpcgarrpdsr llqlhitmpfsspmeaelvrrilsrdaaplprpgavlkdftvsgnllfirltaadhrqlql sis sclqql sllmwitqcflpvflaqapsqqrrQVDEQKLI SEEDLNGVGQDTQEVIWPH SLPFKWVI SAILALWLTI I SLI ILIMLWQKKPR ( SEQ ID NO : 15 ) .

[119] Cell-surface anchored ESOcsl has the following sequence:

METgrLLLlWLLLlWPGSrGgYPYDVPDYAGAQPARmqaegrgtggstgdadgpggpgipd gpggnaggpgeagatggrgprgagaarasgpgggaprgphggaasglngssrsgargpesr llefylampfatpmeaelarrslaqdapplpvpgvllkeftvsgniltirltaadhrqlql sissclqqlsllmwitqcflpvflaqppsgqrrOVDEQKLISEEDLNGVGQDTQEVIWPH SLPFKWVISAILALWLTIISLIILIMLWQKKPR (SEQ ID NO: 16).

[120] Cell-surface anchored ES0cs2 has the following sequence:

METDTLLLWVLLLWVPGSTGDYPYD^'VPDYAGAQ^'PARmqaeqrqtqqstqdadqpqqpqipd gpggnaggpgeagatggrgprgagaarasgpgggaprgphggaasglngccrcgargpesr lief ylampf atpmeaelarrslaqdapplpvpgvll kef tvsgniltirltaadhrqlql sis sslqql sllmwitqsflpvflaqppsqqrrQVDEQKLI SEEDLNGVGQDTQEVIWPH SLPFKWVI SAILALWLTI I SLI ILIMLWQKKPR ( SEQ ID NO : 17 ) .

[121] Cell-surface anchored ES0cs3 has the following sequence:

METDTLLLWVLLLWVPGSTGDYPYD^'VPDYAGAQ^'PARmqaeqrqtqqstqdadqpqqpqipd gpggnaggpgeagatggrgprgagaarasgpgggaprgphggaasglngssrsgargpesr lief ylampf atpmeaelarrslaqdapplpvpgvll kef tvsgniltirltaadhrqlql sis sslqql sllmwitqsflpvflaqppsqqrrQVDEQKLI SEEDLNGVGQDTQEVIWPH SLPFKWVI SAILALWLTI I SLI ILIMLWQKKPR ( SEQ ID NO : 18 ) .

[122] Cell-surface anchored HMGB-I has the following sequence:

METOrLLLlWLLLlWPGSrGgYPYDVPDYAGAQPARmgkgdpkkprgkms syaf fvqtcre ehkkkhpdasvnf sef skkcserwktmsakekgkf edmakadkaryeremktyippkgetk kkf kdpnapkrppsaf f If cseyrpkikgehpglsigdvakklgemwnntaaddkqpyekk aaklkekyekdiaayrakgkpdaakkgvvkaekskkkkeeeedeedeedeeeeedeedede eeddddeQVDEQKLI SEEDLNGVGQDTQEVIWPHSLPFKVWISAILALVVLTI ISLI IL IMLWQKKPR ( SEQ ID NO : 19 ) .

[123] Similarly, the corresponding cDNAs were fused with the leader sequence of the chemokine RANTES to express secreted proteins using methods known in the art. In the following secreted protein sequences, bold indicates a leader from human RANTES precursors; the gene product of interest is in lower case; and double underline indicates a Myc epitope.

[124] Secreted NY-ESO-I has the following sequence:

MKVSAAALAVILIATALCAPASAmqaegrgtggstgdadgpggpgipdgpggnaggpgeag atggrgprgagaarasgpgggaprgphggaasglngccrcgargpesrlIefylampfatp meaelarrslaqdapplpvpgvllkeftvsgniltirltaadhrqlqlsissclqqlsllm witqcflpvflaqppsgqrr (SEQ ID NO:20). [125] Secreted LAGE-I has the following sequence:

MKVSAAALAVILIATALCAPASAmqaegqgtggstgdadgpggpgipdgpggnaggpgeag atggrgprgagaarasgprggaprgphggaasaqdgrcpcgarrpdsrllqlhitmpfssp meaelvrrilsrdaaplprpgavlkdftvsgnllfirltaadhrqlqlsissclqqlsllm witqcflpvflaqapsgqrr (SEQ ID N0:21) .

[126] Secreted ESOcsl has the following sequence:

MKVSAAALAVILIATALCAPASAmqaegrgtggstgdadgpggpgipdgpggnaggpgeag atggrgprgagaarasgpgggaprgphggaasglngssrsgargpesrllefylampfatp meaelarrslaqdapplpvpgvllkeftvsgniltirltaadhrqlqlsissclqqlsllm witqcflpvflaqppsgqrr (SEQ ID NO:22) .

[127] Secreted ES0cs2 has the following sequence:

MKVSAAALAVILIATALCAPASAmqaegrgtggstgdadgpggpgipdgpggnaggpgeag atggrgprgagaarasgpgggaprgphggaasglngccrcgargpesrllefylampfatp meaelarrslaqdapplpvpgvllkeftvsgniltirltaadhrqlqlsissslqqlslim witqsflpvflaqppsqqrrQVDEQKLISEEDL (SEQ ID NO:23).

[128] Secreted ES0cs3 has the following sequence:

MKVSAAALAVILIATALCAPASAmqaegrgtggstgdadgpggpgipdgpggnaggpgeag atggrgprgagaarasgpgggaprgphggaasglngssrsgargpesrllefylampfatp meaelarrslaqdapplpvpgvllkeftvsgniltirltaadhrqlqlsissslqqlslim witqsflpvflaqppsqqrrQVDEQKLISEEDL (SEQ ID NO:24).

[129] Secreted HMGB-I has the following sequence:

MKVSAAALAVILIATALCAPASAmgkgdpkkprgkmssyaffvqtcreehkkkhpdasvnf sefskkcserwktmsakekgkfedmakadkaryeremktyippkgetkkkfkdpnapkrpp saffIfcseyrpkikgehpglsigdvakklgemwnntaaddkqpyekkaaklkekyekdia ayrakgkpdaakkgvvkaekskkkkeeeedeedeedeeeeedeededeeedddde (SEQ ID NO: 25) .

Example 2

Purification of Recombinant Proteins and Preparation of Vaccinia virus Encoding NY-ESO-I

[130] pET-28 (Novagen, Madison, WI) based bacterial expression vectors encoding the full-length NY-ESO-I wild-type protein, ESO1-74, ESO1-95, ESOcsl, ES0cs2, and ES0cs3 were used to transform BL21(DE3) E. coli (Novagen, Madison, WI). Bacteria were grown at 37° C to log phase, then induced for protein production by adding isopropyl β-d-thiogalactoside (IPTG) to a final concentration of 0.5 mM and shaking for about 3 hours. Inclusion bodies of bacterial extract were obtained using an approach as previously described (See Zeng, G. et al. (2000) J. Immunol. 165 : 1153-1159); and proteins were purified by Ni²⁺ affinity chromatography as previously described (See Zeng, G. et al. (2000) J. Immunol. 165:1153-1159). Purified proteins were dialyzed against 50 mM Tris-HCl (pH 8.0) in the presence of 0.5 M free arginine to maintain solubility of the protein.

[131] The Vaccinia viral plasmid encoding NY-ESO-I was constructed on a pSC65 vector, packaged and purified using methods known in the art. See Irvine, KR et al. (1999) Cancer Res. 59(11):2536-40.

Example 3 Antibodies, Cell lines, Transfection of 293 Cells, and Western Blot

[132] Monoclonal antibodies against NY-ESO-I were developed at the NCI

Frederick Development Center by immunizing mice with the full-length recombinant protein emulsified in Complete Freud Adjuvant (CFA) and using methods known in the art. Three NY-ESO-I specific monoclonal antibodies were generated, and were named mAbl31, mAbl32, mAbl34, respectively.

[133] The anti-Flag antibody M2 was a gift from Dr. Sheng Guo at the University of

Pittsburgh Cancer Institute. Antibodies against the c-Myc and hemagglutinin A epitopes were a gift from Dr. Bin Liu at the University of California, Los Angeles, Department of Medicine.

[134] All melanoma cell lines were generated using methods known in the art at the surgery branch of the National Cancer Institute, Bethesda, MD. Tumor cell lines and 293 lines were maintained in DMED medium containing 10% fetal calf serum using methods known in the art. Mouse DC2-4 line was obtained from Dr. Kenneth Rock's laboratory at the University of Massachusetts School of Medicine through Dr. Robert Prins at the University of California, Los Angeles.

[135] Transfection of plasmids into 293 cells was carried out using the FUGENE® transfection system (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. Western blotting was performed as previously described (Zeng, G. et al. (2000) J. Immunol. 165:1153-1159) and was developed using an ECL system (GE Healthcare/ Amersham, Piscataway, NJ). Example 4 Immunization of Animals and Detection of NY-ESO-I Antibodies Present in Serum

[136] Six to 8 week old female C57BL/6 mice were used for immunization studies.

Briefly, wild type NY-ESO-I protein was dissolved in 100 μl of PBS, which were used to inject mice intraperitoneally at the abdominal site. No immune adjuvant was used and only one injection was carried out. Blood from immunized mice were collected through tail veins 2 weeks after immunization and the serum was obtained using a centrifugation serum collection tube. About 3 μg of purified chicken ovalbumin protein (OVA) (Sigma, St. Louis, MO) was also used as controls to immunize the C57BL/6 mice.

[137] For immunization with recombinant Vaccinia virus, 10⁷ viral particles per mouse were injected through the tail vein. For DNA immunization, 100 μg of plasmid DNA (obtained using an endotoxin-free maxiprep kit, Qiagen Inc., Santa Clarita, CA) was injected through intramuscular and intradermal routes per mouse at each time. A total of 3 immunizations were performed with a 2-week interval between every two injections per mouse. Blood from immunized mice were also obtained through the tail vein.

[138] Detection of NY-ESO-I antibodies present in mouse serum was carried out using an ELISA approach as previously described. See Zeng, G. et al. (2000) J. Immunol. 165:1153-1159. The antigen used to coat ELISA plates was ESO 1-74, which contains a B cell epitope that murine antibodies against NY-ESO-I specifically recognize. To analyze the subtypes of antibodies elicited by vaccination, goat-anti- mouse IgGl, IgG2a, and IgM conjugated with horseradish peroxidase (HRP) (CALTAG Laboratories, Burlingame, CA) was used as the secondary antibody, respectively, to substitute the secondary Ab (Zeng, G. et al. (2000) J. Immunol. 165:1153-1159) in the ELISA.

Example 5 Induction of Human Dendritic Cells (DC) From Monocytes, Conjugate FITC with

Monoclonal Antibodies, and the DC Binding Assay

[139] Human DC were generated from adherent monocytes in the presence of 1000 unit/ml granulocyte macrophage colony- stimulating factor (GM-CSF) and 1000 unit/ml IL-4 for 6 days. CD40L-B cells were generated from PBMC by stimulating with 500 ng/ml trimeric CD40 ligand (Immunex Corp., Seattle, WA) and IL-4 (500 unit/ml) for 7 days. Iscove's modified medium (Invitrogen, Carlsbad, CA) supplemented with 10% human male serum (BioCheMed Corp., Charleston, SC) was used for cell culture.

[140] Direct conjugation of fluorescein isothiocyanate (FITC) with NY-ESO- 1 protein failed due to the low solubility of recombinant NY-ESO-I at the concentrations (greater about 2 mg/ml) required carrying out the reaction. Monoclonal antibodies specific for NY-ESO-I, mAbl31 and mAbl32 (Zeng, G. et al. (2000) J. Immunol. 165:1153-1159), were chosen to label with FITC following the manufacturer's instruction (Sigma-Aldrich, St. Louis, MO). The molecular ratios of FITC to both monoclonal antibodies were determined as between 4-6 according to a procedure recommended by the manufacturer. The labeled monoclonal antibodies were diluted to 0.5 μg/ml as the stock; and 0.5 μl of this stock was used for staining 10⁵ cells pulsed with NY-ESO-I .

[141] All protein-DC binding experiments were carried out on ice and in the presence of 5% fetal bovine serum to block non-specific interactions. Unless specified, the protein concentrations used were 10, 30, and 4 μg/ml for NY-ESO-I, gplOO, and ESO 1-74, respectively, in order to maintain substantially equal molarities. After a 30-minute pulse, the cells were washed using cold PBS supplemented with 5% fetal bovine serum. FITC-labeled NY-ESO-I specific monoclonal antibody or first antibody, such as mAb 131 or mAb 132 plus a FITC-labeled goat-anti-mouse secondary antibody (BD Pharmingen, San Diego, CA) were used to stain cells on ice followed by flow cytometry analysis.

Example 6

Effect of NY-ESO-I on Human Immature Dendritic Cells (DC)

[142] A. Amount of F-actin. The amount of F-actin from human immature DC following NY-ESO-I ligation was measured. As shown in Figure IA, the mean fluorescent intensity of F-actin stained with FITC-labeled phalloidin on immature DC treated with various reagents was compared to DC incubated with medium alone. DC were fixed for 15 minutes in the dark, washed in PBS, resuspended in 100 μl of Leucoperm reagent B, with 5 μl of phalloidin-FITC (Sigma, St Louis, MO) and incubated at room temperature for 30 minutes in the dark. Immediately after washing, the cells were analyzed using a Becton Dickinson Facscan (Becton Dickinson, San Jose, CA). [143] The ratios, which represent 4 independent experiments with immature DC from 4 different donors, were plotted. Student t-tests indicated significant difference (p<0.05) between samples treated with NY-ESO-I, LPS, LPS+PK vs. untreated samples.

[144] Figure IB shows images of F-actin stained with FITC-labeled phalloidin and nucleus stained with DAPI on Day-6 immature DC pre-treated with medium, clinical GMP grade gplOO protein at 10 μg/ml, clinical GMP grade NY-ESO-I at 3 μg/ml, NY-ESO-I pretreated (incubated at 37 ⁰C for over 8 hours) with proteinase K (PK), LPS at 100 ng/ml, and LPS pretreated (incubated at 37 ⁰C for over 8 hours) with proteinase K.

[145] As provided in Figures IA and IB, F-actin was significantly increased in immature DC treated with NY-ESO-I as compared to the medium alone or the gplOO protein. The impact of NY-ESO-I on actin re-organization was diminished when NY-ESO-I was pre-treated with proteinase K, thereby indicating that the reorganization of F-actin in immature DC was accounted for by NY-ESO- 1.

[146] B. Redeployment time of F-actin. The time course for the redeployment of F- actin was determined as disclosed herein. It was found that strong F-actin staining started between about 1 and 3 hours and peaked at about 16 hours after ligation with NY-ESO-I. Moreover, NY-ESO-I did not induce apparent F-actin in DC matured by CD40L.

[147] C. Expression of co-stimulatory molecules. Figure 1C shows expression of

CD83 and ICAM (CD54) on the surface of immature DC after incubation with medium (shaded histogram) and the indicated reagents. In these experiments, using methods known in the art, immature human DC on day 6 was co-cultured with the indicated reagent overnight before being stained with FITC-labled antibodies against CD83 and ICAM followed by flow cytometric analysis.

[148] Among the co-stimulatory molecules tested, CD80, CD83, CD86, OX40L,

B7H, B7Hx, only CD83 had apparent upregulation in response to NY-ESO-I ligation. See Figure 1C. Such upregulation was diminished when NY-ESO-I was treated with proteinase K prior to the incubation; whereas proteinase K-treated LPS did not have obvious impact on the level of CD83 expression on DC surface. Up-regulation of cell surface ICAM was also determined as a result of the ligation to NY-ESO-I . See Figure 1C. [149] D. Chemokine and cytokine secretion. Multiplex chemokine/cytokine secretion in the supernatant of human immature DC co-cultured with the indicated reagents. Monocyte-derived immature DC from one healthy donor were used as the target cells. About 2 x 10⁶ cells were incubated in 5 ml OPTI-MEM® medium (Invitrogen, Carlsbad, CA) in a 6-well culture dish for 12 hours before being assayed using SEARCHLIGHT® chemiluminescent ELISA kits (Thermo Fisher Scientific, Inc., Boston, MA). Similar results were obtained using immature DC from a second donor using the protocol as described above.

[150] Furthermore, 16 human cytokines and chemokines were screened to profile the activation of immature DC after ligation with NY-ESO-I . As shown in Figure ID, 5 of 16 were secreted at least about 40% more by immature DC from at least two donors treated with NY-ESO-I as compared to controls that were treated with culture medium alone or NY-ESO-I pre-incubated with proteinase K. The 5 of 16 were ThI- type MIP-Ia, MIP-I β, TARC, TNF-α, and Th2-type IL-6. Other cytokines and chemokines that were tested included ITAC, MDC, MIG, RANTES, IFNα, ILl α, ILlβ, ILlO, IL12P40, and IL12p70. Secretion of RANTES, IL-12, and IL-IO was observed from one experiment but not the other (data not shown). To rule out the possibility of LPS involvement, we showed that NY-ESO-I pretreated (incubated at 37 ⁰C for over 4 hours) with proteinase K abolished induction of cytokines/chemokines from immature DC; whereas LPS pretreated (incubated at 37 0C for over 4 hours) with proteinase K had no apparent effects.

[151] E. Activation blocking by antibodies . Activation of bone-marrow derived immature DC from mouse was also observed and was shown to be blocked by sera from prostate cancer patients who possessed anti-NY -ESO-I antibodies using methods known in the art.

Example 7

Effect of Oligomeric Structure of NY-ESO-I [152] NY-ESO-I forms oligomers held together by intermolecular disulfide bonds.

The effect of the oligomeric structure of NY-ESO-I on its activity was evaluated. [153] NY-ESO-I encoded by bacterial (Zeng, G. et al. (2000) J. Immunol.

165:1153-1159) and mammalian cells, formed oligomers even in the presence of low concentrations of β-mercaptoethanol. As shown in Figure 2A, human 293 cells transfected with a plasmid encoding NY-ESO-I (lane 1) and NY-ESO-I fused with an N-terminal Flag tag (Flag-ESO, lane 3) reacted with mAB131 and M2 monoclonal antibody specific to NY-ESO-I and the Flag epitope, respectively. Lanes 2 and 4 contained lysates from 293 cells transfected with a plasmid encoding the green fluorescent protein. Western blotting methods known in the art were employed.

[154] NY-ESO-I, ESOcsl, ES0cs2, and ES0cs3 were further analyzed to determine the role of cysteines in forming oligomeric structures. After Ni²⁺ affinity chromatography, the polypeptides were analyzed by Western blot using mAB 131. As shown in Figure 2B, NY-ESO-I showed clear formation of dimers, trimers, and even oligomers above about 130 kDa, ESOcsl and ES0cs2 were present as monomers and dimers, and ES0cs3 appeared only as monomers. Thus, the oligomeric structure of NY-ESO-I is due to inter-molecular disulfide bonds.

[155] Figure 2C shows that NY-ESO-I present in cancer cell lines and human embryonic stem cell lines are predominantly tetramers under the conditions employed. See Zeng, G. et al. (2000) J. Immunol. 165:1153-1159. Western blot on melanoma cell lines (lane #1 and #2) and human embryonic stem cells of passage 3, 17, and 70 (lane #3-5) with recombinant NY-ESO-I (lane #6) as control.

[156] Binding and activation of immature DC was found to depend on the oligomeric structure of NY-ESO-I as cysteine to serine substitutions reduced or inhibited its ability to bind immature DC in vitro. See Zeng, G. et al. (2000) J. Immunol. 165:1153-1159. As shown in Figure 2D, NY-ESO-I (1 μg/ml), but not ES0cs3, was able to bind to immature DC. ESOR62H was a control mutant with an Arg to His change at amino acid position 62 of NY-ESO-I. NY-ESO-I pretreated (incubated at 37 ⁰C for over 4 hours) with proteinase K significantly reduced or inhibited its binding to immature DC.

[157] These experiments evidence that modifications to the oligomeric structure or sequence of NY-ESO-I modifies its activity on immature DC.

Example 8

NY-ESO-I Activation of Immature DC is Dependent on TLR4

[158] To determine the mechanism by which immature DC are activated by NY-

ESO-I, NY-ESO-I, ES0cs3, and the secreted version of NY-ESO-I, s-ESO, were studied.

[159] The amount of IL-6 secreted from bone marrow derived immature DC of wild- type, TLR2-/-, and TLR4-/- mice was evaluated. Specifically, DC were cultured in medium from the supernatant of 293 cells transfected with plasmids encoding cell- surface anchored NY-ESO-I, secreted NY-ESO-I, secreted ES0cs3, or green fluorescent protein (GFP), in the same medium containing exogenously added recombinant NY-ESO-I (3 μg/ml), lipopolysacchride (LPS, Sigma, St. Louis, MO) (100 ng/ml), or CPG (oligo DNA, Cell Science, Canton, MA) (1 μM). As provided in Figure 3 A, medium from 293 cells transfected with a plasmid encoding secreted NY- ESO-I, but not secreted ES0cs3 or the cell-surface anchored NY-ESO-I, was able to activate bone marrow-derived immature DC from wild-type C57BL/6 mice and TLR2-/- mice, but not MyD88-/- or TLR4-/- mice.

[160] The amount of secreted NY-ESO-I in the medium was estimated to be less than about 20 ng/ml by Western blot (data not shown), suggesting a powerful immune modulatory effect of the NY-ESO-I protein. DNase treatment of the plasmid pRANTES-ESO prior to transfection into 293 cells significantly reduced or inhibited the activation of immature DC, thereby ruling out the involvement of LPS in the plasmid preparation (data not shown).

[161] Bone marrow DC derived from wild-type, TLR2-/-, and TLR4-/- mice were used for binding to NY-ESO-I, which was subsequently detected by staining with a FITC-labeled anti-ESO Ab (Zeng, G. et al. (2000) J. Immunol. 165:1153-1159). Figure 3B shows that knockout of TLR4, but not TLR2, partially reduced or inhibited binding of NY-ESO-I to bone marrow derived DC in vitro.

[162] Figure 3C shows that TLR4 co-precipitated with NY-ESO-I, but not gplOO control, thereby evidencing a physical interaction between NY-ESO-I and TLR4. In these experiments, membrane fractions of DC2.4 were co-incubated with NY-ESO-I (lane 1) and His-gplOO (lane 2), followed by precipitation with anti-His antibodies. Western blot was conducted with anti-TLR4 Ab (IMGENEX, San Diego, CA) using methods known in the art. Lane 3 is DC2.4 membrane fraction showing TLR4 bands.

[163] These experiments indicate that NY-ESO-I activates immature DC through binding to TLR-4 as a co-receptor.

Example 9 Switch T Helper-Dependent Antibody Responses Through Differential Engagement of TLR4 in vivo

[164] Because NY-ESO-I acts as an antigen and a TLR4 ligand, the effects of TLR4 on adaptive immune responses was further studied by immunizing C57BL/6 mice with reagents as listed in individual figures (ESO = NY-ESO-I, beta gal = β- galactosidase, ESOiv = Vaccinia virus encoding NY-ESO-I, naϊve = no protein (control)).

[165] Ten μg of NY-ESO-I, 45 μg β-galactosidase, or 10⁷ pfu Vaccinia virus encoding NY-ESO-I (wESO) were compared for eliciting Ab responses in HLA- A2.1/Kb mice (gift from Linda Sherman at the Scripps Institute, La Jolla, CA). Proteins were prepared in PBS without adjuvant and were injected via the intraperitoneal route. Two weeks after only one injection, Ab was measured against ESO 1-74 and β-galactosidase using a ELISA protocols known in the art (e.g. Zeng, G. et al. (2000) J. Immunol. 165:1153-1159).

[166] Interestingly, NY-ESO-I in the absence of any adjuvant successfully elicited a high Ab titer against NY-ESO-I after only one immunization at the dosage of 10 μg/mouse. As shown in Figure 4A, the Ab titers elicited by immunization with NY- ESO-I were even higher than that induced by recombinant Vaccinia virus at 10⁷ pfu, which was traditionally regarded as a strong immunogen for mice.

[167] Subsequently, lower doses of recombinant NY-ESO-I (i.e. 3 and 0.3 μg) were used to immunize mice and the subtypes of Ab responses were evaluated. A previously known immunogenic protein for mice, chicken ovalbumin (ova), was used as a control. Specifically, ova and NY-ESO-I free of adjuvant were used at two doses (i.e. 3 and 0.3 μg/mouse for immunization via the intraperitoneal route). Serum samples from two mice in each group were measured for Ab against ESO 1-74 and ova in an ELISA assay. The average OD450 against the control protein was subtracted from the OD against the target protein. Specific secondary Ab against IgGl, IgG2a, and IgM were used for the detection of Ab isotypes using methods known in the art. See Zeng, G. et al. (2000) J. Immunol. 165:1153-1159.

[168] As shown in Figure 4B, recombinant NY-ESO-I was able to induce IgG2a Ab even at 0.3 μg/mouse in the absence of adjuvant. Induction of IgGl Ab was observed at the 3 μg dose in the absence of adjuvant. The Ab was not directed against the polyhistidine tag because the same sera did not react with the his-tagged gplOO protein (data not shown). To exclude the possibility that the antibody was against a contaminating immunogenic molecule in the protein preparation, ELISA plates coated with ESO 1-74 were used as the target in ELISA. Under the same conditions, ova failed to raise any detectable IgG responses. There was only a marginal indication of raising IgM Ab when 3 μg of ova was used in the absence of adjuvant. Induction of Ab of the IgG subtypes, including IgGl and IgG2a rather than IgM, normally required the involvement of both ThI and Th2 types of CD4+ T helper cells, suggesting that wild-type NY-ESO-I effectively engages CD4⁺ T cells through professional antigen presenting cells (APC), such as DC. [169] The wild type and CS variants of NY-ESO-I were also used to immunize c57/B16 mice in an attempt to investigate the ThI and Th2-dependent IgG antibodies induced by various vaccines as a function of the oligomeric structure in Figure 4C, and Figure 4D, respectively, using methods known in the art. See Zeng, G. et al. (2000) J. Immunol. 165:1153-1159.

Example 10 NY-ESO-I Modulates Immune Responses to Allergens

[170] NY-ESO-I engages human and mouse DC through CRT and TLR4, and apparently giving rise to rapid phagocytosis coupled with TLR signaling, thus NY- ESO-I and its variants are potential candidates as molecular adjuvants for polarizing DC towards ThI and/or Th2 responses. It was hypothesized that NY-ESO-I fused with an allergen modulates the ratio of IgGl/IgG2a antibodies against the specific allergen, thereby having an effect on the anaphylactic potential of IgGl antibodies. Therefore, the immune modulatory effects of NY-ESO-I on two different allergens, as fusion protein products was studied.

[171] To facilitate a rapid evaluation, plasmids encoding NY-ESO-I fused with Bet via, the major birch pollen allergen, and Art vl, the major allergen of mugwort pollen, were used as plasmid DNA vaccines and tested in Balb/c mice. See Hochreiter, R., et al., (2003) THl -promoting DNA immunization against allergens modulates the ratio of IgGl/IgG2a but does not affect the anaphylactic potential of IgGl antibodies. J. Allergy and Clin. Immunol. 112(3):579-584; and Himly, M., et al. (2003) Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain 10.1096/fj.02-0472fje. FASEB J. 17(1): 106-108. Specifically, using methods known in the art, 50 μg of plasmid DNA coated on gold particles were delivered through a gene gun (Bio-Rad Laboratories, Hercules, CA) intradermally to the mice and antibodies were analyzed 3 weeks following the immunization.

[172] Total IgG antibodies against the allergen Bet vl were measured following the course of gene gun immunization with the indicated plasmids encoding Bet vl alone or Bet vl fused with NY-ESO-I . As shown in Figure 5 A, the antibody-mediated immune responses to the Bet via allergen when it is fused with the NY-ESO-I protein was reduced or inhibited in mice. Arrows indicated the points of gene gun immunization.

[173] On the other hand, as shown in Figure 5B, immune responses to Art vl were significantly enhanced when a plasmid encoding the fusion protein was used to immunize mice by gene gun. Subclasses of IgG antibodies against Art bl and NY- ESO-I proteins were measured 2 weeks after the 3^rd gene gun immunization.

[174] In these experiments, 3 Balb/c mice were used for each group. Immunizations were done by intramuscular priming and intradermal boosting with the same pcDNA3 (Invitrogen, Carlsbad, CA) based plasmid encoding CA9-ESO. The results are from pools or averages of the 3 mice for each group.

Example 11

NY-ESO-I as a Molecular Adjuvant in Cancer Vaccines [175] The induction of T helper dependent antibodies may result in anti-tumor immunity in hosts bearing CA9-positive cancers. Thus, the ability of NY-ESO-I as a fusion partner to enhance the immune recognition of carbonic anhydrase 9 (C A9), a membrane-anchored TAA with expression in renal cancer and cervical cancer, was evaluated. The polypeptide evaluated, CA9-ESO, was obtained using methods known in the art and has the following amino acid sequence (UPPER CASE indicates the CA9 sequence, bold indicates a linker, lowercase indicates NY-ESO-I): MAPLCPSPWLPLLI PAPAPGLTVQLLLSLLLLVPVHPQRLPRMQEDSPLGGGSSGEDDPLG EEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSLKLEDLPTVEAPGDP QEPQNNAHRDKEGDDQSHWRYGGDPPWPRVS PACAGRFQSPVDIRPQLAAFCPALRPLELL GFQLPPLPELRLRNNGHSVQLTLPPGLEMALGPGREYRALQLHLHWGAAGRPGSEHTVEGH RFPAE IHWHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSAYEQLLSRLEEIAEEGSE TQVPGLDI SALLPSDFSRYFQYEGSLTTPPCAQGVIWTVFNQTVMLSAKQLHTLS DTLWGP GDSRLQLNFRATQPLNGRVIEAS FPAGVDSS PRAAEPVQLNSCLAAGDI LALVFGLLFAVT

SVAFLVQMRRQHRRGTKGGVS YRPAEVAETGAHmqaegrgtggstgdadgpggpgipdgpg gnaggpgeagatggrgprgagaarasgpgggaprgphggaasglngccrcgargpes rl le f ylampf atpmeaelarrs laqdapplpvpgvl lkef tvsgni ltirltaadhrqlql s i s s clqql s llmwitqc flpvflaqppsgqrr ( SEQ I D NO : 2 6 ) . [176] Bal/c mice were immunized with intradermal injection of plasmid DNA vaccines encoding CA9 alone, NY-ESO-I alone and CA9-ES0 with a 2-week interval between immunizations. About 100 μg of plasmid were injected into balb/c mice intramuscularly, followed by 2 injections (intradermally through a gauge 25 needle) of 50 μg plasmid with 2 weeks intervals. After the 3^rd injection, mice immunized with CA9-ESO, but not CA9 alone, developed specific antibodies against CA9 as shown in the Western blot of Figure 5C.

[177] Gpl00:201-220-ESO, which is NY-ESO-I fused with gpl00:201-220 from the human tumor-associated antigen gplOO, was also studied. Gpl00:201-220-ESO was made using methods known in the art and has the following amino acid sequence (UPPER CASE indicates GplOO sequence, bold indicates a histadine tag, lowercase indicates NY-ESO-I):

MGSSHHHHHHSSGLVPRGSHMAHS SSAFTI TDQVPFSVSVSmqaegrgtggstgdadgpgg pgipdgpggnaggpgeagatggrgprgagaarasgpgggaprgphggaasglngccrcgar gpe srllef ylampf atpmeaelarrs laqdapplpvpgvl lkef tvsgni ltirltaadh rqlql si s s clqql s llmwitqc f lpvf laqppsgqrr ( SEQ I D NO : 27 ) . [178] As shown in Figure 5D, gpl00:201-220-ESO, but not the gplOO alone, was cross-presented to human gpl00:208-217 specific CD8+ T cells in vitro. [179] Therefore, these experiments show that NY-ESO-I thus may serve as adjuvant to enable its fusion partners to be cross-presented by professional antigen presenting cells, such as DC, macrophages, and the like.

Example 12

PSMA-ESO and Myc-CaP/ESO Cells

[180] Primary c-myc tumors and Myc-CaP tumor lines share key molecular signatures with the human disease. For example, PSMA (or folate hydrolase 1 , FoIh 1), like other androgen-regulated proteins, is overexpressed more than 4 fold in cancerous versus healthy prostate tissues. See llwood-Yen, K., et al. (2003) Myc- driven murine prostate cancer shares molecular features with human prostate tumors. Cancer Cell 4(3):223-38. PSMA thus confers a valid target for targeted DC vaccines in the Myc-CaP tumor model. Another candidate vaccine is the irradiated Myc-CaP cell line expressing membrane-anchored NY-ESO- 1. In contrast to antigen-specific PSMA-ESO vaccine, Myc-CaP/ESO delivers whole tumor cells with the potential of inducing a robust and broad-based antitumor response. Thus, targeted DC vaccines in the form of ESO-NY-I and PSMA fusion proteins and irradiated Myc-CaP/ESO may induce phagocytosis and DC maturation in vitro. [181] Using methods known in the art, a vector such as pET-28 (Novagen, Madison,

WI) may be used to express a fusion protein having PSMA fused to NY-ESO-I and the PSMA protein, with or without a polyhistidine tag on the N-terminal ends. An example of the fusion protein is PSMA-ESO which has the following amino acid sequence (UPPER CASE indicates PSMA sequence, bold indicates a linker, lowercase indicates NY-ESO-I):

MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAF LDELKAENIKKFLHNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNK THPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTE DFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPD GWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKL LEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAV EPDRYVILGGHRDSWVFGGIDPQSGAAWHEIVRSFGTLKKEGWRPRRTILFASWDAEEFG LLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGF EGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGY PLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADK IYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFL ERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQI

YVAAFTVQAAAETLSEVAHmqaegrgtggstgdadgpggpgipdgpggnaggpgeagatgg rgprgagaarasgpgggaprgphggaasglngccrcgargpesrllefylampfatpmeae larrslaqdapplpvpgvllkeftvsgniltirltaadhrqlqlsissclqqlsllmwitq cflpvflaqppsgqrr (SEQ ID NO:28) .

[182] The resulting polypeptides may be purified using methods known in the art such as metal chelating affinity chromatography followed by ion-exchange chromatography. Any suitable expression system known in the art may be used, but a mammalian expression system is preferred in order to ensure expression of a suitable NY-ESO-I tertiary structure. PSMA may be conjugated to Keyhole Limpet Hemocyanin (KLH) (Sigma, St. Louis, MO) using methods known in the art to serve as a control. Previous experiments showed that KLH (and potentially KLH conjugated proteins) did not possess the DC-binding properties as NY-ESO-I . See Zeng, G., et al. (2006) Dendritic Cell Surface Calreticulin Is a Receptor for NY-ESO- 1 : Direct Interactions between Tumor- Associated Antigen and the Innate Immune System. J Immunol. 177(6):3582-3589. Example 13 PSMA-ESO Binding, Kinetics of Phagocytosis and DC Maturation in vitro

[183] In the following experiments, NY-ESO-I may be used as a positive control and PSMA-KLH may be used for comparison.

[184] A. Binding to DC. Methods known in the art to measure direct interactions between DC and the proteins of interest may be used. See Zeng, G., et al. (2006) Dendritic Cell Surface Calreticulin Is a Receptor for NY-ESO-I : Direct Interactions between Tumor- Associated Antigen and the Innate Immune System. J Immunol. 177(6):3582-3589. The DC binding experiments may be performed on ice with all cells and reagents pre-chilled to minimize the macropinocytosis and phagocytosis of immature DC, which depends on physiological temperature to activate actin assembly and a series of enzymatic activities.

[185] After being pulsed with PSMA-ESO or PSMA on ice, immature DC derived from bone marrow of FVB mice (Taconic, Germantown, NY) or control splenocytes derived from the same donor may be washed twice and then incubated on ice with polyhistidine specific monoclonal Ab labeled with fluorescein isothiocyanate (FITC). Proteins at an increased concentration of 0.1, 0.3, 1, 3, 10, and 30 μg/ml will be co- incubated with 10⁵ DC followed by FITC-labeled polyhistidine specific Ab staining and flow cytometric analysis, which will give rise to an estimated apparent affinity between the protein of interest and DC in vitro.

[186] B. Involvement of TLR4 in the binding to DC . Involvement of TLR4 in the binding between NY-ESO-I and DC was elucidated in the DC binding experiment by comparing binding to bone marrow derived DC from wild-type mice, TLR2 ^{~ ~}, and TLR4 ^{~ ~} knockout mice. The decreased binding affinity to bone marrow derived DC obtained from TLR4 ^{~ ~} mice indicated a role of TLR4 in the interaction between NY- ESO-I and PSMA-ESO with DC.

[187] C Kinetics of phagocytosis by immature DC. The protocol for a "pulse and chase" experiment will be followed. In this experiment, immature DC will be allowed to bind to PSMA-ESO (pulse), washed and followed by intracellular staining of the amount of acquired protein (chase). Since all proteins involved in this experiment possess polyhistidine tag, His-specific monoclonal antibodies will be employed for intracellular staining and flow cytometric analysis at various time points, such as 30 second, 1, 2, 4, 8, 16, 32, 64, and 128 minutes. Confocal microspcopic analysis will be accompanied to ensure the intracellular localized proteins correlate with the data of flow cytometric analysis. It is hypothesized that PSMA-ESO will have rapid kinetics of DC phagocytosis whereas PSMA-KLH will be mainly acquired by a slower process. The potential involvement of CRT in the rapid phagocytosis process will be delineated in a blocking experiment similar to the one discussed in the above paragraph.

[188] D. DC maturation. It is believe that DC undergo a concerted process of phagocytosis coupled with maturation upon ligation with NY-ESO-I . Thus, upregulation of co-stimulatory molecules such as CD83 and ICAM can be used to characterize DC maturation. At the same time, IL-6 and IL- 12 secretion from DC upon ligation with PSMA-ESO can be measured by ELISA. Proteins treated with proteinase K may also be included in the experiment to exclude the possibility that LPS carried over during protein purification is involved in the maturation of DC. NY-ESO-I and PSMA-KLH proteins will serve for comparisons in all above experiments.

[189] In the event that the proximity of two fusion proteins results in one having an adverse effect on the activity of the other, an amino acid linker of a suitable length may be employed between the two fusion partners.

Example 14

Cell-Surface Anchored NY-ESO-I Encoded by Myc-CaP Vaccines in DC Phagocytosis and Maturation

[190] Protein display libraries, pDisplay-ESO and pDisplayLAGE encoding cell- surface anchored NY-ESO-I and LAGE-I, were created using methods known in the art. Sufficient expression of cell-surface NY-ESO-I has been confirmed in 293 cells transiently transfected with the expression vectors.

[191] To facilitate experiments of the kinetics of DC-mediated phagocytosis of irradiated Myc-CaP/ESO (a Myc-CaP cell line expressing cell-surface anchored NY- ESO-I protein) or Myc-CaP/LAGE (a Myc-CaP cell line expressing cell-surface anchored LAGE-I protein) as vaccines, a retrovirus encoding GFP may be used to transduce Myc-CaP, Myc-CaP/ESO, and Myc-CAP/LAGE, to give Myc-CaP/GFP, Myc-CaP/ESO/GFP, Myc-CaP/LAGE/GFP labeled cell lines. These GFP-labeled cell lines will be lethally irradiated at 3500 rad, and used to feed immature DC for phagocytosis experiment as proposed below. Forced cell-surface expression of NY- ESO-I or LAGE-I may directly engage DC surface receptors or may be coated with complement CIq receptor (CRT) from endogenous or exogenous sources, which are known to send DC and macrophage an "eat me signal" and are thus associated with enhanced immunogenicity in vivo. See Ogden, C.A., et al. (2001) CIq and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells. J. Exp. Med, 194(6):781-95; Vandivier, R.W., et al. (2002) Role of surfactant proteins A, D, and CIq in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex. J. Immunol. 169(7):3978-86; and Obeid, M., et al. (2007) Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat. Med. 13(1): 54-61.

[192] Experiments similar to those set forth in Example 13 may be conducted using irradiated Myc-CaP/GFP and Myc-CaP/ESO/GFP labeled cell lines. The direct binding experiments may be conducted by fluorescent microscopic methods known in the art. Surface expression of NY-ESO-I and LAGE-I may lead to binding of irradiated tumor cell vaccines onto DC.

Example 15

Immunological Responses and Anti-Tumor Efficacies in the Transplanted Myc-CaP Prostate Cancer Model in Syngeneic FVB Mice

[193] Subcutaneously injected PSMA-ESO and irradiated Myc-CaP/ESO cell lines may be able to engage DC at the vaccine sites. Since immature DC may efficiently uptake and process vaccines, undergo maturation, and migrate to draining lymph nodes where DC activate effector T helper cells while keeping the CD4⁺CD25⁺ Treg in check to result in efficient cross-priming of CTL and hence anti-tumor efficacies, targeted DC vaccines eliminate the labor-intensive ex vivo DC manipulations.

[194] A. In vivo protective immunity. To compare the protective immunity generated in vivo, immunization experiments known in the art may be conducted with the candidate vaccines. See Wang, H. Y., et al. (2002) Induction of CD4(+) T cell- dependent antitumor immunity by TAT-mediated tumor antigen delivery into dendritic cells. J. Clin. Invest. 109(11): 1463-70; and Dranoff, G., et al. (1993) Vaccination with Irradiated Tumor Cells Engineered to Secrete Murine Granulocyte- Macrophage Colony-Stimulating Factor Stimulates Potent, Specific, and Long- Lasting Anti-Tumor Immunity. PNAS USA 90(8):3539-3543. Generally, the candidate vaccine will be administrated twice with a 2-week interval. The candidate vaccines will be injected via the subcutaneous route under the footpads and tail base; whereas live tumor will be injected via the subcutaneous route on the right back. [195] Various regimen of priming with the irradiated cell vaccine and boosting with the polypeptides of the present invention may be investigated using methods known in the art. [196] For example, on day 0, male FVB mice of 6-8-week of age (Taconic,

Germantown, NY) may be first immunized (primed) with one of the vaccine regimens as provided in Table 1 as follows:

[197] On day 10, each FVB mouse will be inoculated with 2 x 10⁵ Myc-Cap tumor cells suspended in PBS. Under normal conditions with no immunization, tumors will form 3-4 weeks following inoculation, and grow to 1 cm in diameters after another 3- 4 weeks. Second vaccine (boost) dose will be administrated on day 14, 4 days after the tumor inoculation. PSMA-ESO at various amounts, e.g. 5, 10, 20, 40, 80 μg in PBS, as well as irradiated tumor cell vaccines at various amounts, e.g. 2.5 x 10⁵, 5 x 10⁵, 1 x 10⁶, and 2.5 x 10⁶, may be tested to determine the optimal dosage for protecting the 2 x 10⁵ inoculation prior to pursuing the entire experiment.

[198] DC infiltrating the vaccine sites at 12, 24, 48, and 72 hours post immunization may be measured to assess the mechanism of action for the vaccine candidates. Examination of influx of monocytes, granulocytes, and activated lymphocytes and paracortical hyperplasia in the local draining lymph nodes (for example, the bilateral hindlimb popliteal and the inguinal lymph nodes) may also be followed.

[199] B. Therapeutic efficacy . The therapeutic efficacy of DC-targeted vaccines may be evaluated in a 3 -day subcutaneous model using methods known in the art.

[200] In case the proposed vaccines do not exhibit more efficacy in cancer prevention or treatment models than controls, 1) Ab against CTLA-4 may be added after the DC-targeted vaccine based on what have been previously shown to synergize with the GM-CSF-secreting tumor cell vaccines and cell vaccines encoding co- stimulators, and 2) a lung metastases model instead of the subcutaneous model may be used as previous experiments indicate that a lung metastases model is often more responsive to DC-based vaccines.

Example 16 Immune Effectors and Corresponding Surrogate Markers

[201] The immune effectors induced by a candidate vaccine may be determined and immunological markers corresponding to the immune effectors may be used as surrogate markers for future studies.

[202] A. Ab-mediated depletion to determine candidate immune effectors . Methods known in the art may be used to delineate the contributing immune effectors in the prevention and treatment models. For example, 200 μg of anti-CD4 (GKl .5), anti- CD8 (2.43), or control Ab in 500 μl of PBS may be injected intraperitoneally into each KVB mouse on the day before tumor challenge (or before administration of the 1^st vaccine for the therapeutic model), followed by three injections on days 1, 3, and 10 after tumor injection (or vaccine administration in the therapeutic model). Depletion of CD4⁺ or CD8⁺ T cells may be determined by FACS analysis of splenocytes following the last Ab injection in selected mice. The impact of T cell subset depletion on vaccine efficacy may be taken as an indication that such subset plays the effector role in the corresponding tumor model.

[203] In case CD4⁺ T cells are largely responsible for the vaccine effects, further methods known in the art to elucidate Ab involvement may be conducted. For example, adoptive transfer of Ab in vaccinated subjects will be transferred to naive subjects, followed by proper tumor challenge. The transfer of protective immunity will be taken as evidence that Ab contribute to the vaccine effects in vivo.

[204] B. Monitoring the immunologic response relevant to the above effectors induced by vaccines. Due to the lack of defined epitopes from TAA in the Myc-CaP model, one may use cell lines as targets to incubate with ex vivo purified CD8⁺ and CD4⁺ T cells from spleen and regional draining lymph nodes, e.g. the bilateral hindlimb popliteal and the inguinal lymph nodes for vaccines injected through the rear foot pads, using methods known in the art. See Zeng, G., et al. (2000) Identification of CD4+ T cell epitopes from NY-ESO-I presented by HLA-DR molecules. J Immunol. 165(2): 1153-9. IFN-γ ELISPOT assays known in the art may be conducted to assess the frequencies of tumor- specific CTL and T helper cells induced by individual vaccine regimens. [205] PSMA is a membrane-associated protein over-expressed in Myc-CaP comparing to healthy prostate tissues in mice. If PSMA-ESO is found to induce protective or therapeutic immunity, PSMA-specific Ab may likely function as the effector. Extensive PSMA-specific Ab assays using methods known in the art may be conducted. See Zeng, G., et al. (2000) Identification of CD4+ T cell epitopes from NY-ESO-I presented by HLA-DR molecules. J Immunol. 165(2): 1153-9; and Zeng, G., et al. (2001) CD4(+) T cell recognition of MHC class II-restricted epitopes from NY-ESO-I presented by a prevalent HLA DP4 allele: association with NY-ESO-I antibody production. PNAS USA 98(7):3964-9.

[206] To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.

[207] Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.

Claims

We claim:

1. A protein comprising an NY-ESO-I polypeptide having at least one peptide which is not naturally associated with the NY-ESO-I polypeptide fused to the N-terminus or the C- terminus of the NY-ESO-I polypeptide.

2. The protein of claim 1, wherein the NY-ESO-I polypeptide is selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; amino acid residues 74-180 of SEQ ID NO: 1 ; SEQ ID NO:9; SEQ ID NO: 12; and SEQ ID NO: 13, said NY-ESO-I polypeptide may optionally have up to five amino acid residues truncated from the N- terminus, the C-terminus, or both.

3. The protein of claim 1, wherein the peptide is tag, a linker, all or part of a leader sequence, all or part of an antigen, all or part of an allergen, all or part of a transmembrane domain of a receptor, or all or part of an androgen-regulated protein.

4. The protein of claim 3, wherein the leader sequence is an Ig k chain leader sequence or a leader sequence from a human RANTES precursor; the tag is a hamagglutinin A tag, a Flag tag, or a histadine tag, or a Myc epitope tag; a carbonic anhydrase 9 antigen, a human tumor- associated antigen gplOO; the allergen is Bet via, Art vl or PSMA antigen, and the androgen- regulated protein is a platelet derived growth factor receptor transmembrane domain.

5. The protein of claim 1, wherein the peptide is selected from the group consisting of:

MDYKDDDDK (SEQ ID NO: 29); METDTLLLWVLLLWVPGSTGD (SEQ ID NO:30); YPYDVPDYA (SEQ ID NO: 31); GAQPAR (SEQ ID NO:32); QVDEQKLISEEDL (SEQ ID NO:33);

NGVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 34) ;

MKVSAAALAVILIATALCAPASA (SEQ ID NO:35);

MAPLCPSPWLPLLIPAPAPGLTVQLLLSLLLLVPVHPQRLPRMQEDSPLGGGSSGED DPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSLKLEDLPTVE APGDPQEPQNNAHRDKEGDDQSHWRYGGDPPWPRVSPACAGRFQSPVDIRPQLAAFCPAL RPLELLGFQLPPLPELRLRNNGHSVQLTLPPGLEMALGPGREYRALQLHLHWGAAGRPGS EHTVEGHRFPAEIHVVHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSAYEQLLSRLE EIAEEGSETQVPGLDISALLPSDFSRYFQYEGSLTTPPCAQGVIWTVFNQTVMLSAKQLH TLSDTLWGPGDSRLQLNFRATQPLNGRVIEASFPAGVDSSPRAAEPVQLNSCLAAGDILA LVFGLLFAVTSVAFLVQMRRQHRRGTKGGVSYRPAEVAETGA (SEQ ID NO: 36); MGSSHHHHHHSSGLVPRGSHM (SEQ ID NO:37);

AHSSSAFTITDQVPFSVSVS (SEQ ID NO:38); and

MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHN MKAFLDELKAENIKKFLHNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLL SYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYV NYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAP GVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPI GYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYN VIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTIL FASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNL TKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARY TKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDC RDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSN PIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIES KVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA (SEQ ID NO:39), wherein said peptide may optionally have up to five amino acid residues truncated from its N-terminus, C-terminus, or both.

6. The protein of claim 1, wherein the linker comprises 1 to about 20, 1 to about 10, or 1 to about 6 amino acid residues.

7. The protein of claim 1 which has the amino acid sequence set forth in SEQ ID NO:6; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:27; or SEQ ID NO:28.

8. A protein having an amino acid sequence selected from the group consisting of: SEQ ID NO:9; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:19; SEQ ID NO:21; and SEQ ID NO :25.

9. A protein having an amino acid sequence selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; and amino acid residues 74-180 of SEQ ID NO:1.

10. A recombinant cell which expresses the protein according to any one of claims 1 to 9.

11. The recombinant cell of claim 10, wherein the cell is a tumor cell and the protein is expressed on the surface of the cell.

12. A polynucleotide which encodes the protein according to any one of claims 1 to 9.

13. A vector which contains the polynucleotide of claim 12.

14. An expression vector which expresses the protein according to any one of claims 1 to 9.

15. A composition which comprises the protein according to any one of claims 1 to 9, the recombinant cell of claim 10 or 11, the polynucleotide of claim 12, the vector of claim 13, the expression vector of claim 14, or a combination thereof.

16. A method for regulating the NFkB pathway in a cell which comprises binding a toll-like receptor 4 (TLR4) of a dendritic cell with a protein having SEQ ID NO:1; SEQ ID NO:2; or SEQ ID NO:3.

17. A method of inducing dendritic cell activation, maturation, polarization, and/or filamentous actin rearrangement in an immature dendritic cell which comprises contacting the protein according to any one of claims 1 to 9, the recombinant cell of claim 10 or 11, the polynucleotide of claim 12, the vector of claim 13, the expression vector of claim 14, the composition of claim 15, or a combination thereof with a toll- like receptor 4 (TLR4) on the immature dendritic cell or binding the TLR4 according to claim 16.

18. A method of inducing an immune response in a subject which comprises administering to the subject an immunogenic amount of the protein according to any one of claims 1 to 9, the recombinant cell of claim 10 or 11, the polynucleotide of claim 12, the vector of claim 13, the expression vector of claim 14, the composition of claim 15, or a combination thereof or binding the TLR4 according to claim 16.

19. A method of modulating, increasing or decreasing the ability of the protein according to any one of claims 1 to 9 to elicit antibodies, modulate ThI and/or Th2 responses, produce cytokines and/or chemokines, or induce dendritic cell activation, maturation, polarization, filamentous actin rearrangement and/or phagocytosis which comprises increasing or decreasing the number of disulphide bonds in the NY-ESO-I polypeptide.

20. A method of targeting a dendritic cell for phagocytosis or TLR4 signaling which comprises administering to the dendritic cell the protein according to any one of claims 1 to 9, the recombinant cell of claim 10 or 11, the polynucleotide of claim 12, the vector of claim 13, the expression vector of claim 14, the composition of claim 15, or a combination thereof with a toll-like receptor 4 (TLR4) on the immature dendritic cell or binding the TLR4 according to claim 16.

21. A method of reversing, inhibiting, or reducing immune suppression by a cancer in a subject which comprises administering to the subject a TLR4 agonist or the protein according to any one of claims 1 to 9, the recombinant cell of claim 10 or 11, the polynucleotide of claim 12, the vector of claim 13, the expression vector of claim 14, the composition of claim 15, or a combination thereof with a toll-like receptor 4 (TLR4) on the immature dendritic cell or binding the TLR4 according to claim 16.

22. The composition according to claim 15 or the method according to any one of claims 16 to 21, wherein the protein is in the form of an oligomeric structure.

23. The protein according to any one of claims 1 to 9, the composition according to claim 15, or the method according to any one of claims 16 to 21, wherein the protein is covalently linked to a vault protein or a nanoparticle.