The invention relates to colon and prostate tumor associated antigen peptides obtainable from prostate specific G protein-coupled receptor (PSGR), six-transmembrane epithelial antigen of prostate (STEAP) and proteins encoded by genes found overexpressed in colon carcinoma cells, such as human 1-8D interferon induced transmembrane protein 2. The invention further relates to a polynucleotide encoding the tumor associated antigen peptides and to pharmaceutical compositions, which are preferably anti-tumor vaccine compositions, containing a tumor associated antigen, at least one tumor associated antigen peptide thereof, or encoding polynucleotide thereof as an active ingredient. The pharmaceutical compositions can be administered to a patient in need thereof to treat or inhibit the development of colon or prostate cancer.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention provides a tumor associated peptide of eight to ten amino acid residues which is capable of promoting effective binding to a MHC class I-type molecule to elicit a CTL response, such as to colon or prostate cancer cells. The peptide according to the present invention is derived or obtainable from a protein encoded by a polynucleotide overexpressed in human colon cancer cells, where the second residue from the N-terminus and the C-terminal residue are preferably (1) hydrophobic or hydrophilic or (2) neutral, hydrophobic or aliphatic natural or non-natural amino acid residues, or is obtainable from the prostate-restricted antigens, STEAP (six-transmembrane epithelial antigen of prostate) and PSGR (prostate specific G protein-coupled receptor), and specifically those having the amino acid sequences of SEQ ID NO:40, 48, 49, 50, 51, 52 or 53.

The present invention also provides a polynucleotide encoding the tumor associated peptide of the invention and a pharmaceutical composition, that contains at least one tumor associated peptide according to the present invention or at least one encoding polynucleotide thereof as an active ingredient.

Another aspect of the present invention is more specifically directed to a pharmaceutical composition which contains the tumor associated antigen (TAA) encoded by a human 1-8D interferon inducible gene, an at least one eight to ten residue TAA peptide thereof, a polynucleotide comprising the coding sequence of a 1-8D gene, or a polynucleotide encoding at least one 1-8D TAA peptide. When containing at least one tumor associated peptide, the pharmaceutical compositions of the present invention are preferably vaccine compositions, which may be cellular vaccine compositions.

Further aspects of the present invention are directed to a method for treating or for inhibiting the development of colon or prostate cancer by administering the pharmaceutical composition of the present invention to a patient in need thereof, to a method for treating or for inhibiting the development of colon cancer by administering an antibody specific for the TAA encoded by a human 1-8D interferon inducible gene, and to a method for determining overexpression of human 1-8D interferon induced transmembrane protein 2.

The present invention is also directed to a human 1-8D interferon induced transmembrane protein 2 which includes the amino acid sequence of SEQ ID NO:61 and to an encoding polynucleotide which includes the nucleotide sequence of SEQ ID NO:60.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns peptides and pharmaceutical and vaccine compositions including some which can be used to treat or to inhibit the development of cancer, both primary tumors and metastases. Specifically, the present invention provides potent tumor associated antigen (TAA) peptides derived or obtainable from prostate-specific G protein-coupled odorant receptor (PSGR), six transmembrane epithelial antigen of prostate (STEAP), and proteins encoded by polynucleotides overexpressed in colon cancer cells, which can be used in anti-tumor vaccines to treat or to inhibit the development of prostate or colon cancers, and carcinomas in particular, or any other tumor expressing the above listed proteins.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Only a small number of CTL epitopes has been defined in colon cancer patients, derived mainly from her-2/neu, CEA and Ep-CAM (Melief et al., 2000 and Nagorsen et al., 2000). Since the repertoire of peptides eluted from surface MHC class I molecules is highly similar between normal colon and colon tumors (Savoie et al., 1998), the laboratory of the present inventors focused on identification of novel colon carcinoma TAAs derived from overexpressed genes. Utilizing the data of Zhang L. et al comparing transcripts of colon tumor and normal tissue samples from the same patients (Zhang et al., 1997), a set of 26 genes overexpressed at least 5 fold in tumor was obtained.

To discriminate between immunologically relevant and irrelevant MHC class I restricted peptides, D.sup.bX.beta.2 microglobulin (.beta.2m) null mice transgenic for a recombinant HLA-A2.1/D.sup.b-.beta.2m single chain (HHD mice) was used by the laboratory of the present inventors. These mice combine classical HLA transgenesis with selective destruction of murine H-2. Therefore, unlike the classical HLA transgenics, these mice mount only HLA-A2.1-restricted CTL responses (lysis is HLA-A2 restricted) and were demonstrated as a useful biological tool for identifying potential TAA HLA-A2.1 restricted epitopes and to establish hierarchy in their anti-tumor efficacy among these peptides (Carmon et al., 2000 and Firat et al., 1999).

Over 500 putative TAA peptides were screened with anti-colon carcinoma CTL, and seven peptides were shown to be antigenic and immunogenic in HHD mice as detailed in Example 1 herein. Three of the seven peptides were derived from human 1-8D interferon induced transmembrane protein 2 (SEQ ID NO:59; accession no. BC009696) encoded by "human 1-8D interferon inducible gene (1-8D gene)" (nucleotides 31-426 of SEQ ID NO:58).and identified as peptides 1-6 (SEQ ID NO:11), 3-5 (SEQ ID NO:25), and 3-7 (SEQ ID NO:27), and were found to react both in vitro and in vivo against a colon carcinoma cell line. One of the peptides (peptide 3-7 with the amino acid sequence of SEQ ID NO:27), shared by all members of the 1-8 interferon inducible gene family, was highly immunogenic in human PBMCs. These results highlight 1-8D gene and its family as putative colon carcinoma associated antigens. The other four antigenic and immunogenic TAA peptides are identified in Table 3 (see Original Patent) in Example 1 as peptides 1-11 (SEQ ID NO:16), 2-3 (SEQ ID NO:20), 3-1 (SEQ ID NO:21), and 3-2 (SEQ ID NO:22). The laboratory of the present inventors also discovered the presence of a sequence polymorphism in 1-8D isolated from colon cancer samples (nucleotide sequence SEQ ID NO:60 and amino acid sequence SEQ ID NO:61) where when compared to SEQ ID NO:58 and 59 (accession no. BC009696 for human 1-8D interferon induced transmembrane protein 2), nucleotide 122 is a T instead of a C and changes amino acid residue 41 from Thr to Met, nucleotide 171 is a G instead of a C with no change in amino acid residue, nucleotide 234 is a G instead of a C with no change in amino acid residue, and nucleotide 362 is an A instead of a G and changes amino acid residue 121 from Val to Ile. It is intended that 1-8D nucleotide and amino acid sequences used according to the present invention for TAA peptides encompass the 1-8D nucleotide sequences of SEQ ID NOs: 58 and 60 and amino acid sequences of SEQ ID NOs: 59 and 60.

Using the HHD model, which was shown to be an effective biological tool for screening of putative novel peptides as well as for assessment of their ability to elicit powerful antitumor CTL response, prostatic acid phosphatase (PAP) derived peptide 3 (PAP-3; SEQ ID NO:46), six-transmembrane epithelial antigen of prostate (STEAP) derived peptide 3 (STEAP-3; SEQ ID NO:41) as well as PSGR derived peptides (PSGR 1-7; SEQ ID NOs: 49-55) were shown to be immunogenic in HHD mice. Importantly, testing the peripheral blood of healthy individuals has shown the existence of peripheral CTL precursors for peptide PAP-3 (2 out of 2 donors) and for peptide STEAP-3 (1 out of 2 donors) peptides. In addition, peptide STEAP-3 has activated peripheral CTL precursors in PBMC derived from CaP individual.

In accordance with another aspect of the present invention, there is provided a pharmaceutical composition, more preferably a vaccine composition, which includes at least one tumor associated antigen peptide derived or obtainable from PSGR, STEAP, or proteins encoded by polynucleotides overexpressed in colon cancer cells, which are tumor associated antigens such as the human 1-8D interferon induced transmembrane protein 2.

A further aspect of the present invention relates more particularly to a pharmaceutical composition, preferably a vaccine composition, that includes the human 1-8D interferon inducible gene, or at least one 8-10 residue TAA peptide thereof. The at least one 8-10 residue TAA peptide can specifically fit one type of HLA class I molecule, i.e., HLA-A2.1 or another HLA haplotype, or can be multiple peptides that are immunogenic in different HLA haplotypes. Thus, the human 1-8D interferon induced transmembrane protein 2 or TAA peptides thereof serve as inducer and target for cellular immunity, including CTL (CD8+ cells) and helper T cells (CD4+ cells). It is well recognized in the art that many tumor associated antigens (TAAs) from which peptides were defined for one type of HLA class I molecule, i.e., HLA-A2.1, can be shown to have immunogenic peptides that bind to other types of HLA class I molecules. Table 1 (see Original Patent) presents a representative list of tumor associated antigens with TAA peptides that bind to different types of HLA class I molecules.

While it may be preferred that, in the pharmaceutical composition according to the present invention, the at least one TAA peptide of human 1-8D interferon induced transmembrane protein 2 binds to the same HLA class I molecule as is present in a patient to which the pharmaceutical composition is to be administered (as can be readily determined, i.e., by HLA tissue typing beforehand, in order to tailor the TAA peptide or peptides to the individual patient), a pharmaceutical composition containing a mixture of TAA peptides that can bind to different types of HLA class I molecules may be alternatively used. For instance, a combination of about 5-6 peptides, each of which binds to a different HLA haplotype (i.e., a total of about 5-6 different HLA haplotypes) would cover about 95% of the world population.

Furthermore, many tumor associated antigens that induce cellular immunity can also induce humoral immunity, i.e., antibodies. Representative examples of TAAs reported to induce CTL and antibodies are tyrosinase (Fishman et al., 1997), MUC1 (Moase et al., 2001; Reddish et al., 1998), p53 (Govorko et al., 2001), CEA (Neithammer et al., 2002; Behr et al., 2002 Ullenhag et al., 2002), pmel/gp100 (Huang et al., 1998), ErbB-2 (Yum et al., 2002), MAGE-A1 (Chames et al., 2000), NY-ESO-1 (Jager et al., 1999), and TRP-2 (Okamoto et al., 1998). Accordingly, the pharmaceutical composition of the present invention that includes human 1-8D interferon induced transmembrane protein 2 can be used as a vaccine composition not only to induce cellular immunity but also to induce humoral immunity. Antibodies induced by a TAA (i.e., 1-8D) bind to tumors where the TAA is a cell surface molecule and the killing of tumor cells occurs by a number of mechanisms.

According to another aspect of the present invention, there is provided a method of vaccination for treating or inhibiting the development of colon or prostate cancer. The method is effected by administering to a patient in need thereof a vaccine composition containing at least one tumor associated antigen peptide derived or obtainable from STEAP, PSGR, or a protein encoded by a polynucleotide overexpressed in human colon cancer cells. In the case of 1-8D, the entire protein as a tumor associated antigen or at least one antigenic fragment thereof, such as a TAA peptide, may be administered according to the method of the present invention.

Immunotherapy by in vivo DNA transfer of DNA coding for TAA is based on the rationale of quality or quantity increased peptide presentation leading to activation of an immune response against these peptides. Gene or DNA vaccination results in the intracellular processing and presentation of immunogenic peptides (Spooner et al., 1995). Initial reports on DNA vaccination showed that "naked" DNA injected into the muscle tissue of a mouse is expressed efficiently (Ulmer et al., 1993). Embryonically expressed TAA such as CEA was tested (Conry et al., 1994). Immunization of mice with CEA expressing plasmid DNA was indeed found to protect 100% of these mice against a challenge with CEA-expressing colon carcinoma cells (Conry et al., 1995). Both cellular and humoral responses have been reported after DNA vaccination in mice. In other studies, a MUC-1 tandem repeat array was used for DNA vaccination of mice and 30% of these mice were protected from a tumor challenge with MUC-1 transfected murine tumor cells (Acres et al., 1993). DNA vaccination may also be used to elicit immune responses against predefined peptide epitopes. Several groups now exploit the string-bead approach to link multiple different CTL or helper epitopes together on the DNA level (Whitton et al., 1993). In some cases the string-bead of peptide coding DNA is built into a vaccinia virus as a delivery vehicle. Recently, it was shown that such a vaccinia virus recombinant poly-epitope vaccine was able to protect mice against several virus infections and a tumor challenge (Thomson et al., 1996). The authors showed that all 10 minimal peptide epitopes encoded by the string-bead are expressed and recognized by the appropriate T cell clones (Thomson et al., 1998). RNA was also shown to confer anti-tumor immunity. Vaccination with RNA to ovalbumin induced CTL in mice (Boxzkowski et al., 1996). In conclusion, multiple studies have shown the efficacy of DNA vaccines in anti-viral and anti-tumor immunity.

Thus, according to yet another aspect of the present invention, there is provided a DNA vaccine composition which includes at least one polynucleotide encoding a tumor associated antigen, such as 1-8D, or a tumor associated antigen peptide of the present invention. The at least one polynucleotide can be a part of a longer polynucleotide designed to encode a fused protein product from which the tumor associated antigen peptide is cleavable by a protease.

The polynucleotide is preferably DNA in a form of, or contained in, for example, naked DNA, plasmid, retroviral vector, adenoviral vector, vaccinia viral vector, herpes viral vector, lenti virus vector, EBV vector, CMV vector, polio virus vector, sindbis viral vector, semliki forest virus vector, parvo virus vector, adeno-associated virus vector, virus like particle (VLP) vector. Alternatively, the polynucleotide can be in the form of RNA. Aside from delivery by a vector, the polynucleotide can also be delivered in a non-viral and non-plasmid delivery system, such as, for example, but not limited to, in liposomes, in complex with cationic reagents, or with a polycation, such as poly-lysine. The polynucleotide can also be delivered by mechanical means, such as, but not limited to, a gene-gun, by electrical means, or in bacterial vectors like BCG. Such methods are described in many standard laboratory manuals.

When a vector is used, the vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotide may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

Preferred vectors carry cis-acting control regions that direct expression of the polynucleotide encoded TAA or TAA peptides. Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host. Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors as are well known in the art.

The DNA insert should be operatively linked to an appropriate promoter, such as the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.

Transcription of the DNA encoding the TAA or TAA peptides may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

There is increasing evidence that peptide vaccination may be much more effective when the peptides are introduced together with an antigen presenting cell (APC) (Mayordomo et al., 1995). In previous studies of a murine lung carcinoma, the laboratory of the present inventors have shown that vaccination with a defined TAA peptide (MUT-1) loaded on APC result in long term survival of mice bearing lung metastases (Mandelboim et al., 1994 and 1995). The most common cells used to load antigens are bone marrow and peripheral blood derived dendritic cells (DC), as these cells express costimulatory molecules that help with activation of CTL. Preliminary clinical trials have been performed. In one trial, HLA-A1 melanoma patients have been treated with autologous DC loaded with a MAGE-1 peptide. CTL activity was increased in tumor infiltrated lymphocytes (Mukherji et al., 1995). In another study, five patients with advanced pancreatic carcinoma were treated with a K-ras derived peptide loaded on DC. As a mutation of K-ras at codon 12 is frequently found in pancreatic carcinoma, three differently mutated peptides, 12-Asp, 12-Arg and 12-Val (non-mutated sequence is 12-Gly) were used for vaccination, matched to the mutation in the patient's tumor. Two of the patients showed a specific CTL response and prolonged survival (Gjertsen et al., 1996). A phase I clinical trial in 51 prostate cancer patients compared a soluble peptide, derived from PSMA, to a DC based peptide in HLA-A2 patients. Only 7 patients that received DC based vaccines with this peptide responded by decreased levels of serum PSA (Murphy et al., 1996). In animal studies, a number of groups showed that macrophages loaded with peptides constitute efficient vaccines, yet the number of cells used for vaccination is 10 fold higher than equivalent DC vaccines. Recently, in a murine lung carcinoma model, the efficacy of syngeneic fibroblasts treated with a proteasome inhibitor to decrease levels of endogenous peptides and loaded with synthetic MUT peptides as vaccines was tested. Effective protection was found against metastatic spread of lung carcinoma.

Thus, the present invention is further directed to a cellular vaccine composition which contains an antigen presenting cell presenting at least one tumor associated antigen peptide. The antigen presenting cell can, for example, be a dendritic cell, a macrophage, a B cell and a fibroblast. Presenting the at least one tumor associated antigen peptide of the present invention can be effected by a method selected from the group consisting of (a) transducing the antigen presenting cell with at least one polynucleotide (e.g., DNA) encoding the at least one tumor associated antigen peptide; (b) loading the antigen presenting cell with at least one polynucleotide (e.g., RNA) encoding the at least one tumor associated antigen peptide; (c) loading the antigen presenting cell with the at least one tumor associated antigen peptide (e.g., synthetic); and (d) loading the antigen presenting cell with at least one polypeptide (e.g., purified) that includes the at least one tumor associated antigen peptide. Loading can be external or internal. The polynucleotide, peptide or polypeptide can be fused to internalizing sequences, antennapedia sequences or toxoid sequences or to helper sequences, such as, but not limited to, heat shock protein sequences.

While it is clear that CD8+ class-I restricted CTL recognize and destroy tumor cells in vitro and in vivo, animal models often show a requirement of CD4+ MHC-class-II restricted T cell help for optimal responses (Ciccodicola et al., 1987). Helper T cell epitopes can contribute to induction of cellular immune responses by class I peptide vaccines, as seen by the synergistic tumor protection upon simultaneous vaccination with T helper and CTL epitopes (Qi et al., 1994). The "help" to CTL is most often provided via the production of specific cytokines. Helper epitopes can be specific and derived from a tumor antigen (Kuniyasu et al., 1991). They can also broadly crossreact with a number of MHC class II molecules, and may be either pathogen-derived or comprised of sequences not found in nature (Saeki et al., 1992; Freiss et al., 1994; and Byrne et al., 1998). More specifically, a sequence containing a T helper epitope can be linked to a CTL epitope to create one immunogenic entity. Alternatively, a mixture of two or more separate entities, corresponding to CTL and T helper epitopes can be administered to elicit the desired CTL response. T helper epitopes can also be conjugated to other molecules or compounds which increase their biological activity.

As used herein, the term "tumor associated antigen" also refers to tumor specific antigen and the term "peptide" refers to native peptides (either degradation products or synthetically synthesized peptides) and further to peptidomimetics, such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body, or more immunogenic. Such modifications include, but are not limited to, cyclization, N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH.sub.2--NH, CH.sub.2--S, CH.sub.2--S.dbd.O, O.dbd.C--NH, CH.sub.2--O, CH.sub.2--CH.sub.2, S.dbd.C--NH, CH.dbd.CH or CF.dbd.CH, backbone modification and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein.

Also as used herein, the term "amino acid" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including for example hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids.

The tumor associated antigen peptides of the present invention can be of 8, 9 or 10 amino acid residues in length with peptides of 9 or 10 amino acid residues in length desirable, more preferably 9 residues in length. Thus, the following positions (P1-P9) are represented in a 9-mer peptide: P1-P2-P3-P4-P5-P6-P7-P8-P9

The P2 and P9 positions include the anchor residues which are the main residues participating in binding to MHC class 1 molecules, more specifically HLA-A2. Amino acid residues engaging positions P2 and P9 in HLA-A2.1 and some other haplotypes, but not in all haplotypes, are hydrophobic or hydrophilic natural amino acids or non-natural amino acids. The discussion below is directed with particular reference to the HLA-A2.1 haplotype. Examples of hydrophobic or hydrophilic natural amino acids being Ala, Cys, Gln, Glu, Ile, Leu, Met, Ser, Thr and Val. These residues may preferably be neutral, hydrophobic, aliphatic and more preferably Val, Leu and Ile. Examples of non-natural amino acids being norleucine (Nle), norvaline (Nva), aminobutyric acid preferably .alpha.-aminobuytric acid. These residues may preferably be non-charged and more preferably aliphatic. P9 can also be an aliphatic amino acid of the general formula --HN(CH.sub.2).sub.nCOOH, wherein n=2-5, as well as by branched derivatives thereof, such as, but not limited to, -- see Original Patent.

Positions P1 and P3 are also known to include amino acid residues which participate or assist in binding to MHC molecules, however, these positions can include any amino acids, natural or non-natural.

The N-terminal residue (position P1) can also be positively charged aliphatic carboxylic acids, such as, but not limited to, H.sub.2N(CH.sub.2).sub.nCOOH, wherein n=2-5 and H.sub.2N--C(.dbd.NH)--NH(CH.sub.2).sub.nCOOH, wherein n=2-4, hydroxy Lysine, N.sup..epsilon.-methyl lysine, N.sup..epsilon.-ethyl lysine, N.sup..epsilon.-propyl lysine or ornithine (Orn). Additionally, the N-terminal residue can be aromatic residues, such as, but not limited to, phenyl glycine, p-aminophenyl alanine, p-guanidinophenyl alanine or pyridinoalanine (Pal). These latter residues may form hydrogen bonding with the OH-moieties of the tyrosine residues at the MHC-1 N-terminal binding pocket, as well as to create, at the same time aromatic-aromatic interactions.

The other positions P4-P8 are engaged by amino acid residues which typically do not participate in binding to MHC molecules, rather these amino acids are presented to the immune cells. Further details relating to the binding of peptides to MHC molecules can be found in Parker et al. (1994). See Table V (see Original Patent) thereof, in particular.

Amino acid residue engaging positions P4-P8 can include any natural or non-natural amino acid residues. These residues may optionally be phosphorylated and/or glycosylated. Indeed residues which have been phosphorylated or glycosylated have been shown in some cases to enhance the binding to the T cell receptor.

Cyclization can engage positions P4-P8, preferably positions P6 and P7. Cyclization can be obtained through amide bond formation, e.g., by incorporating Glu, Asp, Lys, Orn, di-amino butyric (Dab) acid, di-aminopropionic (Dap) acid at various positions in the chain (--CO--NH or --NH--CO bonds). Backbone to backbone cyclization can also be obtained through incorporation of modified amino acids of the formulas H--N((CH.sub.2).sub.n--COOH)--C(R)H--COOH or H--N((CH.sub.2).sub.n--NH.sub.2)--C(R)H--COOH, wherein n=1-4, and further wherein R is any natural or non-natural side chain of an amino acid.

Cyclization via formation of S--S bonds through incorporation of two Cys residues is also possible. Additional side-chain to side chain cyclization can be obtained via formation of an interaction bond of the formula --(--CH.sub.2--).sub.n--S--CH.sub.2--C(.dbd.O)--, wherein n=1 or 2, which is possible, for example, through incorporation of Cys or homocys and reaction of its free SH group with, e.g., bromoacetylated Lys, Orn, Dab or Dap.

In longer peptides, such as in a 10 mer peptide in which the second anchor amino acid is at position P10, the amino acid engaging P9 may include most L-amino acids. In some cases, shorter peptides, such as 8 mer peptides, are also applicable, in which the C-terminal acid P8 may serve as the second anchor residue. All the options described for the anchor amino acid residues engaging positions P2 and P9 in a 9 mer peptide may apply likewise to the anchor amino acid residues engaging positions P2 and P10 in a 10 mer peptide and P2 and P8 in an 8 mer peptide.

The amino acids may be modified as is necessary to provide certain characteristics such as greater immunogenicity, more stability or improved pharmacological properties. The peptides can be for instance subject to changes such as the replacement of one or more amino acid residues whether dissimilar or similar.

Modification of the peptides may also be by decreasing, e.g., in a 10 mer peptide, or extending, e.g. in an 8 mer peptide, the amino acid sequence, for example, by deletion or addition of amino acids. It will be appreciated that preferably anchor amino acids should not be deleted.

Peptide bonds (--CO--NH--) within the peptide may be replaced by N-alkylated bonds such as N-methylated (--N(CH.sub.3)--CO--), ester bonds (--C(R)H--C--O--O--CH(R)--N--), ketomethylene bonds (--CO--CH.sub.2--), .alpha.-aza bonds (--NH--N(R)--CO--), wherein R is hydrogen or any alkyl, e.g., methyl carba bonds (--CH.sub.2--NH--), hydroxyethylene bonds (--CH(OH)--CH.sub.2--), thioamide bonds (--CS--NH--), olefinic double bonds (--CH.dbd.CH--), retro amide bonds (--NH--CO--), and peptide derivatives (--N(R)--CH.sub.2--CO--), naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time. Preferably, but not in all cases necessary, these modifications should exclude anchor amino acids.

For amino acid residue engaging positions other than the second residue from the N-terminus and the end residue at the C-terminus, natural aromatic amino acids, Trp, Tyr and Phe, may be replaced by synthetic non-natural amino acid such as TIC, naphthylalanine (Nal), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.

As used herein, the term "transduced" refers to the result of a process of inserting nucleic acids into cells. The insertion may, for example, be effected by transformation, viral infection, injection, transfection, gene bombardment, electroporation or any other means effective in introducing nucleic acids into cells. Following transduction the nucleic acid is either integrated in all or part, to the cell's genome (DNA), or remains external to the cell's genome, thereby providing stably transduced or transiently transduced cells.

As used herein, phrase "derived from a protein" refers to peptides derived from the specified protein or proteins and further to homologous peptides derived from equivalent regions of proteins homologous to the specified proteins of the same or other species, provided that these peptides are effective as anti-tumor vaccines. The term further relates to permissible amino acid alterations and peptidomimetics designed based on the amino acid sequence of the specified proteins or their homologous proteins.

The term "anti-tumor vaccines" refers to vaccines effective in treating or inhibiting the development of cancer, including primary tumor and/or metastases, which include inhibiting, slowing or reversing the progression of a disease, substantially ameliorating clinical symptoms of a disease or substantially preventing the appearance of clinical symptoms of a disease.

The phrase as used herein "loading" refers to exposing, adding or introducing a substance into or onto a cell or vesicle or part thereof as is well known to those of skill in the art.

According to yet another preferred embodiment of the present invention the composition further comprises a carrier. Usually the tumor associated antigen peptide(s) are presented in context of the carrier. The carrier can be a proteinaceous carrier to which the peptides are linked. Methods of linking short peptides to carriers are well known in the art of vaccination. The carrier can alternatively be a particulate adjuvant, an oil or emulsifier based adjuvant, a gel based type adjuvant, or an adjuvant based on specific targeting of antigen, such as, but not limited to, antibody-liposome conjugates. The carrier can also be a protein or a recombinant protein produced, for example in bacteria, yeast or in mammalian cells, including cytokines, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon-.alpha., interferon-.beta., interferon-.gamma. and others. The carrier can also be an antigen presenting cell, such as, but not limited to, a dendritic cell, a macrophage, a B cell or a fibroblast. The cell selected is either an autologous or non-autologous HLA matching cell. Optionally, the cell can be a cultured cell, a cell treated by various reagents (e.g., by early and/or late acting cytokines), transduced by genes, and/or irradiated or radiated.

The pharmaceutical composition according to the present invention, which is preferably a vaccine composition, is effective in treating or inhibiting the development of cancer and/or cancer metastases. In other words, the composition is effective for primary tumors, secondary tumors and metastases thereof in the same organ or in another organ, provided that the tumor expresses the above listed tumor associated proteins. According to a preferred embodiment of the present invention, the cancer being treated or whose development is inhibited via the administration of the vaccine composition is a carcinoma, i.e., a malignant tumor composed of epithelial tissue, of the colon or prostate.

For therapeutic or prophylactic anti-tumor treatment, the vaccine composition according to the present invention may include thickeners, carriers, excipients, buffers, diluents, surface active agents, auxiliary agents, preservatives, and the like, all as well known in the art. The composition may also include one or more active ingredients, such as, but not limited to, anti-inflammatory agents, anti-microbial agents, anesthetics and the like.

The vaccine composition may be administered in either one or more ways. Administration may be effected topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip or intraperitoneal, intravesical, subcutaneous, or intramuscular injection.

Compositions for topical administration can include, but are not limited to, lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Formulations for parenteral administration can include, but are not limited to, sterile aqueous solutions which may also contain buffers, diluents, adjuvant and other suitable additives. The adjuvant is preferably of a type allowed for use in treating human beings, such as BCG adjuvant.

Dosing is dependent on responsiveness, but will normally be one or more doses per week or month, with course of treatment lasting from several weeks to several months. Persons ordinarily skilled in the art can easily determine optimum dosages, dosing methodologies and repetition rates.

The present invention provides novel tumor associated antigen peptides effective in eliciting CTL response which can therefore be effective therapeutic agents to combat cancer.

The present invention further provides a method for treating or for inhibiting the development of colon cancer which involve administering to a patient in need thereof a molecule which includes the antigen-binding portion of an antibody specific for the tumor associated antigen, human 1-8D interferon induced transmembrane protein 2, to treat or inhibit the development of colon cancer in the patient, such as by direct cytotoxicity of the antibody, e.g., as mediated by complement (CDC) or by effector cells (ADCC).

A still further aspect of the present invention relates to a method for determining overexpression of human 1-8D interferon induced transmembrane protein 2 in human colon cells, involving immunohistochemistry, such as contacting a sample of colon cells from a patient with a molecule which includes the antigen-binding portion of an antibody specific for human 1-8D interferon induced transmembrane protein 2, then detecting binding of the molecule to the colon cells and determining the level of expression of human 1-8D interferon induced transmembrane protein 2 by the colon cells from the patient sample.

It should be understood that when the term "antibody" is used, this is intended to include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic fragments thereof such as the Fab or F(ab')2 fragments. Furthermore, the DNA encoding the variable region of the antibody can be inserted into other antibodies to produce chimeric antibodies (see, for example, U.S. Pat. No. 4,816,567) or into T-cell receptors to produce T-cells with the same broad specificity (see Eshhar, et al, 1990 and Gross et al, 1989). Single-chain antibodies can also be produced and used. Single-chain antibodies can be single-chain composite polypeptides having antigen binding capabilities and comprising a pair of amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain (linked VH-VL or single-chain FV). Both VH and VL may copy natural monoclonal antibody sequences or one or both of the chains may comprise a CDR-FR construct of the type described in U.S. Pat. No. 5,091,513 (the entire content of which is hereby incorporated herein by reference). The separate polypeptides analogous to the variable regions of the light and heavy chains are held together by a polypeptide linker. Methods of production of such single-chain antibodies, particularly where the DNA encoding the polypeptide structures of the VH and VL chains are known, may be accomplished in accordance with the methods described, for example, in U.S. Pat. Nos. 4,946,778, 5,091,513 and 5,096,815, the entire contents of each of which are hereby incorporated herein by reference.

An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.

Monoclonal antibodies (mAbs) are a substantially homogeneous population of antibodies to specific antigens. MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler et al, (1975); U.S. Pat. No. 4,376,110; Harlow et al, (1988); and Colligan et al, (2002), the entire contents of which references are incorporated entirely herein by reference. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof. The hybridoma producing the mAbs of this invention may be cultivated in vitro or in vivo. High titers of mAbs can be obtained by in vivo production where cells from the individual hybridomas are injected intraperitoneally into pristane-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs. MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.

Chimeric antibodies are molecules, the different portions of which are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies are primarily used to reduce immunogenicity during application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric or humanized mAbs are used. Chimeric and humanized antibodies and methods for their production are well-known in the art, such as Cabilly et al (1984), Morrison et al (1984), Boulianne et al (1984), European Patent 0 125 023, Neuberger et al, European Patent 0 171 496, European Patent 0 173 494, WO 8601533, European Patent 0 184 187, Sahagan et al (1986); WO 9702671 (1987), Liu et al (1987), Sun et al (1987), Better et al (1988), and Harlow et al (1988). These references are hereby incorporated herein by reference.

A "molecule which includes the antigen-binding portion of an antibody," is intended to include not only intact immunoglobulin molecules of any isotype and generated by any animal cell line or microorganism, or generated in vitro, such as by phage display technology for constructing recombinant antibodies, but also the antigen-binding reactive fraction thereof, including, but not limited to, the Fab fragment, the Fab' fragment, the F(ab')2 fragment, the variable portion of the heavy and/or light chains thereof, and chimeric or single-chain antibodies incorporating such reactive fraction, or molecules developed to deliver therapeutic moieties by means of a portion of the molecule containing such a reactive fraction. Such molecules may be provided by any known technique, including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques. A recent paper by Lev et al. (2002) is representative of the state of the art with respect to human recombinant antibodies directed towards expressed tumor T cell epitopes.
 

Claim 1 of 15 Claims

1. An isolated tumor associated antigen (TAA) peptide of eight to ten amino acid residues, which is capable of promoting effective binding to a MHC class I molecule to elicit a CTL response and which is encoded by a polynucleotide overexpressed in human colon carcinoma cells, selected from the group consisting of the amino acid sequence of SEQ ID NO:27, SEQ ID NO:11; SEQ ID NO:25, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22.

 

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