KR20020008134A - Novel compounds - Google Patents

Abstract

The present invention relates to methods of producing such polypeptides by CASB619 polypeptides and polynucleotides and recombinant techniques. The present invention also relates to methods of using CASB619 polypeptides and polynucleotides in diagnostics and vaccines for the prevention and treatment of cancers, in particular ovarian and colon cancers, autoimmune diseases, and related diseases.

Description

Compound {NOVEL COMPOUNDS}

Polypeptides and polynucleotides of the invention are specific for tumors because they can be specifically expressed or targeted by antigen-specific immune mechanisms that are highly over-expressed in tumors relative to normal cells resulting in the destruction of tumor cells. It is considered an important immunogen for prophylactic or therapeutic immunization. They can also be used to diagnose the development of tumor cells. In addition, their inappropriate expression in certain circumstances can induce autoimmune, inappropriate immune responses, which may be treated via appropriate vaccination using the same polypeptide or polynucleotide. The most important biological activity for this purpose in this regard is the antigenic and immunogenic activity of the polypeptides of the invention. In addition, the polypeptides of the present invention may exhibit one or more other biological activities of the CASB619 polypeptide, which may be adapted as a target for therapeutic or prophylactic interventions other than those associated with an immune response.

The present invention relates to polynucleotides (herein referred to as CASB619 polynucleotides), thereby encoded polypeptides (herein referred to as CASB619 polypeptides), recombinants and methods of producing them. In another aspect, the present invention relates to methods of using the polypeptides and polynucleotides, including cancer, in particular colon cancer, and autoimmune diseases and other related diseases. In a further aspect, the present invention relates to a method for identifying agonists and antagonists / inhibitors using a substance provided by the present invention and for treating diseases associated with CASB619 polypeptide imbalance using the identified compounds. In a further aspect, the present invention relates to a diagnostic assay for detecting a disease associated with inappropriate CASB619 polypeptide activity or level.

In a first aspect, the present invention relates to a CASB619 polypeptide. The peptide is at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably 95% over the full length of SEQ ID NO: 2 An isolated polypeptide comprising an amino acid sequence having at least the same identity, most preferably at least 97% to 99% identity. The polypeptide includes a polypeptide comprising the amino acid of SEQ ID NO: 2.

Further peptides of the invention are at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably with the amino acid sequence of SEQ ID NO: 2 over the full length of SEQ ID NO: 2 Includes an isolated polypeptide that is an amino acid sequence having at least 95% identity, most preferably at least 97% to 99% identity. The polypeptide comprises a polypeptide of SEQ ID NO: 2.

Additional peptides of the invention include an isolated polypeptide encoded by a polynucleotide comprising a sequence comprised in SEQ ID NO: 1.

The present invention also provides immunogenic fragments of CASB619, which fragments are proximal portions of the CASB619 polypeptide having the same or similar immunogenic properties to the polypeptide comprising the amino acid sequence of SEQ ID NO: 2. That is, the fragment (when coupled with a carrier if necessary) can increase the immune response recognizing the CASB619 polypeptide. Such immunogenic fragments include, for example, CASB619 polypeptides, transmembrane domains, or C-terminal anchor domains that lack an N-terminal leader sequence. In a preferred embodiment the immunogenic fragment of CASB619 according to the invention is substantially at least 70% identity, preferably at least 80% identity, more preferably 90 over the full length of SEQ ID NO: 2 It includes all of the extracellular domains of a polypeptide having at least% identity, even more preferably at least 95% identity, most preferably at least 97% to 99% identity.

Peptide fragments incorporating an epitope of CASB619 will typically comprise at least 7, preferably at least 9 or 10 contiguous amino acids from SEQ ID NO: 2. Preferred epitopes are shown in SEQ ID NO: 5 to SEQ ID NO: 68.

Peptides incorporating the epitope form a preferred aspect of the present invention. Immunogens comprising mimotopes having the same characteristics as the epitopes and the mimotopes that produce an immune response that cross-reacts with the epitope in connection with the CASB619 molecule are also part of the present invention.

Therefore, the present invention includes isolated peptides comprising such epitopes themselves, and any mimotopes thereof. The meaning of mimotof is sufficiently similar to the original CASB619 epitope so that it can be recognized by an antibody that recognizes the original molecule; When coupled to a suitable carrier, it is defined as a whole capable of increasing antibodies that cross-react with the original molecule.

Peptide mimotopes of the epitopes identified above can be designed for specific purposes by addition, deletion or substitution of selected amino acids. Thus, the peptides of the present invention may be modified for the purpose of easy conjugation with a protein carrier. For example, it may be desirable for some chemical conjugation methods to include terminal cysteines in the epitope. Additionally, it may be desirable to include a hydrophobic end distal from the conjugated end of the peptide to conjugate the peptide to the protein carrier, with the unconjugated free end of the peptide bound to the surface of the carrier protein. This reduces the morphological degree of freedom of the peptide, thereby increasing the likelihood that the peptide will exist in a form that most closely resembles that of the peptide as found in relation to the entire molecule. For example, the peptide can be altered to have an N-terminal cysteine and a C-terminal hydrophobic amidated tail. In addition, addition or substitution of the D-stereoisomers of one or more amino acids can be performed, for example, to produce beneficial derivatives for enhancing the stability of the peptide. Those skilled in the art will appreciate that such modified peptides, or mimotopes, may be mimotopes in which the constituent moiety is in whole or in part nonpeptidic, which is not necessarily limited to the 20 naturally occurring amino acids. In addition, they can be cyclized by techniques known in the art to constrain the peptide in a form very similar to its shape when the peptide sequence relates to the entire molecule. Preferred methods of cyclizing the peptide include the addition of a pair of cysteine residues to allow to form a disulfide bridge.

In addition, those skilled in the art will appreciate that the mimotopes or immunogens of the present invention may be larger than the epitopes identified above and may thus comprise the sequences described herein. Thus, the mimotopes of the present invention may consist of the addition of N and / or C terminal extensions of numerous other natural residues at one or both ends. Peptide mimotopes may also be reversed from the retro sequence of the native sequence, ie the sequence orientation; Alternatively, the sequence may be, in whole or at least in part, a D-stereoisomeric amino acid (inverso sequence). In addition, the peptide sequence may be characteristically retro-inverso, ie opposite in sequence direction and in the form of D-stereoisomers of amino acids. Such retro or retro-inverso peptides have the advantage of being nonmagnetic, and thus can overcome the problem of self-resistance in the immune system.

Peptide mimotopes can also be identified using antibodies that can bind themselves to epitopes of the invention using techniques such as phage display techniques (EP 0 552 267 B1). The technique thus produces numerous peptide sequences that mimic the structure of the original peptide and can bind to anti-native peptide antibodies, but cannot necessarily share significant sequence homology with the original peptide by itself. . This approach has the advantage of being able to identify peptides with improved immunogenic properties, or can overcome any potential self-antigen resistance problems that may be associated with the use of the original peptide sequence. The technique also allows for the identification of recognition patterns for each native peptide by its shared chemical nature among the recognized mimotopes sequences.

Covalent coupling of peptides to immunogenic carriers can be performed by methods well known in the art. Thus, for example, carbodiimide, glutaraldehyde or (N- [γ) using conventional commercially available heteroobifunctinal linkers (using manufacturer's instructions) such as CDAP and SPDP for direct covalent coupling. It is possible to use maleimidobutyryloxy] succinimide esters After the coupling reaction, the immunogen can be easily isolated and purified by dialysis method, gel filtration method, fractionation method and the like.

The type of carrier used for the immunogen of the present invention will be readily known to those skilled in the art. The function of the carrier is to provide a helper cytokine to help induce an immune response against the peptide. The list of carriers that can be used in the present invention includes inactivated bacteria such as keyhole limpet Haemocyanin (KLH), serum albumin such as bovine serum albumin (BSA), tetanus or diphtheria toxin (TT and DT). Toxins, or recombinant fragments thereof (eg, domain 1 of fragment C of TT, or translocation domain of DT), or purified protein derivatives (PPDs) of tubulin (PPD). Mimotopes or epitopes may also be conjugated directly to liposome carriers which may further comprise an immunogen capable of providing helper T-cells. Preferably the ratio of mimotope to carrier is from about 1: 1 to 20: 1, preferably each carrier should carry 3 to 15 peptides.

Preferred carriers in an embodiment of the invention are protein D from Haemophilus influenzae (EP 0 594 610 B1). Protein D is an IgD-binding protein from Haemophilus influenzae and has been patented by Forsgren (WO 91/18926, EP 0 594 610 B1). In some circumstances, for example, it is desirable to use fragments of protein D, such as fragments of protein D 1/3 ^rd (including N-terminal 100-110 amino acids of protein D (GB 9717953.5)) in a recombinant immunogen expression system. .

Another preferred method of providing the peptides of the present invention relates to recombinant fusion molecules. For example, EP 0 421 635 B describes the use of chimeric hepadnavirus core antigen particles to provide foreign peptide sequences in virus-like particles. As such, the immunogens of the invention may comprise peptides provided in chimeric particles composed of hepatitis B core antigen. In addition, recombinant fusion proteins may include mimoto and carrier proteins of the invention, such as NS1 of influenza virus. For any recombinantly expressed protein that forms part of the present invention, nucleic acids encoding immunogens form an aspect of the present invention.

Peptides used in the present invention can be easily synthesized by solid phase methods well known in the art. Suitable synthesis can be carried out by the "T-boc" or "F-moc" method. Cyclic peptides can be synthesized by the well-known "F-moc" method and solid phase methods using polyamide resins in fully automated devices. In addition, those skilled in the art will know the necessary laboratory methods for carrying out the method by hand. Techniques and methods for solid phase synthesis are described in 'Solid Phase Peptide Synthesis: A Practical Approach' by E. Atherton and RC Sheppard, Published by IRL at Oxford University Press (1989). Peptides can also be produced by recombinant methods, including expressing nucleic acid molecules encoding mimotopes in bacterial or mammalian cell lines, and then purifying the expressed mimotopes. Techniques for recombinant expression of peptides and proteins are known in the art and described in Maniatis, T., Fritsch, EF and Sambrook et al., Molecular cloing, a laboratory manual , 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

Polypeptides or immunogenic fragments of the invention may be in the form of "mature" proteins or may be part of larger proteins such as precursors or fusion proteins. It is often advantageous to include secretory or leader sequences, pro-sequences, sequences that aid in purification such as multiple histidine residues, or additional sequences for stability during recombinant production. In addition, addition of polynucleotide sequences to increase the immunogenic potential of exogenous polypeptides or lipid tails or final molecules has been contemplated.

In one aspect, the invention provides a constant region of the heavy or light chain of a genetically engineered soluble fusion protein comprising a polypeptide of the invention, or a fragment thereof, and immunoglobulins (IgE, IgM, IgA, IgE) of various subclasses. It is about the various parts of. As immunoglobulins, the constant portion of the heavy chain of human IgG, in particular IgG1, is preferred, where fusion takes place in the hinge region. In certain embodiments, the Fc moiety can be simply removed by incorporation of a cleavage sequence that can be cleaved using blood coagulation factor Xa. The present invention also relates to methods for producing fusion proteins by genetic engineering and their use for drug screening, diagnosis and treatment. A further aspect of the present invention also relates to polynucleotides encoding the fusion protein.

Examples of fusion protein engineering can be found in International Patent Applications Nos. WO94 / 29458 and WO94 / 22914.

The protein may be expressed as a recombinant fusion protein that allows for increased levels produced in the expression system compared to proteins that are chemically conjugated or unfused. Fusion partners may help to provide T helper epitopes, preferably T helper epitopes that are recognized by humans (immunological fusion partners), or may help to express proteins in higher yield than native recombinant proteins (expression). Enhancer). Preferably the fusion partner will be both an immunological fusion partner and an expression enhancement partner.

Fusion partners include Protein D from Haemophilus influenza B and NS1 (hemaglutinin), a nonstructural protein from influenza viruses. Another immunological fusion partner is a protein known as LYTA. Preferably the C terminal portion of the molecule is used. Lyta is encoded by N-acetyl-L-alanine amidase, an amidase LYTA [lytA gene, a self-degrading enzyme that specifically cleaves specific binding of the peptideglycan backbone. Gene, 43 (1986) page 265-272), which is derived from Streptococcus pneumoniae . The C-terminal domain of the LYTA protein is responsible for affinity with choline, or some choline analogs such as DEAE. This property has been used for the development of E. coli C-LYTA to express plasmids useful for the expression of fusion proteins. Purification of a hybrid protein comprising a C-LYTA fragment at its amino terminus is described in Biotechnology: 10, (1992) pages 795-798. Repeating portions of the Lyta molecule found at residues C178 at the beginning of residues 178 can be used, such as residues 188-305.

The invention also encompasses variants of polypeptides other than those indicated by the aforementioned polypeptides, namely conserved amino acid substituents, whereby the residues are substituted by another residue with similar characteristics. Such typical substituents include one of Ala, Val, Leu and Ile; One of Ser and Thr; One of the acidic residues Asp and Glu; One of Asn and Gln; And one of the basic residues Lys and Arg; Or aromatic residues Phe and Tyr. It is particularly preferred that several, 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 amino acids are substituted, deleted or added in any combination.

Polypeptides of the invention can be prepared by any suitable method. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well known in the art.

In a further aspect, the present invention relates to a CASB619 polynucleotide. Such polynucleotides have at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably 95 with the amino acid sequence of SEQ ID NO: 2 over the full length of SEQ ID NO: 2 An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least% identity. In this regard, polypeptides having at least 97% identity are highly preferred, but polypeptides having at least 98% to 99% identity are more preferred, and polypeptides having at least 99% identity are most preferred. The polynucleotide comprises a polynucleotide comprising a nucleotide sequence contained within SEQ ID NO: 1 encoding a polypeptide of SEQ ID NO: 2.

Further polynucleotides of the invention include at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more than the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 over the entire coding region. Preferably isolated polynucleotides comprising nucleotide sequences having at least 95% identity. In this regard, polynucleotides having at least 97% identity are very preferred, but polynucleotides having at least 98% to 99% identity are more preferred, and polynucleotides having at least 99% identity are most preferred.

Further polynucleotides of the present invention have at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably with SEQ ID NO: 1 over the full length of SEQ ID NO: 1 Isolated polynucleotides comprising nucleotides having at least 95% identity. In this regard, polynucleotides having at least 97% identity are very preferred, but polynucleotides having at least 98% to 99% identity are more preferred, and polynucleotides having at least 99% identity are most preferred. Such polynucleotides include polynucleotides comprising the polynucleotide of SEQ ID NO: 1 as well as the polynucleotide of SEQ ID NO: 1. The polynucleotide can be inserted into an appropriate plasmid or recombinant microorganism and used for immunization. Wolff et. al., Science 247: 1465-1468 (1990); Corr et. al., J. Exp. Med. 184: 1555-1560 (1996); Doe et. al., Proc. Natl. Acad. Sci. 93: 8578-8583 (1996). The present invention also provides polynucleotides that are complementary to all of the polynucleotides described above.

The invention also provides fragments of CASB619 polynucleotides having the same immunogenic properties as the polynucleotides of SEQ ID NO: 1 when administered to a subject.

The present invention also provides a polynucleotide encoding an immunological fragment of the CASB619 polypeptide as defined above.

The nucleotide sequence of SEQ ID NO: 1 showed homology with Homo sapiens No. 1 chromosome clone RP4-641D22 map p13.1-13.3 (Accession No. AL157901). The nucleotide sequence of SEQ ID NO: 1 comprises a polypeptide encoding sequence (nucleotides 1 to 3043) encoding a polypeptide of amino acid 1013 which is a cDNA sequence and is a polypeptide of SEQ ID NO: 2. The nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 may be the same as the polypeptide encoding the sequence contained in SEQ ID NO: 1 or may be different from the sequence contained in SEQ ID NO: 1, which is As a result of duplication (degeneracy), the polypeptide of SEQ ID NO: 2 is also encoded. The polypeptide of SEQ ID NO: 2 is not related to any other protein of known function.

Preferred polypeptides and polynucleotides of the invention are believed to have biological functions / characteristics particularly similar to their homologous polypeptides and polynucleotides. In addition, preferred polypeptides, immunological fragments and polynucleotides of the invention have the activity of at least one of SEQ ID NO: 1 or SEQ ID NO: 2 as suitable.

The invention also relates to partial or other incomplete polynucleotides and polypeptides first identified prior to the determination of the corresponding full length sequence of SEQ ID NO: 1 and SEQ ID NO: 2.

Thus, in a further aspect, the present invention

(a) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity, over the full length of SEQ ID NO: 3, Even more preferably it comprises a nucleotide sequence having at least 97% to 99% identity;

(b) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity, over SEQ ID NO: 3 over the full length of SEQ ID NO: 3, Even more preferably have a nucleotide sequence having at least 97% to 99% identity;

(c) the polynucleotide of SEQ ID NO: 3; or

(d) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, more preferably at least 95% over the amino acid sequence of SEQ ID NO: 4 over the full length of SEQ ID NO: 4 Nucleotide sequences encoding polypeptides having identity, more preferably 97-99% or more identity; Also provided is an isolated polynucleotide, which is the polynucleotide of SEQ ID NO: 3.

The present invention

(a) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, more preferably at least 95% over the amino acid sequence of SEQ ID NO: 2 over the full length of SEQ ID NO: 4 Comprises an amino acid sequence having identity, most preferably at least 97-99% identity;

(b) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, more preferably at least 95% over the amino acid sequence of SEQ ID NO: 2 over the full length of SEQ ID NO: 4 Amino acid sequences that are identical, most preferably at least 97-99% identical;

(c) comprises the amino acid of SEQ ID NO: 4;

(d) a polypeptide that is a polypeptide of SEQ ID NO: 4; Also provided is a polypeptide that is a polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO: 3.

SEQ ID NO: 3 and the nucleotide sequence and peptides encoded thereby are derived from the EST (expressed sequence tag) sequence. It is known by those skilled in the art that there will be some nucleotide sequence reading error in the EST sequence (Adams, MD et al , Nature 377 (supp) 3, 1995). Thus, the nucleotide sequence of SEQ ID NO: 3 and the peptide sequence encoded therefrom are subject to the same genetic limitations in sequence accuracy. In addition, the peptide sequence encoded by SEQ ID NO: 3 comprises regions of identical or close homology and / or close structural similarity (eg, conservative amino acid differences) to the nearest homologous or structurally similar protein.

Polynucleotide of the invention may be obtained using standard cloning and screening techniques from a cDNA library derived from mRNA in human colon cancer cells (see: Sambrook et al, Molecular Cloning: . A Laboratory Manual, 2 nd Ed,. Cold Spring harbor Laboratory Press, Cold Spring harbor, NY (1989). The polynucleotides of the invention can also be obtained from natural sources such as genome DNA libraries or synthesized using well known and commercially available techniques.

When a polynucleotide of the invention is used for recombinant production of a polypeptide of the invention, the polynucleotide alone comprises a coding sequence for a mature polypeptide; Or mature polypeptide in a reading frame with other coding sequences, such as a leader or secretory sequence, a pre- or pro- or pre-pro-protein sequence, or a sequence encoding another fusion peptide moiety. For coding sequences. For example, marker sequences may be encoded that facilitate purification of the fusion polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is provided in a pQE vector (Qiagen, Inc) and described in Gentz et al ., Proc Natl Acad Sci USA (1989) 86: 821-824. Hexa-histidine peptide as shown, or HA tag. Polynucleotides can also include non-coding 5 'and 3' sequences, such as sequences that are transcribed and not translated, splicing and polyadenylation signals, ribosomal binding sites, and sequences that stabilize mRNA.

Further embodiments of the invention include polynucleotides encoding polypeptide variants comprising the amino acid sequence of SEQ ID NO: 2, wherein several, such as from 5 to 10, from 1 to 5, from 1 to 3 , 1 to 2 or 1 amino acid residues are substituted, deleted or added in any combination.

Full length cDNA and polypeptides of the invention, using polynucleotides identical or sufficiently identical to the nucleotide sequence contained in SEQ ID NO: 1, as hybridization probes for cDNA and genome DNA or as primers for nucleic acid amplification (PCR) reactions Isolating genome clones encoding and encoding other genes (paralog from human sources and orthologs and paralogs from species other than human) with high sequence similarity to SEQ ID NO: 1 CDNA and genome clones) (including genes) can be isolated. Typically these nucleotide sequences are 70%, preferably 80%, more preferably 90%, most preferably 95% identical to the nucleotide of the indicator. Probes or primers will generally comprise at least 15 nucleotides, preferably at least 30 nucleotides, and may have at least 50 nucleotides. Particularly preferred probes will have 30 to 50 nucleotides. Particularly preferred primers will have 20 to 25 nucleotides. In particular, polypeptides or polynucleotides derived from sequences originating in cognate animals may be used as immunogens to obtain cross-reactive immune responses against human genes.

Polynucleotides encoding polypeptides of the invention, including homologues from species other than human, are screened for appropriate libraries using labeled probes having sequences of SEQ ID NO: 1 or fragments thereof under stringent hybridization conditions ; And isolating a genome clone having full length cDNA and said polynucleotide sequence. Such hybridization techniques are well known to those skilled in the art. Preferred stringent hybridization conditions are 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5xdenhardt solution, 10% dextran sulfate and 20 μg / ml Incubation at 42 ° C. overnight in a solution containing the sheared salmon sperm DNA and then washing the filter in 0.1 × SSC at about 65 ° C. The present invention therefore encompasses polynucleotides that can be obtained by screening appropriate libraries using labeled probes having sequences of SEQ ID NO: 1 or fragments thereof under stringent hybridization conditions.

In many cases, those skilled in the art will appreciate that an isolated cDNA sequence will be incomplete, ie the coding region for the polypeptide will be short at the 5 'end of the cDNA.

Several methods based on the method of obtaining full-length cDNAs or elongating short cDNAs, such as rapid amplification of the cDNA ends (RACE) (Frohman et al., PNAS USA 85, 8998-9002, 1988), are useful and useful to those of skill It is well known. A recent variant of this technology, illustrated by Marathon ^™ technology (Clontech Laboratories Inc.), has significantly simplified the search for longer cDNAs. In Marathon® technology, cDNA is prepared from mRNA extracted from selected tissues and 'adapter' sequences are linked to each end. Nucleic acid amplification (PCR) is then performed to amplify the 'deleted' 5 'end of the cDNA by using a combination of gene specific and adapter specific oligonucleotide primers. The PCR reaction is then subjected to 'nested' primers, i.e. primers designed to anneal in the amplified product (typically adapter specific primers that anneal additional 3 'in the adapter sequence and additional 5' in known gene sequences). Is repeated using a gene specific primer to anneal. The product of the reaction can then be analyzed by DNA sequencing to produce full length cDNA by binding the product directly to an existing cDNA to provide a complete sequence, or to obtain new sequence information for the design of the 5 'primer. Can be used to perform individual full length PCR.

Recombinant polypeptides of the invention can be prepared by methods known in the art from genetically engineered host cells comprising an expression system. Thus, in a further aspect, the present invention relates to the production of an expression system comprising the polypeptide of the invention, a genetically engineered host cell having said expression system and the production of a polypeptide of the invention by recombinant technology. Cell-free translation systems can also be used to generate such proteins using RNA derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for the polynucleotides of the invention. Introduction of polynucleotides into host cells is described in Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al ., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)] can be accomplished by the methods described in many standard laboratory manuals. Preferred such methods include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrap loading, ballistics Introduction or injection.

Preferably the protein of the invention is co-expressed with thioredoxin in trans. Co-expression of thioretoxin in trans to cis preferably maintains the antigenic freedom of thioredoxin without the need for a protease. Thioredoxin co-expression facilitates solubilization of the proteins of the invention. Thioredoxin co-expression also has a significant effect on protein purification yield, purified protein solubility and quality.

Representative examples of appropriate host include streptococcus (Streptococci), Stability Philo nose kusu (Staphylococci), E. coli, Streptomyces (Streptomyces) and Bacillus subtilis (Bacillus subtilis) bacteria cells such as cell; Yeast cells and Aspergillus (Aspergillus) fungal cells, such as cells; Insect cells such as Drosophila S2 and Spodoptera Sf9 cells; CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; And plant cells.

A wide variety of expression systems are for example chromosomes, episomes and virus-derived systems such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, baculoviruses, papova viruses such as SV40 , Such as vectors derived from viruses such as vaccinia virus, adenovirus, foul fox virus, Pseudorabies virus and retroviruses, and plasmids and those derived from bacteriophage genetic elements such as cosmids and phagemids A vector derived from the combination of can be used. Expression systems may include control regions that regulate expression as well as cause expression. In general, any system or vector capable of maintaining, propagating or expressing a polynucleotide that produces a polypeptide in a host can be used. Suitable nucleotide sequences can be inserted into the expression system by any of a variety of well known and universal techniques, such as those described in Sambrook et al ., Molecular Cloning, A Laboratory Manual (supra). Appropriate secretion signals can be incorporated into the desired polypeptide to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasm, or the extracellular environment. Such signals may be endogenous to the polypeptide or they may be heterologous signals.

The expression system can also be a live recombinant microorganism such as a virus or bacterium. The gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Inoculation and in vivo infection using such live vectors will result in in vivo expression of the antigen and induction of an immune response. Viruses and bacteria used for this purpose include, for example: poxviruses (e.g. vaccinia, foulpox, canarypox), alphaviruses (syndbis virus, semliki forest virus, venezuelan equine encephalitis virus), adenoviruses , Adeno-associated viruses, picornaviruses (polioviruses, rhinoviruses), herpes viruses (such as varicella zoster virus), Listeria, Salmonella, Shigella, BCG. Such viruses and bacteria can be toxic or attenuated in various ways to obtain live bexin. Such live bexins also form part of the present invention.

Polypeptides of the invention include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography It can be recovered and purified from recombinant cell culture by well known methods. Most preferably, ionic metal affinity chromatography (IMAC) is used for purification. If the polypeptide is denatured during intracellular synthesis, isolation and / or purification, well known techniques for refolding the protein for regeneration into the active form can be used.

Another important aspect of the invention is a mammal using a fragment or whole polypeptide or polynucleotide of the invention suitable for generating an antibody and / or T cell immune response for the prevention or treatment of cancer and autoimmune diseases and related diseases. The present invention relates to a method for inducing, enhancing or regulating an immunological response in a mammal, including inoculating a. Another aspect of the invention is directed to a vector or cell that is responsible for the expression of a polynucleotide in vivo and that encodes a polypeptide to induce an immunological response to generate an immune response for the prevention or treatment of the mammal from a disease. A method of inducing, enhancing, or regulating an immunological response in a mammal, including delivering a polypeptide of the present invention therethrough.

A further aspect of the invention relates to an immunological / vaccine formulation (composition) that, when introduced into a mammalian host, induces, enhances or modulates an immunological response in said mammal against a polypeptide of the invention. Wherein the composition comprises a polypeptide or polynucleotide of the present invention or an immunological fragment thereof as previously defined herein. Vaccine formulations may further comprise a suitable carrier. Since the polypeptide can be destroyed in the stomach, it is preferably administered parenterally (eg, subcutaneously, intramuscularly, intravenously or intradermal infusion). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile infusion solutions, which may include antioxidants, buffers, fungicides and solvents to make the formulation isotonic with the blood of the recipient; And aqueous and non-aqueous sterile suspensions which may include suspending agents and enhancing agents. Formulations may be provided in unit-dose or multi-dose containers such as sealed ampoules and vials and stored in a freeze-dried state requiring only the addition of a sterile liquid carrier immediately before use.

A further aspect of the present invention uses cells from the immune system of a mammal to reinject these activated immune cells for the treatment of a disease, thereby fragments or whole polypeptides or polynucleotides of the invention or polypeptides or polys of the invention. It relates to inducing an immune response to a molecule comprising a nucleotide in vitro. Activation of cells from the immune system is performed by in vitro incubation with the entire polypeptide or polynucleotide of the present invention or molecules comprising the polypeptide or polynucleotide of the present invention in the presence or absence of various immunoregulatory molecules. A further aspect of the invention relates to the immunization of mammals by administration of modified antigen presenting cells by in vitro loading with a polypeptide comprising part or all of the invention or a molecule comprising a polypeptide of the invention and by immunological methods Administration was in vivo. In addition, antigen presenting cells are transfected in vitro and immunological methods using a vector comprising a fragment comprising or a polynucleotide of the present invention or a molecule comprising a polynucleotide of the present invention, such as to express a corresponding polypeptide. Can be administered in vivo.

Vaccine formulations of the present invention may also include an adjuvant system to enhance the immunogenicity of the formulation. Preferably the adjuvant system preferentially causes a TH1 type reaction.

Immune responses can be broadly divided into two extreme categories: humoral or cell mediated immune responses (traditionally characterized by cell effector mechanisms for antibodies and protection, respectively). This category of response is referred to as TH1-type response (cell-mediated response), and TH2-type immune response (humoral response).

Extreme TH1-type immune responses can be characterized by the generation of antigen specific, haplotype limited cytotoxic T lymphocytes, and natural killer cell responses. TH1-type responses in mice are often characterized by the production of antibodies of the IgG2a subtype, but in humans they correspond to IgG1 type antibodies. The TH2-type immune response is characterized by the generation of a broad range of immunoglobulin isotypes including IgG1, IgA and IgM in mice.

Inducibles behind the development of these two types of immune responses can be thought of as cytokines. High levels of TH1-type cytokines tend to induce cell mediated immune responses against certain antigens, while high levels of TH2-type cytokines tend to induce humoral immune responses to antigens.

The distinction between TH1 and TH2-type immune responses is not absolute. In practice the individual will support the described immune response, such as predominant TH1 or predominant TH2. Often, however, Mosmann and Coffman ( Mosmann, TR and Coffma, RL (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties.Annual Review of Immunology, 7, p145 -173 ), it is convenient to think of families of cytokines as described in murine CD4 + ve T cell clones. Traditionally, TH1-type responses have been associated with the production of INF-γ and IL-2 cytokines by T-lymphocytes. Other cytokines, often directly related to the induction of TH1-type immune responses, are not produced by T-cells such as IL-12. In contrast, TH2-type responses are associated with the secretion of IL-4, IL-5, IL-6 and IL-13.

It is known that certain vaccine adjuvants are particularly suitable for the stimulation of TH1 or TH2-type cytokine responses. Traditionally, the best indicator of the TH1: TH2 balance of the immune response after vaccination or infection is a direct measurement of the production of TH1 or TH2 cytokines by T lymphocytes in vitro after restimulation with antigen, and / or antigen specific antibodies. Measurement of the IgG1: IgG2a ratio of the response.

Thus, TH1-type adjuvant preferentially stimulates isolated T-cell populations to produce high levels of TH1-type cytokines when restimulated with antigen in vitro, and is associated with TH1-type isotypes. Promotes the development of both CD8 + cytotoxic T lymphocytes and antigen specific immunoglobulin responses.

Adjuvants capable of preferentially stimulating the TH1 cell response are described in International Patent Applications Nos. WO 94/00153 and WO 95/17209.

3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is known from GB 2220211 (Ribi). Chemically it is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is prepared by Ribi Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid A is described in European Patent No. 0 689 454 B1 (SmithKline Beecham Biologicals SA).

Preferably, the particles of 3D-MPL are small enough to be sterile filtered through a 0.22 micron membrane (European Patent 0 689 454).

3D-MPL will be present in the range of 10 μg to 100 μg, preferably 25 μg to 50 μg, where the antigen will typically be in the range of 2 μg to 50 μg per dose.

Another preferred adjuvant includes Q21, an Hplc purified non-toxic fraction derived from the shell of Quillaja Saponaria Molina. Optionally it can be mixed with 3 De-O-acylated monophosphoryl lipid A (3D-MPL), optionally with a carrier.

The production method of QS21 is described in US Pat. No. 5,057,540.

Non-reactogenic adjuvant formulations containing QS21 have been previously described (WO 96/33739). Such formulations containing QS21 and cholesterol have been found to be successful TH1 stimulatory adjuvants when formulated with antigen.

Additional adjuvants that are preferential stimulators of TH1 cell responses include immunoregulatory oligonucleotides, such as unmethylated CpG sequences as described in WO 96/02555.

Combinations of different TH1 stimulatory adjuvants as mentioned above were also considered in providing an adjuvant, which is a preferential stimulator of TH1 cell responses. For example, QS21 can be formulated with 3D-MPL. The ratio of QS21: 3D-MPL is typically about 1:10 to 10: 1; Preferably 1: 5 to 5: 1 and often substantially 1: 1. The preferred 3D-MPL: QS21 range for optimal synergy is 2.5: 1 to 1: 1.

Preferably the carrier is present in the vaccine composition according to the invention. This carrier may be an oil-in-water emulsion, or an aluminum salt such as aluminum phosphate or aluminum hydroxide.

Preferred oil-in-water emulsions include metabolizable oils such as squalene, alpha tocopherol and tween 80. In a particularly preferred aspect, the antigen in the vaccine composition according to the invention binds QS21 and 3D-MPL in the emulsion. In addition, the oil-in-water emulsion may comprise span 85 and / or lecithin and / or tricapryline.

Typically for human administration QS21 and 3D-MPL will be present in the vaccine in the range of 1 μg to 200 μg, such as 10 μg to 100 μg, preferably 10 μg to 50 μg per dose. Typically oil-in-water will comprise 2 to 10% squalene, 2 to 10% alpha tocopherol and 0.3 to 3% Tween 80. Preferably the ratio of squalene: alpha tocopherol is less than or equal to 1 so that it provides a more stable emulsion. Span 85 may also be present at a level of 1%. In some cases it is advantageous for the vaccines of the invention to further comprise stabilizers.

Non-toxic oil-in-water emulsions preferably comprise non-toxic oils such as squalane or squalene, emulsifiers such as Tween 80 in an aqueous carrier. This aqueous carrier can be, for example, phosphate buffered saline.

Particularly potent adjuvant formulations involving QS21, 3D-MPL and tocopherol in oil in water are described in WO 95/17210.

The invention also provides a multivalent vaccine composition comprising the vaccine formulation of the invention in combination with other antigens, in particular antigens useful for treating cancer, autoimmune diseases and related diseases. Such multivalent vaccine compositions may comprise TH-1 inducible adjuvants as described above.

The invention also relates to the use of polynucleotides in the form of primers derived from polynucleotides of the invention, as diagnostic reagents, and to the use of polypeptides in antibody or reagent specific forms for the polypeptides of the invention.

Identification of genetic or biochemical markers in the blood or tissue will allow detection of early changes along the path of cancer that help to determine the best treatment for the patient. Tumor marker substitutes, such as polynucleotide expression, can be used to diagnose different forms and conditions of cancer. Identification of the expression levels of the polynucleotides of the present invention will be useful both in determining the stage of cancer disease and in classifying the properties of cancer tissue. The determination of the stages of the process monitors the progress of the cancer and is determined by the presence of malignant tissue in the biopsy area. The polynucleotides of the present invention can help complete the determination of the stages of the process by identifying markers for cancer aggregation, such as the presence of different regions of the body. The classification of cancer describes how similar a tumor is to its normal type of normal tissue and is evaluated by other markers for its cell morphology and differentiation. The polynucleotides of the present invention may be useful for determining tumor grade as they may help determine the differentiation status of the cells of the tumor.

Diagnostic assays are methods for diagnosing or determining susceptibility to cancer, autoimmune diseases and related diseases through diagnosis by a method comprising determining abnormally reduced or increased levels of polypeptide or mRNA from a sample derived from a patient. To provide. This diagnostic method is known as differential expression. Expression of specific genes was compared between diseased tissues and normal tissues. Differences in polynucleotide-related genes, mRNAs or proteins in the two tissues may, for example, be attributed to molecular weight, amino acid or nucleotide sequence, or relative abundance indicating changes in the gene or genes that control it in the tissues of a diseased person. Are compared against.

Reduced or increased expression can be measured at the RNA level. PolyA RNA is first isolated from two tissues and detection of mRNA encoded by a gene corresponding to the differentially expressed polynucleotide of the present invention can be achieved by, for example, in situ hybridization in tissue sections, Northern including polyA + mRNA. It can be detected by reverse transcriptase-PCR using blots, or any other direct or indirect RNA detection method. Increased or decreased expression of certain RNA in diseased tissue relative to normal tissue suggests that transcripts and / or expressed proteins play a role in the disease. Thus, detection of high or low levels of mRNA corresponding to SEQ ID NO: 1 or SEQ ID NO: 3 in connection with normal levels indicates the presence of cancer in the patient.

MRNA expression levels in a sample can be determined by generation of a library of expressed sequence tags (EST) from the sample. The relative representation of EST in the library can be used to assess the relative representation of gene transcripts in the starting sample. The EST analysis of the test can then be compared to the EST analysis of the reference sample to determine the level of state expression of the polynucleotide of interest.

Other mRNA analyzes can be performed on methods of continuous analysis of gene expression (Velculescu et.al. Science (1995) 270: 484), differential display methods (eg, US Pat. No. 5,776,683) or hybrids that depend on the specificity of nucleotide interactions. It can be performed using a quantitative analysis.

In addition, the comparison can be performed at the protein level. Protein size in both tissues can be compared using antibodies to detect polypeptides in western blots of protein extracts from both tissues. Expression levels and subcell positioning may also be detected immunologically using antibodies to the corresponding protein. Additional assay techniques that can be used to determine the level of a protein, such as a polypeptide of the invention, in a sample derived from a host are well known to those skilled in the art. Increased or decreased levels of polypeptide expression in diseased tissues compared to expression levels of the same protein in normal tissues suggest that the expressed protein may be associated with the disease.

In the assays of the present invention, the diagnosis can be determined by the detection of gene product expression levels encoded by one or more sequences beginning with SEQ ID NO: 1 or SEQ ID NO: 3. Comparison of mRNA or protein levels in diseased versus normal tissue can also be used to watch for disease progression or alleviation.

Numerous polynucleotide sequences in a sample can be assayed using polynucleotide arrays. They can be used to determine gene function by examining differential expression of genes. For example, the array of polynucleotide sequence SEQ ID NO: 1 or SEQ ID NO: 3 can be used to determine whether any polynucleotide is differentially expressed between normal and cancer cells. In one embodiment of the present invention, an array of oligonucleotides comprising SEQ ID NO: 1 or SEQ ID NO: 3 nucleotide sequences or fragments thereof can be prepared, for example, to perform efficient screening of genetic mutations. . Array engineering methods are well known and have general applicability and can be used to address various problems in molecular genetics, including gene expression, genetic association, and genetic diversity. M. Chee et al., Science, Vol 274, pp 610-613 (1996).

As used herein, "diagnosis" includes the determination of the subject's susceptibility to a disease, the determination of whether the subject is currently suffering from the disease, and the prognosis of the diseased subject.

The invention further relates to a diagnostic kit for performing a diagnostic assay comprising:

(a) a polynucleotide of the invention, preferably the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3SEQ ID NO: 1 or SEQ ID NO: 3, or a fragment thereof;

(b) a nucleotide sequence complementary to the sequence of (a);

(c) a polypeptide of the invention, preferably a polypeptide of SEQ ID NO: 2 or a fragment thereof; or

(d) an antibody of the polypeptide of the invention, preferably the polypeptide of SEQ ID NO: 2.

The nucleotide sequence of the invention is also important for chromosomal positioning. Such sequences can be specifically targeted to and hybridized to specific locations on individual human chromosomes. The mapping of related sequences to chromosomes according to the invention is an important first step in correlating these sequences with gene related diseases. Once the sequences are mapped to elaborate chromosomal locations, the physical locations of the sequences on the chromosomes can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on-line through Johs Hopkins University Welch Medical Library). Then, the relationship between the gene and the disease mapped to the same chromosomal region was confirmed by binding analysis (cogeneity of physically adjacent genes). It is also possible to determine differences in cDNA or genome sequences between affected and unaffected individuals.

Polypeptides of the invention or fragments thereof or analogs thereof, or cells expressing them can also be used as immunogens for generating antibodies specific for the polypeptide of the invention. The term "immunospecific" means that the antibody has an affinity for the polypeptide of the invention that is substantially greater than that for other related polypeptides in the prior art.

In a further aspect the invention provides an antibody specific for a polypeptide according to the invention or an immunological fragment thereof as defined above. Preferably the antibody is a monoclonal antibody.

Antibodies generated against the polypeptides of the invention can be obtained by administering a polypeptide or epitope-bearing fragment, analog or cell to an animal, preferably an animal other than a human, using a universal protocol. For the preparation of monoclonal antibodies, techniques for providing antibodies produced by continuous cell line culture can be used. Examples include hybridoma technology (Kohler, G. and Milstein, C., Nature (1975) 256: 495-497), trioma technology, human B-cell hybridoma technology (Kozbor et al ., Immunology Today (1983) 4:72 and EBV-Hybridoma technology (Cole et al ., Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies as described in US Pat. No. 4,946,778 may also be suitable for generating single chain antibodies against the polypeptides of the invention. In addition, transgenic mice, or other organisms including other mammals, can be used to express humanized antibodies.

The antibodies described above can be used to isolate or identify clones expressing the polypeptide or to purify the polypeptide by affinity chromatography. The antibodies of the invention can also be used to prevent or treat cancer, in particular uterine and colon cancers, autoimmune diseases and related diseases.

Another aspect of the invention is an immunological in a mammal comprising inoculating the mammal with a polypeptide of the invention suitable for generating an antibody and / or T cell immune response that prevents or alleviates the symptoms or progression of the disease. It relates to a method for inducing or controlling a response. Another aspect of the invention relates to the delivery of a polypeptide of the invention through a vector expressing a polynucleotide in vivo and encoding the polypeptide to induce an immunological response to produce an antibody preventing the animal from disease. It relates to a method for inducing or regulating an immunological response in a mammal.

It will therefore be appreciated that the present invention provides a method for treating abnormal diseases such as cancer and autoimmune diseases, in particular uterine cancer and colon cancer, associated with, for example, the presence, excess, or down-expression of CASB619 polypeptide activity.

The present invention provides methods for screening compounds to identify compounds that stimulate or inhibit the function of the CASB619 polypeptide. In general, agonists or antagonists can be used for therapeutic and prophylactic purposes for the diseases as mentioned above. Compounds can be identified from a variety of sources, such as cells, cell free preparations, chemical libraries, and natural product mixtures. The agent, antagonist or inhibitor so identified may be a natural or modified substance, ligand, receptor, enzyme, etc., as may be a polypeptide; May be structural or functional mimics thereof (Coligan et al ., Current Protocols in Immunology 1 (2): Chapter 5 (1991)). Screening methods will be known to those skilled in the art. Additional screening methods are described, for example, in D. Bennett et al ., J Mol Recognition, 8: 52-58 (1995); and K. Johanson et al ., J Biol Chem, 270 (16): 9459-9471 (1995) and references therein.

The present invention therefore provides a screening method for identifying compounds that stimulate or inhibit the function of a polypeptide of the invention, comprising a method selected from the group consisting of:

(a) measuring the binding of a candidate compound to a polypeptide (or cell or membrane with the polypeptide) or a fusion protein thereof by a label directly or indirectly associated with the candidate compound;

(b) measuring the binding of a candidate compound to a polypeptide (or cell or membrane with the polypeptide) or a fusion protein thereof in the presence of a labeled competitor;

(c) using a detection system suitable for the cell or cell membrane with the polypeptide to test whether the candidate compound results in a signal produced by activation or inhibition of the polypeptide;

(d) mixing the candidate compound with the solution containing the polypeptide of claim 1 to form a mixture, measuring the activity of the polypeptides in the mixture, and comparing the activity of the mixture against a standard; or

(e) A method for detecting the effect of a candidate compound on the production of mRNA encoding the polypeptide and the polypeptide in the cell, such as using an ELISA assay.

Polypeptides of the invention can be used to identify membrane-bound or soluble receptors through standard receptor binding techniques known in the art. Well-known screening methods can also be used to identify agents and antagonists of the polypeptides of the invention that allow the polypeptides of the invention to fully bind their receptors.

Thus, in another aspect, the invention provides a kit for screening for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, and the like, for a polypeptide of the invention; Or (a) a polypeptide of the invention; (b) recombinant cells expressing a polypeptide of the invention; (c) cell membranes expressing a polypeptide of the invention; Or (d) a compound that reduces or enhances the production of said polypeptide, comprising an antibody against a polypeptide of the invention, wherein the polypeptide is preferably a polypeptide of SEQ ID NO: 2.

In addition, the polypeptide of the present invention (a) first determines the three-dimensional structure of the polypeptide; (b) infer a three-dimensional structure for the site that is likely to be the reaction or binding site (s) of the agent, antagonist or inhibitor; (c) synthesizing a candidate compound that is predicted to bind or react to the inferred binding or reaction site; (d) It will be readily understood by those skilled in the art that the candidate compounds can be used in methods for structure-based design of agents, antagonists or inhibitors of polypeptides by testing whether they are actual agents, antagonists or inhibitors.

Gene therapy can also be used to influence the endogenous production of CASB619 polypeptides by appropriate cells in a subject. For an overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and AP Read, BIOS Scientific Publishers Ltd (1996). See

Vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol. 61 Vaccine Design-the subunit and adjuvant approach, edited by Powell and Newman, Plenurn Press, 1995. New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, USA 1978). Encapsulation in liposomes is described, for example, in US Pat. No. 4,235,877 to Fullerton. Conjugation of proteins to polymers is described, for example, in US Pat. No. 4,372,945 by Lechite and US Pat. No. 4,474,757 by Armor et al.

The amount of protein in each vaccine dose is chosen as the amount that induces the immunoprophylactic response without causing significant adverse side effects for typical vaccinates. This amount will vary as the specific immunogen is used. In general, each dosage will comprise from 1 to 1000 μg of protein, preferably from 2 to 100 μg, most preferably from 4 to 40 μg. The optimal amount for a particular vaccine can be assessed by standard studies related to the observation of antibody titers and other responses in the subject. Following initial vaccination, subjects can be boosted within about 4 weeks.

"Isolated" means changed from its natural state "by the hand of man." When an "isolated" composition or material occurs in nature, it is changed or removed from, or changed and removed from its original environment. For example, a polynucleotide or polypeptide that is naturally present in a living animal is one that is not "isolated", but the same polynucleotide or polypeptide that is isolated from its coexisting material in its natural state is "isolated" as used herein. .

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, including single and double stranded regions, or modified RNA or DNA.

A “variant” refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but has essential properties. Typical variants of polynucleotides differ in nucleotide sequence from another reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, and fusions within the polypeptide encoded by the reference sequence, as discussed below. Typical variants of the polypeptide differ from the amino acid sequence from another reference polypeptide. In general, the differences are limited such that the sequences of the reference polypeptides and variants are very similar throughout and identical in many regions. Variants and reference polypeptides may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The substituted or inserted amino acid residue may or may not be an amino acid encoded by the genetic code. Variants of polynucleotides or polypeptides may occur naturally, such as allelic variants, or may be naturally occurring unknown variants. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis techniques or direct synthesis.

As known in the art, “identity” is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing sequences. Also in the art, "identity" refers to the degree of sequence relevance between polypeptide or polynucleotide sequences, as determined by consensus between a sequence of sequences. "Identity" and "similarity" are described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Seqeunce Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), can be readily calculated by known methods, including but not limited to. Preferred methods of determining identity are designed to provide the largest agreement between the sequences tested. Methods of determining identity and similarity are aggregated into publicly available computer programs. Preferred computer programs for determining identity and similarity between two sequences include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN and FASTA [see Atschul, SF et al., J. Molec. Biol. 215: 403-410 (1990)]. The BLAST X program includes NCBI and other sources, see BLAST Manual, Altschul, S., et al ., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al ., J. Mol. Biol. 215 : 403-410 (1990). The well known Smith Waterman algorithm can also be used to determine identity.

The preferred algorithm used is FASTA. Preferred parameters for polypeptides or polynucleotides using this algorithm include:

Gap Penalty: 12

Gap Extension Penalty: 4

Word size: 2, up to 6

Preferred parameters for polypeptide sequence comparison using other methods include:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)]

Comparative Matrix: BLOSSUM 62, Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992).

Gap Penalty: 12

Cap Length Penalty: 4

Useful programs using these parameters are publicly available as "gap" programs from the Genetics Computer Group (Madison WI). The parameters described above are the default parameters for polypeptide comparison (without penalty for terminal gap).

Preferred parameters for polynucleotide comparisons include:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)]

Comparison Matrix: Match = + 10, Mismatch = 0

Gap Penalty: 50

Gap Length Penalty: 3

Useful programs using these parameters are publicly available, such as the "gap" program from the Genetics Computer Group. The parameters described above are default parameters for polynucleotide comparison.

By way of example, the polynucleotide sequence of the present invention may comprise a nucleotide change equal to, or 100% identical to, the reference sequence of SEQ ID NO: 1 or less than a certain integer as compared to the reference sequence. Such changes are selected from the group consisting of substitutions, or insertions, including one or more nucleotide deletions, transitions and transversions, wherein the changes are individually 5 'or 3' terminal positions of the reference nucleotide sequence or within the reference sequence or reference sequence. It may occur at any position between these terminal positions disposed between nucleotides of one or more adjacent groups. The number of nucleotide changes is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the number percent of each percent identity (divided by 100) and subtracting the product from the total number of nucleotides in SEQ ID NO: 1:

_{n n ≤ x n - (x} n · y),

Wherein n _n is the number of nucleotide changes, x _n is the total number of nucleotides in SEQ ID NO: 1, y is, for example, 0.70 at 70%, 0.80 at 80%, 0.85 at 85%, If the 90% 0.90, 0.95 or the like if the 95%, in which any non-integer product of x _n and y is in the cut fraction to the nearest integer before removing it from x _n. A change in the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 can change the polypeptide encoded by the polynucleotide following the change by generating a nonsense, missense or frameshift mutation in this coding sequence.

Similarly, a polypeptide sequence of the present invention may comprise an amino acid change of less than a certain integer as compared to a reference sequence that is identical to the reference sequence of SEQ ID NO: 2, ie 100% identical or less than 100% identity. Can be. Such changes are selected from the group consisting of one or more amino acid deletions, substitutions comprising conservative and non-conservative substitutions, or insertions, wherein said changes are at the amino-terminal or carboxy-terminal positions of the reference polypeptide sequence or the reference sequence individually Or at any position between the terminal positions disposed between amino acids in one or more contiguous groups in the reference sequence. The number of amino acid changes for a given percent identity is multiplied by the total number of amino acids in SEQ ID NO: 2 by the numeric percentage of each percent identity (divided by 100) and then the total number of amino acids in SEQ ID NO: 2 Determined by subtracting the product from:

_{n a ≤ x a - (x} a · y),

Wherein n _a is the number of amino acid changes, x _a is the total number of amino acids in SEQ ID NO: 2, y is, for example, 0.70 at 70%, 0.80 at 80%, 0.85 at 85%, and the like. , Where any non-integer product of x _a and y is truncated to the nearest integer before subtracting it from x _a .

"Homolog" is a genetic term used in the art to refer to a polynucleotide or polypeptide sequence that has a high degree of sequence association with the subject sequence. This association can be quantified by determining the degree of identity and / or similarity between the compared sequences as described above. Within these genetic terms the term "ortholog", which refers to a polynucleotide or polypeptide which is a functional equivalent of a polynucleotide or polypeptide within another species, and "paralog," which refers to a functionally similar sequence when considered within the same species (paralog) ".

Example 1

Real-Time RT-PCR Analysis

Real-time RT-PCR (U. Gibson. 1996. Genome Research: 6,996) was used to compare the mRNA transcript abundance of candidate antigens in matched tumors and normal colon tissues from several patients. In addition, mRNA levels of candidate genes in a panel of normal tissues were evaluated by this approach.

Total RNA from normal and tumor colons was extracted from snap frozen biopsies using TriPure reagent (Boehringer). Total RNA from normal tissues was purchased from InVitrogen or extracted from snap frozen biopsies using TriPure reagents. Poly-A ⁺ mRNA was purified from total RNA after DNase treatment with oligo-dT magnetic beads (Dynal). Quantification of mRNA was performed by SpectroFluormetry (VersaFluor, BioRad) using SybrII dye (Molecular Probes). Primers for real time PCR amplification were designed using Perkin-Elmer Primer software using default options for TaqMan amplification conditions.

Real time responses were evaluated according to standard PCR protocol using 2 ng of purified mRNA for each reaction. SybrI dye (Molecular Probes) was added at final dilution 1/75000 for real time detection. Amplification (40 cycles) and real time detection were performed on a Perkin-Elmer Biosystems PE7700 system using universal instrument settings. Ct values were calculated using PE7700 sequence detection software. Two Ct values were obtained for each patient sample: tumor Ct (CtT) and matched normal colon Ct (CtN). The Ct values obtained by real time PCR were log-linear with respect to the copy number of the target template. Since the efficiency of PCR amplification under general experimental conditions is similar to the theoretical amplification efficiency, 2 ^(CtN-CtT) is an estimate of relative transcript level in both tissues (ie, mRNA over-expression fold in tumors). Real time PCR reactions were performed on biopsies from 24 patients. The level of mRNA over-expression was calculated as described for each patient. The average level of mRNA over-expression for the candidate antigen and the proportion of patients over-expressing the candidate antigen were then calculated from this data set. Individual values were normalized to actin in the same sample (ratio) as shown in FIG. 1. Thus a value of 1 corresponds to the same level of actin expression. Results are shown on a logarithmic scale.

In addition, a series of 81 normal tissue samples representing 28 different tissues were tested by the same method. The Ct values for the candidate antigens were compared to the Ct values of the actin obtained using the same tissue sample. Standardized results with respect to actin are shown in FIG. 2.

Real-Time PCR Results in Colon Cancer / Normal Colon Samples

summary

% Of patients over-expressing CASB619 in colon tumors Average level of over-expression in colon tumors (fold) 16/24 (67%) 208

Results: CASB619 was overexpressed at an average rate of nearly 200-fold in 67% of colon cancer samples for normal adjacent colon. Normal tissue expression was limited mainly by organs and testes as well as other digestive system tissues.

Example 2.

DNA microarray

DNA micro-arrays were used to examine mRNA expression profiles of large collections of genes in various samples. This information was used to supplement the data obtained by real-time PCR to provide independent measurement of gene expression levels in tumors and normal tissues.

Examples of current engineering for the generation of DNA micro-arrays include: 1) Affymetrix “GeneChip” arrays, wherein oligonucleotides are synthesized on the surface of the chip by solid state chemical synthesis using photolithographic methods. DNA spotting engineering involves depositing a small amount of DNA solution by a robot and then immobilizing on a surface of a solid phase (eg, glass). In both examples, the chip is hybridized with cDNA or cRNA extracted from the tissue of interest (eg, normal tissue, tumor, etc.) and labeled by a radioactive or fluorescent reporter molecule. The labeled material was hybridized with the chip and the amount of probe bound to each sequence on the chip was determined using a specialized scanner. This experiment may be initiated using a single fluorescent reporter (or radioactive), or alternatively, may be performed using two fluorescent reporters. In the latter case, each of the two samples was labeled with one of the reporter molecules. These two labeled samples were then hybridized competitively with the sequences on the DNA chip. The ratio of the two fluorescent signals was determined for each of the sequences on the chip. This ratio was used to calculate the relative abundance of transcripts in both samples. Detailed protocols are described in "DNA Microarrays: A practical approach. Schena M. Oxford University Press 1999" and the World Wide Web (http://cmgm.stanford.edu/pbrown/protocols/index.html, http: / /arrayit.com/DNA-Microarray-Protocols/) and professional distributors (eg Affymetrix).

Example 3.

EST profile

A complementary approach for characterizing experimental antigenic tissue expression is to search a human "expressed sequence tag" (EST) database. EST is a small fragment of cDNA made from a collection of mRNA extracted from patient tissues and cell lines. This database currently provides a tremendous amount of EST (10 ⁶ ) from hundreds of cDNA tissue libraries, including tumor tissue from diseases of various types and conditions. By an informatics tool (Blast), a comparative search of CASB616 was performed to further understand tissue expression.

EST distribution of CASB619

DbEST accession number ATG lib ID Explanation NCBI: 1202616 937 NCI_CGAP_Co2 (Colonary Villi Adenoma) NCBI: 1202659 937 NCI_CGAP_Co2 (Colonary Villi Adenoma) NCBI: 1208269 935 NCI_CGAP_Pr4.1 (prostate epithelial tumor) NCBI: 1152744 888 NCI_CGAP_Pr6 (prostate epithelial tumor) NCBI: 1157532 910 NCI_CGAP_Pr22 (normal prostate) NCBI: 1178873 882 NCI_CGAP_Co3 (12 pooled tumor colons) NCBI: 1040601 628 Testicular NHT NCBI: 1056221 628 Testicular NHT NCBI: 1298131 910 NCI_CGAP_Pr22 (normal prostate) NCBI: 976517 715 Epididymis NCBI: 1618753 417 Parathyroid tumor NCBI: 1737305 895 NCI_CGAP_Br2 (Pooled Breast Tumor Tissue) NCBI: 1738 600 895 NCI_CGAP_Br2 (Pooled Breast Tumor Tissue) NCBI: 1767617 628 Testicular NHT NCBI: 1889992 424 Fetal heart NCBI: 1907671 628 Testicular NHT NCBI: 2074937 1076 NCI_CGAP_Lu5 View stats NCBI: 2147596 1461 NCI_CGAP_Ut1 NCBI: 2147632 1461 NCI_CGAP_Ut1 NCBI: 2308815 1462 NCI_CGAP_Ut2 NCBI: 2441896 1410 NCI_CGAP_Pr28 NCBI: 2447648 1463 NCI_CGAP_Ut3 NCBI: 2381271 1461 NCI_CGAP_Ut1 NCBI: 2582781 1461 NCI_CGAP_Ut1 NCBI: 2583713 1461 NCI_CGAP_Ut1 NCBI: 2585593 1410 NCI_CGAP_Pr28

NCBI: 2587322 1463 NCI_CGAP_Ut3 NCBI: 2601136 1461 NCI_CGAP_Ut1 NCBI: 2824305 1463 NCI_CGAP_Ut3 NCBI: 3007221 1461 NCI_CGAP_Ut1 NCBI: 3078572 2301 NCI CGAP Pit1 NCBI: 3086989 1461 NCI_CGAP_Ut1 NCBI: 3087235 1461 NCI_CGAP_Ut1 NCBI: 3045696 1661 NCI_CGAP_Lu19 NCBI: 3218161 2467 NCI CGAP Co20 NCBI: 3291383 2508 NCI_CGAP_Sub3 NCBI: 3028913 2107 BT130 NCBI: 3655706 1447 NCI_CGAP_Co14 NCBI: 2553046 1728 Soares_Dieckgraefe_Colon_NHCD NCBI: 979345 781 Endometrial tumor NCBI: 3289738 2508 NCI_CGAP_Sub3 NCBI: 614598 464 ? NCBI: 978076 781 Endometrial tumor NCBI: 979060 781 Endometrial tumor NCBI: 1616799 417 Parathyroid tumor NCBI: 978445 781 Endometrial tumor

In summary, 93% of ESTs consistent with CASB619 originated from tumor tissue, fetal tissue, or normal reproductive organs. This suggests that the gene is more frequently expressed in these tissues.

Example 4

Northern-Southern Blot Analysis

Limited amounts of mixed tumors and matched normal colon cDNA were amplified by Advantage PCR (see above). In addition, messenger RNAs from several normal tissues were amplified using the same method. Amplified cDNA (1 μg) was electrophoresed on 1.2% agarose gel and transferred onto nylon membrane. This membrane was hybridized with probes prepared using fragments of candidate TAA cDNA (AlkPhos Direct System). Northern-Southern analysis provided information on transcript size, the presence of splice variants, and transcript abundance in tumors and normal tissues.

Example 5

Northern blot analysis

Northern blots were performed according to standard protocol using 1 μg poly A + mRNA. Radioactive probes were prepared using a Ready-to-Go system (Pharmacia).

Example 6

Identification of Full Length cDNA Sequences

Colon tumor cDNA libraries were prepared from 5 μg polyA + mRNA using the Lambda Zap II system (Stratagene). Following the protocol provided, SuperscriptII (LifeTechnologies) was used for the reverse transcription step. Oligo dT-primed libraries and random-primed libraries were prepared. About 1.5 × 10 6 independent phages were plated for each screening of the library. Phage plaques were transferred onto nylon filters and hybridized using cDNA probes labeled with AlkPhos Direct. Positive phage was detected by chemiluminescence. Positive phages were excised from agar plates, eluted in 500 μL SM buffer and confirmed by gene-specific PCR. Eluted phage was converted to single stranded M13 bacteriophage by in vivo excision. This bacteriophage was then converted to double stranded plasmid DNA by infection with E. coli . Infected bacteria were plated and used for the second round of screening using cDNA probes. Plasmid DNA was purified from positive bacterial clones and sequenced on both strands. If the full length gene could not be obtained directly from the cDNA library, the missing sequence was isolated using RACE engineering (Marathon Kit, ClonTech). This approach relies on reverse transcription of mRNA into double-stranded cDNAs, linking linkers to the ends of the cDNA and amplifying the desired ends of the cDNA using one of gene-specific primers and linker oligonucleotides. Marathon PCR products were cloned into plasmids (pCRII-TOPO, InVitrogen) and sequenced.

The obtained sequence (SEQ ID NO: 1) had a putative open reading frame of 1013 amino acids (SEQ ID NO: 2). The deduced protein sequence was used to predict the algorithm for intracellular positioning (PSORT: http://psort.nibb.ac.jp/ and TopPred: http: //www.biokemi.su.se/~server /toppred2/toppred_source.html). CASB619 seems to have a peptide signal and one to three transmembrane domains (low confidence prediction). No other motifs or domains were found.

Example 7:

7.1 Expression and Purification of Tumor-Specific Antigens

Expression in a microbial host, or alternatively in vitro transcription / translation, is used to generate the antigens of the present invention for vaccine purposes and to characterize or purify naturally expressed proteins by immunohistochemistry, followed by Protein fragments or whole proteins were generated for rapid purification and production.

Recombinant proteins can be expressed in two microbial hosts: E. coli and yeast (such as Saccharomyces cerevisiae or Pichia pastoris ). Pichia. This allows the selection of the expression system with the best characteristics for this specific antigen production. In general, recombinant antigens would be expressed in E. coli and reagent proteins were expressed in yeast.

Expression strategies first relate to the design of the primary structure of the recombinant antigen. In general, the expression fusion partner (EFP) is located at the N terminus to enhance the level of expression that may include antigens, regions useful for modulating the immunogenic properties of the immune fusion partner (IFP). In addition, affinity fusion partners (AFPs) useful for facilitating further purification are included at the C-terminus. Where recombinant strains were available, recombinant products were characterized by evaluation of the level of expression by analysis of patterns in crude extracts and prediction of further solubility of the protein.

After growing on appropriate culture medium and inducing recombinant protein expression, the entire extract was analyzed by SDS-PAGE. Recombinant proteins were visualized on stained gels and confirmed by Western blot analysis using specific antibodies.

Comparative evaluation of the different versions of the expressed antigen will allow selection of the most promising candidates to be used for further purification and immunological evaluation.

The purification scheme followed a classical approach based on the presence of His affinity tails in the recombinant protein. In a typical embodiment the disrupted cells were filtered and the cell-free extracts were loaded on ionic metal affinity chromatography (IMAC; Ni ⁺⁺ NTA from Qiagen) to specifically retain the recombinant protein. Residual protein was eluted by a 0-500 mM imidazole gradient (possibly in the presence of detergent) in phosphate buffer.

7.2 Antibody Production and Immunohistochemistry

By using a small amount of relatively purified protein to generate an immunological tool, a) detecting expression by immunohistochemistry in normal or cancerous tissue sections; b) detecting the expression followed by protein detection during the purification process (ELISA / Western blot); c) Purified protein was characterized / quantified (ELISA).

7.2.1 Polyclonal Antibodies:

Immunization

Two to three rabbits were immunized intramuscularly (I.M.) three times at three week intervals using 100 μg of protein formulated in adjuvant 3D-MPL / QS21. Blood samples were taken three weeks after each immunization and antibody titers were estimated in serum by ELISA using the protein with antigen coating according to standard protocols.

ELISA

96 well microplates (maxisorb Nunc) were coated using 5 μg protein at 4 ° C. overnight. After 1 hour saturation at 37 ° C. with 1% PBS NCS, serial dilutions of rabbit serum were added at 37 ° C. for 1 hour 30 minutes (starting at 1/10). After three washes in PBS tween, anti-rabbit biotinylated antiserum (Amersham) was added (1/5000). The plate was washed and peroxidase coupled streptavidin (1/5000) was added at 37 ° C. for 30 minutes. After washing, 50 μl TMB (BioRad) was added for 7 minutes and then the reaction was stopped using 0.2 M H ₂ SO ₄ . OD can be measured at 450 nm and median dilution was calculated by SoftmaxPro.

7.2.2 Monoclonal Antibodies:

Immunization

5 BALB / c mice were immunized three times at three week intervals using 5 μg purified protein. Blood collection was performed 14 days after II and one week after 3 weeks. This serum was tested by ELISA on purified proteins used as coated antigens. Based on this result (medium dilution> 10000), one mouse was selected for fusion.

Fusion / HAT selection

Splenocytes were fused with SP2 / 0 myeloma following standard protocol using 40% PEG and 5% DMSO. The cells were then seeded in 96 well plates at 2.5 × 10 ⁴ to 2.5 × 10 ⁵ cells / well and resistant clones were selected in HAT medium. Supernatants of these hybridomas were tested for their content in specific antibodies and, if positive, used for 2 cycles of defined dilution. After two rounds of screening, three hybridomas were selected for ascites production.

7.2.3 Immunohistochemistry

If antibodies are available, by performing immunostaining on normal or cancerous tissue sections:

The level of expression of the antigen of the invention in cancerous tissues relative to normal tissue or

◇ Proportion of specific types of cancer expressing antigen

When other cancer types also express antigens

The proportion of cells expressing antigen in cancer tissues was determined.

Tissue Sample Preparation

After incision, tissue samples were placed on cork discs in OCT compounds and rapidly frozen in isopentane, previously supercooled in liquid nitrogen (-160 ° C.). This block was then stored at -70 ° C until use. 7-10 μm sections were realized in a cryostat chamber (-20, −30 ° C.).

dyeing

Tissue sections were dried for 5 minutes at room temperature (RT), fixed in acetone for 10 minutes at RT, dried again and saturated with PBS 0.5% BSA 5% serum. Direct or indirect staining was performed using antigen specific antibodies after 30 minutes at RT. Direct staining resulted in better specificity but less intense staining whereas indirect salivary glands resulted in stronger but less specific staining.

7.3 Analysis of Human Intracellular Immune Response to Antigens of the Invention

The immunological relevance of the antigens of the invention can be assessed by in vitro priming of human T cells. All T cell lymphocyte lines and dendritic cells were derived from PBMCs (peripheral blood mononuclear cells) of healthy donors (preferably HLA-A2 subtypes). HLA-A2.1 / K ^b transgenic mice were also used for the screening of HLA-A2.1 peptides.

Newly discovered antigen-specific CD8 + T cell lines were elevated and maintained by weekly in vitro stimulation. Lysis activity and γ-IFN production of CD8 lines in response to antigen or antigen-induced-peptide were tested using standard methods.

Two strategies were used to elevate CD8 + T cell lines: peptide-based approach and whole gene-based approach. Both approaches require the full-length cDNA of the newly discovered antigen in the correct reading frame to be cloned in an appropriate delivery system or used to predict the sequence of the HLA binding peptide.

Peptide-Based Approach

Parker's algorithm (Parker, KC, MA Bednarek, and JE Coligan. 1994. Scheme for ranking potential HLA-A2 binding peptide based on independent binding of individual peptide side-chains. J. Immunol. 152: 163 and http://bimas.dcrt.nih.gov/molbio/hla_bind/) or the Rammensee method (see Rammensee, Friede, Stevanovic, MHC ligands and peptide motifs: 1st listing, Immunogenetics 41, 178-228, 1995; Rammensee, Bachmann, Stevanovic: MHC ligands and peptide motifs.Landes Bioscience 1997, and http://134.2.96.221/scripts/hlaserver.dll/home.htm). Peptides were then screened in a HLA-A2.1 / K ^b transgenic mouse model (Vitiello et al.).

a) predicted epitope binding HLA_A0201 allele:

a.1) HLA-A ^* 0201 hexamer

Estimate the half-life of the separation of molecules containing these subsequences

a.2) HLA A02_01 depolymer

Estimate the half-life of the separation of molecules containing these subsequences

Briefly, transgenic mice are immunized with an adjuvanted HLA-A2 peptide and those unable to induce a CD8 response (as defined by the effective lysis of peptide-pulsed autologous spleen cells) are further added in the human system. Will be analyzed.

Human dendrites (incubated according to Romani et al.) Were used to pulsate with peptides and stimulate CD8-sorted T cells (by Facs). After several weekly stimulation, CD8 lines were first tested on peptide-pulsed autologous BLCLs (EBV-B transformed cell lines). To demonstrate proper in vivo processing of the peptides, CD8 lines were tested on cDNA-transfected tumor cells (HLA-A2 transfected LnCaP, Skov3 or CAMA tumor cells).

Whole Gene-Based Approach

CD8 + T cell lines were gene-gun transfected dendritic cells, retrovirally transduced B7.1-transfected fibroblasts, recombinant pox virus (Kim et al.) Or adenoviruses Butterfield et al. Infected dendritic cells were used to prime and stimulate. Virus infected cells are highly efficient at providing antigen peptides because the antigens are expressed at high levels but can only be used once to avoid over-growth of the viral T cell line.

After alternating stimulation, CD8 + lines were tested on cDNA-transfected tumor cells as indicated above. Immunological efficacy was confirmed by determining peptide specificity and identity.

references

Vitiello et al. (L. Sherman), J. Exp. Med., J. Exp. Med, 1991, 173: 1007-1015.

Romani et al., J. Exp. Med., 1994, 180: 83-93.

Kim et al., J. Immunother., 1997, 20: 276-286.

Butterfield et al., J. Immunol., 1998, 161: 5607-5613.

All publications, including but not limited to patents and patent applications cited in this application, are hereby incorporated by reference as if individually and individually indicated to be incorporated herein by reference as if each publication were the entire described.

Claims (37)

An isolated polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence of SEQ ID NO: 2 over the full length of SEQ ID NO: 2. The isolated polypeptide of claim 1, wherein the amino acid sequence has at least 95% identity with SEQ ID NO: 2. The polypeptide of claim 1 comprising the amino acid sequence of SEQ ID NO: 2. Isolated polypeptide of SEQ ID NO: 2. A polypeptide comprising an immunogenic fragment of a polypeptide according to any one of claims 1 to 4, wherein the immunogenic activity of the immunogenic fragment is substantially the same as the polypeptide of SEQ ID NO: 2. 6. A polypeptide according to any one of claims 1 to 5, wherein the polypeptide is part of a large fusion protein. The polypeptide of claim 1, wherein the polypeptide is chemically conjugated with a carrier protein. An isolated polynucleotide encoding a polypeptide according to any one of claims 1 to 6. An isolated polynucleotide comprising a nucleotide sequence that encodes a polypeptide having at least 70% identity with the amino acid sequence of SEQ ID NO: 2 over the full length of SEQ ID NO: 2; Or a nucleotide sequence complementary to this isolated polynucleotide. An isolated polynucleotide comprising a nucleotide sequence having at least 70% identity with a nucleotide sequence encoding a polypeptide of SEQ ID NO: 2 over the entire coding region; Or a nucleotide sequence complementary to this isolated polynucleotide. An isolated polynucleotide comprising a nucleotide sequence having at least 70% identity with the sequence of SEQ ID NO: 1 over the full length of SEQ ID NO: 1; Or a nucleotide sequence complementary to this isolated polynucleotide. 12. The isolated polynucleotide according to any one of claims 8 to 11, wherein the identity is at least 95%. (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide of SEQ ID NO: 2; (b) a polynucleotide of SEQ ID NO: 1; And (c) polynucleotides that can be obtained by screening appropriate libraries using labeled probes having sequences of SEQ ID NO: 1 or fragments thereof under stringent hybridization conditions, the polynucleotides having a protein of SEQ ID NO: 2 Encoding a protein having similar immunogenic properties) or an nucleotide sequence complementary to the isolated nucleotide. An expression vector or live recombinant microorganism comprising the isolated polynucleotide according to claim 8. A host cell comprising the expression vector of claim 14 or the isolated polynucleotide of claims 8-13. A method of producing a polypeptide of claim 1, comprising culturing the host cell of claim 15 under conditions sufficient to produce the polypeptide and recovering the polypeptide from the culture medium. A vaccine comprising an effective amount of the polypeptide of any one of claims 1 to 7 and a pharmaceutically acceptable carrier. A vaccine comprising an effective amount of the polynucleotide of any of claims 8 to 13 and a pharmaceutically effective carrier. An effective amount of antigen presenting cells and pharmaceuticals modified by in vitro loading with a polypeptide of any one of claims 1 to 7, or genetically modified in vitro to express the polypeptide of claim 1. Vaccine containing an effective carrier. 20. The vaccine of any one of claims 17-19, further comprising a TH-1 inducible adjuvant. The vaccine of claim 20, wherein the TH-1 inducible adjuvant is selected from the group of adjuvants comprising 3D-MPL, QS21, a mixture of QS21 and cholesterol and CpG oligonucleotides. An antibody that is immunospecific to a polypeptide or immunological fragment according to any one of claims 1 to 5. (a) measuring the binding of a candidate compound to a polypeptide (or a cell or membrane with the polypeptide) or a fusion protein thereof by a label that is directly or indirectly bound to the candidate compound; (b) measuring the binding of a candidate compound to a polypeptide (or cell or membrane with the polypeptide) or a fusion protein thereof in the presence of a labeled competitor; (c) using a suitable detection system on the cell or cell membrane with the polypeptide to test whether the candidate compound produces a signal generated by activation or inhibition of the polypeptide; (d) a method of mixing a candidate compound with a solution containing the polypeptide of any one of claims 1 to 7 to form a mixture, measuring the activity of the polypeptide in the mixture, and comparing the activity of the mixture with a standard. ; or (e) using a method selected from the group consisting of methods for detecting the effect of mRNA encoding an polypeptide in a cell and the candidate compound on producing the polypeptide, for example using an ELISA assay. A screening method for identifying a compound that stimulates or inhibits the function of the polypeptide of claim 5. The polypeptide of any one of claims 1 to 7 or the polynucleotide of any one of claims 8 to 13 is incubated with cells from the immune system of a mammal in vitro and thus activated so as to treat the disease. A method of treating a subject by immunoprevention or treatment, comprising inducing an immune response to the molecule of any one of claims 1 to 5 by reinjecting the cells into a mammal. The method of claim 24, wherein the treatment is for ovarian cancer or colon cancer. An agent or antagonist for the polypeptide of claim 1. (a) an agent or antagonist to the polypeptide of claims 1 to 5; (b) the isolated polynucleotide of claims 8-13; or (c) A therapeutic compound that is a nucleic acid molecule that modulates the expression of a nucleotide sequence encoding a polypeptide of any one of claims 1-5. Diagnosing susceptibility to a disease or disorder in a subject associated with expression or activity of the polypeptide of any one of claims 1 to 5 in a subject, including analyzing the presence or amount of the polypeptide in a sample derived from the subject. How to. Susceptibility to a disease or condition in a subject associated with the expression or activity of the polynucleotide of any one of claims 8 to 13 in the subject, including analyzing the presence or amount of polynucleotide in the sample derived from the subject How to diagnose. Presence of colorectal cancer in a subject or susceptibility to colon cancer associated with expression or activity of the polypeptide of any one of claims 1 to 5 in a subject, including analyzing the presence or amount of the polypeptide in a sample derived from the subject. How to diagnose. The presence or absence of colon cancer in a subject associated with the expression or activity of the polynucleotide of any one of claims 8-13 in the subject, including analyzing the presence or amount of polynucleotide in the sample derived from the subject. How to diagnose sensitivity to. (a) an isolated polynucleotide comprising a nucleotide sequence having at least 70% identity with SEQ ID NO: 3 over the full length of SEQ ID NO: 3; (b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 3; (c) an isolated polynucleotide selected from the group consisting of polynucleotides of SEQ ID NO: 3. Live vaccine composition comprising the expression vector according to claim 14 or the live recombinant microorganism. Use of the polynucleotide of any one of claims 8 to 13 for the manufacture of a medicament for use in the treatment of carcinoma. Use of the polynucleotide of any one of claims 8 to 13 for the manufacture of a medicament for use in the treatment of colon carcinoma. Use of the polynucleotide of any one of claims 1 to 7 for the manufacture of a medicament for use in the treatment of carcinoma. Use of the polypeptide of any one of claims 1 to 7 for the manufacture of a medicament for use in the treatment of colon carcinoma.