ES2355129T3 - NEW COMPOUNDS. - Google Patents

Abstract

An immunogenic composition comprising a CASB7439 polypeptide which comprises an amino acid sequence having at least 70% identity in relation to the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 10 along the total length of SEQ ID NO: 2 or SEQ ID NO: 10, or an immunogenic fragment of CASB7439 in which the fragment comprises an amino acid sequence of SEQ ID NO: 16 to SEQ ID NO. : 33, and a pharmaceutically acceptable vehicle, for use in medicine.

Description

The present invention relates to pharmaceutical compositions and methods for inducing immune responses against tumor related antigens. More specifically, the disclosure refers to polynucleotides, herein referred to as CASB7439 polynucleotides, polypeptides encoded by them (referred to herein as CASB7439 polypeptides), recombinant materials and methods for their production. In another aspect, the disclosure relates to methods for using such polypeptides and polynucleotides, which include the treatment of cancer, more particularly colorectal cancer, and autoimmune diseases and other related conditions. In another aspect, the disclosure relates to pharmaceutical compositions containing CASB7439 polypeptides and polynucleotides, to methods of making such compositions and their use in medicine. In a further aspect, the disclosure refers to the procedures for identifying agonists and antagonists / inhibitors that use the materials provided by the disclosure, and that treat conditions associated with the imbalance of CASB7439 polypeptide with the identified compounds. In a further aspect, the disclosure refers to diagnostic tests to detect diseases associated with inappropriate activity or levels of CASB7439 polypeptide.

It is believed that the polypeptides and polynucleotides of the present disclosure are important immunogens for specific prophylactic or therapeutic immunization against tumors, because they are specifically expressed or highly overexpressed in tumors compared to normal cells and therefore can be selected as a target by Antigen-specific immune mechanisms that lead to the destruction of the tumor cell. They can also be used to diagnose the appearance of tumor cells. In addition, its inappropriate expression in some circumstances may cause induction of inappropriate, autoimmune immune responses, which could be corrected through appropriate vaccination using the same polypeptides or polynucleotides. In this regard, the most important biological activities for our purpose are the antigenic and immunogenic activities of the polypeptide of the present disclosure. A polypeptide of the present disclosure may also exhibit at least one other biological activity of a CASB7439 polypeptide, which could qualify it as a target for therapeutic or prophylactic intervention other than that related to the immune response. 25

In a first aspect, the present disclosure relates to CASB7439 polypeptides. Such peptides include isolated polypeptides, which comprise an amino acid sequence which has an identity of at least 70%, preferably an identity of at least 80%, more preferably an identity of at least 90%, even more preferably an identity of at least 95%, most preferably an identity of at least 97-99%, to SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID #: 14 along the full length of SEQ ID NO: 2, 30 SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14 respectively, with the condition that said isolated polypeptide is not SEQ ID NO: 2, SEQ ID NO: 12, or SEQ ID NO: 14. Such polypeptides include those comprising the amino acid of SEQ ID NO: 10.

Additional peptides of the present invention include isolated polypeptides, in which the amino acid sequence has an identity of at least 70%, preferably an identity of at least 80%, more preferably an identity of at least 90%, even more preferably an identity of at least 95%, most preferably an identity of at least 97-99%, to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, along the full length of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14 respectively, with the proviso that said polypeptide is not SEQ ID NO: 2, SEQ ID NO: 12, or SEQ ID NO: 14. Such polypeptides include the polypeptides of the SEQ ID NO: 10. 40

Preferably the aforementioned polypeptides are produced recombinantly. Most preferably, the polypeptides according to the invention are purified, and are substantially free of any other proteins or contaminating material of host origin.

Additional peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 1. 45

The disclosure also provides an immunogenic fragment of a CASB7439 polypeptide, which is a contiguous portion of the CASB7439 polypeptide that has the same or similar immunogenic properties as the polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID N .º: 10, SEQ ID NO: 12, or SEQ ID NO: 14. That is, the fragment (if necessary when coupled to a carrier or as part of a larger condensation protein) is capable of eliciting an immune response that recognizes the CASB7439 polypeptide. Such an immunogenic fragment 50 may include, for example, the CASB7439 polypeptide lacking an N-terminal leader sequence, a transmembrane domain or a C-terminal assurance domain. In a preferred aspect of the immunogenic fragment of CASB7439 according to the invention it substantially comprises the entire extracellular domain of a polypeptide having an identity of at least 70%, preferably an identity of at least 80%, more preferably an identity of at minus 90%, even more preferably an identity of at least 95%, most preferably an identity of at least 97-99%, to that of SEQ ID NO: 2, SEQ ID NO: 10 , SEQ ID NO: 12, or SEQ ID NO: 14, along the full length of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12 , or SEQ ID

No.: 14 respectively. Preferably, an immunogenic fragment according to the invention comprises at least one epitope.

Peptide fragments incorporating an epitope of CASB7439 will typically comprise at least 7, preferably 9 or 10 contiguous amino acids derived from SEQ ID NO: 2. Preferred epitopes are shown in SEQ ID NO: 16 to SEQ ID. No .: 33. 5

Peptides incorporating these epitopes form a preferred aspect of the present disclosure. Mimotopes that have the same characteristics as these epitopes, and immunogens comprising such mimotopes that generate an immune response that cross-reacts with an epitope in the context of the CASB7439 molecule, are also part of the present disclosure.

Therefore, the present invention includes isolated peptides that encompass these epitopes themselves, and any mimotope thereof. The meaning of mimotope is defined as an entity that is sufficiently similar to the native CASB7439 epitope so that it is capable of being recognized by antibodies that recognize the native molecule; (Gheysen, H.M., et al., 1986, Synthetic peptides as antigens. Wiley, Chichester, Ciba foundation symposium 119, p130-149; Gheysen, H.M .. 1986, Molecular Immunology, 23, 7, 709-715); or are capable of producing antibodies, when coupled to a suitable carrier, whose antibodies react with the native molecule. fifteen

Peptide mimotopes of the epitopes identified above may be designed for a particular purpose by adding, removing or substituting the chosen amino acids. Consequently, the peptides of the present invention can be modified for purposes of ease of conjugation to a protein carrier. For example, it may be desirable for some chemical conjugation procedures to include a terminal cysteine to the epitope. In addition, it may be desirable for peptides conjugated to a protein carrier to include a distal hydrophobic end 20 of the conjugated end of the peptide, such that the free unconjugated end of the peptide remains associated with the surface of the transporter protein. This reduces the conformational degrees of freedom of the peptide, and thus increases the probability that the peptide is present in a conformation that closely resembles that of the peptide as found in the context of the entire molecule. For example, the peptides can be altered to have an N-terminal cysteine and a C-terminal hydrophobic nested tail. Alternatively, the addition or substitution of a stereoisomer D form of one or more of the amino acids can be performed to create a beneficial derivative. , for example, to improve the stability of the peptide. Those skilled in the art will realize that such modified peptides or mimotopes could be a completely or partially non-peptide mimotope in which the constituent residues are not necessarily confined to the naturally occurring amino acids. In addition, these can be recycled by techniques known in the art to restrict the peptide into a conformation that closely resembles its shape when the peptide sequence is in the context of the entire molecule. A preferred method of recycling a peptide comprises the addition of a pair of cysteine residues to allow the formation of a disulfide bridge.

In addition, those skilled in the art will realize that the mimotopes or immunogens of the present invention may be greater than the epitopes identified above and as such may comprise the sequences described herein. In accordance with the foregoing, the mimotopes of the present disclosure may consist of the addition of N-terminal and / or C-terminal extensions of a certain number of other natural residues at one or both ends. Peptide mimotopes can also be backsequences of natural sequences, because sequence orientation is reversed; or alternatively the sequences may completely or at least in part consist of amino acids of stereoisomer D (reverse sequences). Also, the peptide sequences can be retro-reversed in character, because the sequence orientation is reversed and the amino acids are in the form of the stereoisomer D. Such retro or reverse-reverse peptides have the advantage of being non-independent, and as such can overcome problems of self-tolerance in the immune system.

Alternatively, peptide mimotopes can be identified using antibodies that are capable of binding themselves to the epitopes of the present invention using techniques such as phage display technology 45 (EP 0 552 267 B1). This technique generates a large number of peptide sequences that mimic the structure of native peptides and, therefore, are capable of binding anti-native peptide antibodies, but may not necessarily by themselves share a significant sequence homology to the native peptide This approach can have significant advantages by allowing the possibility of identifying a peptide with improved immunogenic properties, or it can overcome any potential antigen self-tolerance problem that may be associated with the use of the native peptide sequence. Additionally, this technique allows the identification of a recognition pattern for each native peptide in terms of its chemical properties shared among the recognized mimotope sequences.

Covalent coupling of the peptide to the immunogenic carrier can be carried out in a manner well known in the art. Therefore, for example, for direct covalent coupling it is possible to use a carbodiimide, glutaraldehyde or ester of (N- [γ-maleimidobutyryloxy] succinimide, using common commercially available heterobifunctional linkers such as CDAP and SPDP (using the manufacturers instructions ).

After the coupling reaction, the immunogen can be easily isolated and purified by means of a dialysis procedure, a gel filtration procedure, a fractionation procedure, etc.

The types of vehicles used in the immunogens of the present invention will be readily known to those skilled in the art. The function of the vehicle is to provide cytokine support in order to help induce an immune response against the peptide. A non-exhaustive list of carriers that can be used in the present invention include: California Barnacle Hemocyanin (KLH), serum albumins such as bovine serum albumin (BSA), inactivated bacterial toxins such as tetanus or diphtheria toxins (TT and DT), or recombinant fragments thereof (eg, Domain 1 of Fragment C of TT, or the transposition domain of DT), or the derivative of purified tuberculin protein (PPD). Alternatively, the mimotopes or epitopes can be directly conjugated to liposome carriers, which may additionally comprise immunogens capable of providing support for T cells. Preferably the proportion of carrier mimotopes is of the order of 1: 1 to 20: 1, and preferably each Carrier should carry between 3-15 peptides.

In one embodiment of the disclosure a preferred carrier is Protein D derived from Haemophilus influenzae (EP 0 594 610 B1). Protein D is an Ig-D binding protein derived from Haemophilus influenzae and has been patented by Forsgren (WO 91/18926, EP 0 594 610 B1 granted). In some circumstances, for example in recombinant immunogen expression systems it may be desirable to use protein D fragments, for example, Protein D 1/3 (comprising 100-110 N-terminal amino acids of protein D (GB document 9717953.5)).

Another preferred method of presenting the peptides of the present disclosure is in the context of a recombinant condensation molecule. For example, EP 0 421 635 B describes the use of chimeric hepadnavirus core antigen particles to present foreign peptide sequences in a virus-like particle. As such, the immunogens of the present invention may comprise peptides presented in chimeric particles consisting of hepatitis B core antigen. Additionally, recombinant condensation proteins may comprise the mimotopes of the present invention and a transporter protein, such as NS1. of the influenza virus. For any recombinantly expressed protein that is part of the present invention, the nucleic acid encoding said immunogen also forms an aspect of the present disclosure.

The peptides used in the present disclosure can be easily synthesized by solid phase procedures well known in the art. Suitable syntheses can be carried out using "T-boc" or "F-moc" procedures. The cyclic peptides can be synthesized by the solid phase method which employs the well known "F-moc" method and the polyamide resin in the fully automated apparatus. Alternatively, those skilled in the art will know the laboratory procedures necessary to carry out the process manually. The techniques and procedures for solid phase synthesis are described in 'Solid Phase Peptide Synthesís: A Practical Approach' by E. Atherton and R.C. Sheppard, published by IRL in the Oxford University Press (1989). Alternatively, the peptides can be produced by recombinant methods, which include the expression of nucleic acid molecules encoding mimotopes in a bacterial or mammalian cell line, followed by purification of the expressed mimotope. Techniques for recombinant expression of peptides and proteins are known in the art, and are described in Maniatis, T., Fritsch, E.F., and Sambrook, et al., Molecular cloning, a laboratory manual, 2nd Ed; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

In a further embodiment of the invention there is provided a method of producing a polypeptide as described herein. The process of the invention can be carried out by conventional recombinant techniques such as those described in Maniatis et al., Molecular Cloning-A Laboratory Manual; Cold Spring Harbor, 1982-1989. In accordance with the foregoing, a process for producing a polypeptide according to the disclosure is provided, which comprises culturing a host cell under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture medium. In particular, the process of the invention may preferably comprise the steps of:

i) preparing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence encoding the protein or an immunogenic derivative thereof;

ii) transforming a host cell with said vector; fifty

iii) culturing said transformed host cell under conditions that allow the expression of said DNA polymer to produce said protein; Y

iv) recover said protein.

The polypeptides or the immunogenic fragment of the disclosure may be in the form of a "mature" protein or may be part of a larger protein such as a precursor or a condensation protein. It is often 55

It is advantageous to include an additional amino acid sequence containing secretory or leader sequences, pro-sequences, sequences that aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production. In addition, the addition of the exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential of the final molecule is also considered. 5

In one aspect, the disclosure relates to genetically modified soluble condensation proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the heavy or light chain constant regions of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). Preferred as immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgG 1, where condensation takes place in the hinge region. In a particular embodiment, the part of Fc can be removed simply by the incorporation of a cleavage sequence that can be segmented with blood coagulation factor Xa. In addition, this disclosure refers to the processes for the preparation of these condensation proteins by genetic engineering, and their use for drug screening, diagnosis and therapy. A particularly preferred aspect of the disclosure relates to the use of a polypeptide or polynucleotide in the preparation of a vaccine to immunotherapeutically treat a patient suffering from or susceptible to carcinoma, especially colon cancer or other tumors or diseases. associated with the colon. An additional aspect of the disclosure also relates to polynucleotides encoding such condensation proteins. Examples of condensation protein technology can be found in International Patent Applications No. W094 / 29458 and W094 / 22914.

Proteins can be chemically conjugated, or expressed as recombinant condensation proteins that allow improved levels to be produced in an expression system compared to uncondensed protein. The condensation partner can help to provide helper T epitopes (immune condensation partner), preferably human helper T epitopes, or help express the protein (expression enhancer) at levels higher than the native recombinant protein. Preferably, the condensation partner will be both an immunological condensation partner and an expression improvement partner.

Condensation partners include protein D derived from Haemophilus influenza B and non-structural protein derived from influenza virus, NS1 (hemagglutinin). Another immunological condensation partner is the protein known as LYTA. Preferably, the C-terminal portion of the molecule is used. Lyta is derived from Streptococcus pneumoniae that synthesizes an N-acetyl-L-alanine amidase, LYTA amidase (encoded by the lytA gene {Gene, 43 30 (1986) page 265-272), an autolysin that specifically degrades some bonds in the structure main peptidoglycan The C-terminal domain of the LYTA protein is responsible for the affinity to choline or some choline analogs such as DEAE This property has been exploited for the development of E. C-LYTA expression plasmids. coli useful for the expression of condensation proteins The purification of hybrid proteins containing the C-LYTA fragment at its amino terminus has been described {Biotechnology: 10, (1992) page 795-798} It is possible to use the portion of repeating the Lyta molecule found at the C-terminal end that begins at residue 178, for example, residues 188-305.

The present disclosure also includes xenogeneic forms (also called orthologous forms) of the aforementioned polypeptides, said xenogenic forms referring to an antigen that has substantial sequence identity to the human antigen (also called autologous antigen) that serves as a reference antigen but 40 which is derived from a different non-human species. In this context, substantial identity refers to the concordance of an amino acid sequence with another amino acid sequence or a polynucleotide sequence with another polynucleotide sequence when such sequences are arranged in a better alignment in any number of sequence alignment proteins known in The matter. Substantial identity means at least 70-95% and preferably at least 85-95%, most preferably at least 90-95%, of sequence identity between the compared sequences. Therefore, according to the invention, the xenogenic CASB7439 polypeptide will be a CASB7439 polypeptide that is xenogeneic with respect to human CASB7439, in other words that is isolated from a species other than human. In a different embodiment, the polypeptide is isolated from mouse, rat, pig, or rhesus monkey, most preferably from mouse or rat. In accordance with the foregoing, the present invention also provides a method for inducing an immune response against human CASB7439 having a 50 amino acid sequence as set forth in any of the sequences SEQ ID NO: 2 or SEQ ID NO: 10 in a human being, comprising administering to the patient an effective dose of a composition comprising a xenogeneic form of said human CASB7439 as described herein. A preferred embodiment is a method for inducing an immune response against human CASB7439 using the xenogeneic CASB7439 isolated from mouse, rat, pig or rhesus monkey. Another preferred method of inducing an immune response according to the present invention is to use an antigenic composition that includes a live viral expression system that expresses said xenogenic antigen. The preferred xenogeneic CASB7439 polypeptide has the sequence set forth in SEQ ID NO: 12 (mouse) or in SEQ ID NO: 14 (rat).

The isolated xenogeneic CASB7439 polypeptide will generally share substantial sequence affinity, and will include

isolated polypeptides comprising an amino acid sequence which has an identity of at least 70%, preferably an identity of at least 80%, more preferably an identity of at least 90%, even more preferably an identity of at least 95%, which more preferably an identity of at least 97-99%, to SEQ ID NO: 12 or to SEQ ID NO: 14, along the full length of SEQ ID NO: 12 or the SEQ ID NO: 14. According to the foregoing, the xenogenic polypeptide will comprise an immunogenic fragment of the polypeptide of SEQ ID 5: 12 or SEQ ID NO: 14 in which the immunogenic activity of the fragment Immunogenic is substantially equal to the polypeptide of SEQ ID NO: 12 or of SEQ ID NO: 14. In addition, the xenogenic CASB7439 polypeptide may be a fragment of at least about 20 consecutive amino acids, preferably about 30, more preferably of about 50, even more preferably te of about 100, most preferably about 150 contiguous amino acids selected from the sequences of 10 amino acids as shown in SEQ ID NO: 12 or in SEQ ID NO: 14. More particularly the fragments of Xenogenic CASB7439 will maintain some functional property, preferably an immunological activity, of the major molecule set forth in SEQ ID NO: 12 or in SEQ ID NO: 14, and are useful in the procedures described herein (eg , in pharmaceutical and vaccine compositions, in diagnoses, etc.). In particular, the fragments will be capable of generating an immune response against the human homologue, such as the generation of cross-reactive antibodies that react with the human antologous form derived from CASB7439 as set forth in any of SEQ ID NO. : 2. In a specific embodiment, the xenogeneic polypeptide of the disclosure may be part of a larger condensation, comprising the xenogeneic CASB7439 polypeptide or fragment thereof and a heterologous protein or part of a protein that acts as a condensation partner as It was described previously. twenty

The present disclosure also includes variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative amino acid substitutions, in which one residue is replaced by another with similar characteristics. Such typical substitutions are found between Ala, Val, Leu, and Ile; between Ser and Thr: between the acid residues Asp and Glu; between Asn and Gln; and between the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred variants are those in which several 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, eliminated, or added in any combination.

The polypeptides of the present disclosure can be prepared in any suitable manner. Such polypeptides include naturally occurring isolated polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these procedures. The means for preparing such polypeptides are well understood in the art. 30

In a further aspect, the present disclosure relates to polynucleotides of CASB7439. Such polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide having an identity of at least 70%, preferably an identity of at least 80%, more preferably an identity of at least 90%, even more preferably an identity of at least 95%, to the amino acid sequence of SEQ ID NO: 2 or of SEQ ID NO: 10, along the full length of SEQ ID NO: 2, SEC ID No.: 35 10, respectively. In this regard, encoded polypeptides having an identity of at least 97% are highly preferred, while those with an identity of at least 98-99% are more highly preferred, and those with an identity of at least 99% are the most highly preferred

Additional polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence having an identity of at least 70%, preferably an identity of at least 40 80%, more preferably an identity of at least 90%, even more preferably an identity of at least 95%, to a nucleotide sequence encoding a polypeptide of SEQ ID NO: 2 or of SEQ ID NO: 10, throughout the entire coding region. In this regard, polynucleotides that have an identity of at least 97% are highly preferred, while those with an identity of at least 98-99% are more highly preferred, and those with an identity of at least 99% are the most highly preferred. Four. Five

Additional polynucleotides of the present disclosure include isolated polynucleotides comprising a nucleotide sequence having an identity of at least 70%, preferably an identity of at least 80%, more preferably an identity of at least 90%, even more preferably an identity of at least 95%, to SEQ ID NO: 1, SEQ ID NO: 8, or to SEQ ID NO: 9, along the entire length of said sequences, or the coding sequence of SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9 along the entire length of said said coding sequence of SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9. In this regard, polynucleotides having an identity of at least 97% are highly preferred, while those with an identity of at least 98-99% are more highly preferred, and those with at least 99% identity are the most highly preferred. Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9, as well as the polynucleotide of SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 9 or the coding sequence of SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 9.

The present disclosure also provides a nucleic acid encoding the aforementioned xenogenic proteins of the present invention and their use in medicine. In a preferred embodiment, the CASB7439 polynucleotide

Xenogenic for use in pharmaceutical compositions has the sequence set forth in SEQ ID No. 13 (mouse) or in SEQ ID No. 15 (rat). The xenogeneic CASB7439 polynucleotides isolated according to the disclosure can be single stranded (coding or antisense) or double stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but do not require, be present within a polynucleotide of the present invention. In other related embodiments, the present invention provides polynucleotide variants that have substantial identity with the sequences described herein in SEQ ID No. 13 or in SEQ ID No. 15, for example those comprising an identity sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a polynucleotide sequence of this disclosure using the procedures described herein (eg, BLAST analysis using standard parameters). In a related embodiment, the xenogeneic polynucleotide isolated from the disclosure will comprise a nucleotide sequence encoding a polypeptide having an identity of at least 90%, preferably 95% and higher, with the amino acid sequence of SEQ ID N .12 or SEQ ID No. 14, along the entire length of SEQ ID No. 12 or SEQ ID No. 14, or a nucleotide sequence complementary to said isolated polynucleotide.

The disclosure also provides polynucleotides that are complementary to all the polynucleotides described above.

Such polynucleotides can be inserted into a plasmid, a recombinant microorganism vector or a suitable recombinant living microorganism and is used for immunization (see for example 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)). In accordance with the foregoing, a recombinant live expression or microorganism vector is provided in the present disclosure comprising said polynucleotides as defined above.

The disclosure also provides a fragment of a CASB7439 polynucleotide that when administered to a patient has the same immunogenic properties as the polynucleotide of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9 , SEQ ID NO: 13 or SEQ ID NO: 15.

The disclosure also provides a polynucleotide encoding an immune fragment of a CASB7439 polypeptide as defined above.

The fragments have an immunogenic activity level of at least about 50%, preferably at least about 70%, and more preferably at least about 90% of the level of immunogenic activity of a polypeptide sequence set forth in SEQ ID NO. #: 2, or in SEQ ID NO: 10 or a polypeptide sequence encoded by a polynucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID 30 : 9, SEQ ID NO: 13 or SEQ ID NO: 15.

Polypeptide fragments according to the disclosure preferably comprise at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all intermediate lengths, of a polypeptide composition set forth herein. , such as those set forth in SEQ ID NO: 2, or SEQ ID NO: 10, or those encoded by a polynucleotide sequence set forth in a sequence of SEQ ID 35: 1, SEQ ID NO. º: 8, SEQ ID NO: 9, SEQ ID NO; 13 or SEQ ID NO: 15.

The nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence comprising a sequence encoding polypeptides (nucleotide 545 to 1126) encoding a 193 amino acid polypeptide, the polypeptide of SEQ ID NO: 2. Nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 may be identical to the sequence encoding polypeptides contained in SEQ ID NO: 1 or may be a different sequence than that contained in SEQ ID NO. º: 1, which, as a result of the redundancy (degeneration) of the genetic code, also encodes the polypeptide of SEQ ID NO: 2. The polypeptide of SEQ ID NO: 2 is structurally related to other proteins of the achaete scute family, and it is also called "human Achaete Scute homolog 2" (HASH2) (accession number NP_005161 and AAB86993).

The homologous gene 2 of Human Achaete Scute (HASH2), officially designated human ASCL2 (Achaete 45 Scute type 2 complex) is a homologue of the Dchaeophila Achaete and Scute genes. Human ASCL2 is expressed only in extravellosal trophoblasts of the developing placenta and maps on chromosome 11p15 near IGF2 and H 19. The homologous-2 mouse achaete-scute (MASH2) gene encodes a transcription factor that plays a role in the development of the trophoblast. The Mash2 gene is subject to paternal imprinting in the mouse, and the lack of expression of human ASCL2 in non-malignant hydatidiform moles (androgenesis) indicates that human Ascl2 also undergoes imprinting in humans.

Alders et al. (1997) Human Molecular Genetics 6 (6): 859-867 reveals that HASH2 is expressed only in extravellosal trophoblasts of the developing placenta.

The Ascl2 genes are members of the basic helix-loop-helix (BHLH) family of transcription factors. Activate transcription by joining box E (5'-CANNTG-3 '). Dimerization with other BHLH proteins is required for a

effective DNA binding. They are involved in the determination of neuronal precursors in the peripheral nervous system and the central nervous system in Drosophila melanogaster, and probably also in mammals.

The complementary strand of the nucleotide sequence of SEQ ID NO: 1 is the polynucleotide sequence of SEQ ID NO: 6. This strand also comprises two other polypeptide coding sequences. The first polypeptide coding sequence (nucleotide 1184 to 399 of SEQ ID NO: 1, nucleotide 608 to 1393 of SEQ ID 5: 6) encodes a 262 amino acid polypeptide, the polypeptide of SEQ ID N .º: 3. The second polypeptide coding sequence (nucleotide 840 to 262 of SEQ ID NO: 1, nucleotide 952 to 1530 of SEQ ID NO: 6) encodes a 193 amino acid polypeptide, the polypeptide of SEQ ID NO: 11. The nucleotide sequence encoding the polypeptides of SEQ ID NO: 3 and of SEQ ID NO: 11 may be identical to the polypeptide coding sequence contained in the SEQ ID NO: 6 or it may be a different sequence from that contained in SEQ ID NO: 6, which, as a result of the redundancy (degeneration) of the genetic code, also encodes the polypeptides of SEQ ID N .º: 3 and 11. The polypeptide of SEQ ID NO: 3 is structurally related to other proteins of the family of protein coactivadoras of ayuste, having homology and / or structural similarity with the coactivadora subunit of ayuste of Homo sapiens srm300 (access to the genetic bank AAF21439). The polypeptide of SEQ ID NO: 11 is not related to any known protein. Polypeptide sequences as set forth in SEQ ID NO: 3 and SEQ ID NO: 11, and polynucleotide sequences as set forth in SEQ ID NO: 6 are novel and also part of the revelation.

It is expected that the preferred polypeptides and polynucleotides of the present invention will have, among other things, biological functions / properties similar to those of their homologous polypeptides and polynucleotides. In addition, preferred polypeptides, immunological fragments and polynucleotides of the present invention have at least one activity of either SEQ ID NO: 1 or SEQ ID NO: 2 as appropriate.

The present disclosure also relates to other incomplete polynucleotide and polypeptide sequences that were first identified before the determination of the corresponding complete sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

The present invention is provided for a polypeptide that:

(a) comprises a nucleotide sequence having an identity of at least 70%, preferably an identity of at least 80%, more preferably an identity of at least 90%, even more preferably an identity of at least 95 %, most preferably an identity of at least 97-99%, to that of SEQ ID NO: 2, along the full length of SEQ ID NO: 2

(b) has a nucleotide sequence that has an identity of at least 70%, preferably an identity of at least 80%, more preferably an identity of at least 90%, even more preferably an identity of at least 95%, most preferably an identity of at least 97-99%, to the 15 amino acid sequence of SEQ ID NO: 2 along the full length of SEQ ID NO: 2 and

(c) comprises the amino acid of SEQ ID NO: 2. 35

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

When the polynucleotides of the present invention are used for recombinant production of polypeptides of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro-prepro-protein sequence, or other 45 peptide portions of condensation For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In some preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Nati Acad. Sci. USA (1989) 86: 821-824, or is a HA label. The polynucleotide may also contain 5 'and 3' non-coding sequences, such as untranslated, transcribed sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize the mRNA.

Additional embodiments of the present invention include polynucleotides encoding polypeptide variants which comprise the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 15 and in which substitute, eliminate or add, in any combination, several amino acid residues, for example, from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1. 55

Polynucleotides that are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 or in SEQ ID NO: 6, can be used as hybridization probes for cDNA and genomic DNA or as primers for a Nucleic acid amplification reaction (PCR), to isolate full-length cDNAs and genomic clones encoding polypeptides of the present disclosure and to isolate cDNA and genomic clones from other genes (including genes encoding paralogues derived from human and orthologous sources and paralogs derived from 5 different species from human) that have a high sequence similarity to SEQ ID NO: 1 or SEQ ID NO: 6. Typically these nucleotide sequences are 70% identical, preferably 80% identical , more preferably identical to 90%, most preferably identical to 95% to that of the referent. The 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 between 30 and 50 nucleotides. Particularly preferred primers will have between 20 and 25 nucleotides. In particular, polypeptides or polynucleotides derived from sequences of homologous animal origin could be used as immunogens to obtain a reactive immune response to the human gene.

A polynucleotide encoding a polypeptide of the present disclosure, including homologues derived from species other than human, can be obtained by a process comprising the steps of tracking an appropriate library 15 under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or SEQ ID NO: 6 or a fragment thereof; and isolating the full length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to the skilled worker. Preferred stringent hybridization conditions include overnight incubation at 42 ° C in a solution comprising: 50% formamide, 5 x SSC (150 mM NaCl NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate 20 (pH 7.6), 5x Denhardt solution, 10% dextran sulfate, and 20 micrograms / ml denatured sheared salmon sperm DNA; followed by washing the filters in 0.1 x SSC at approximately 65 ° C. Consequently, the present disclosure also includes polynucleotides obtainable by examining an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or SEQ ID NO: 6 or a fragment thereof. 25

The person skilled in the art will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, because the coding of the region for the polypeptide is short at the 5 'end of the cDNA.

There are several procedures available and well known to those skilled in the art for obtaining full-length cDNAs, or extending short cDNAs, for example, those based on the Rapid Amplification procedure of cDNA ends (RACE) (see, for example , Forman et al., PNAS USA 85, 8998-9002, 1988). The recent 30 modifications of the technique, exemplified by Marathón ™ technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for larger cDNAs. In Marathón ™ technology, cDNAs have been prepared from mRNA extracted from a selected tissue and an 'adapter' sequence linked to each end. Nucleic acid amplification (PCR) is then carried out to amplify the 5 '"lost" end of the cDNA using a combination of gene-specific and adapter-specific oligonucleotide primers. The PCR reaction is then repeated using "nested" primers, that is, primers designed to fuse within the amplified product (typically an adapter-specific primer that fuses the additional 3 'into the adapter sequence and a gene-specific primer that fuses the additional 5 'in the known gene sequence). The products of this reaction can then be analyzed by sequencing DNA and full length cDNA constructed either by binding the product directly to the existing cDNA to give a complete sequence, or by carrying out a separate full length PCR using the new sequence information to 5 'primer design.

The recombinant polypeptides of the present disclosure can be prepared by processes well known in the art from genetically modified host cells comprising expression systems. In accordance with the foregoing, in a further aspect, the present disclosure relates to an expression system which comprises a polynucleotide of the present invention, to host cells that are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins that use RNAs derived from the DNA constructs of the present invention.

For recombinant production, host cells can be genetically modified to incorporate expression systems or portions thereof for polynucleotides of the present disclosure. The introduction of polynucleotides into host cells can be accomplished by the procedures described in many standard laboratory manuals, such as 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). Such preferred methods include, for example, calcium phosphate transfection, DEAE-dextran-mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, ladle loading, ballistic introduction or infection.

Preferably the proteins of the invention are coexpressed with thioredoxin in trans (TIT). Coexpression is preferred

of thioredoxin in trans versus cis in order to keep the antigen free of thiodoxin without the need for protease. The coexpression of thioredoxin facilitates the solubilization of the proteins of the invention. The coexpression of thioredoxin also has a significant impact on the production of protein purification, on the solubility and quality of the purified protein.

Representative examples of appropriate hosts include bacterial cells, such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila Sf2 cells and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowles melanoma; and plant cells.

A wide variety of expression systems can be used, for example, chromosomal, episomal, and virus-derived systems, for example, vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, chromosomal elements of yeast, viruses such as baculovirus, papovavirus, such as SV40, vaccine virus, adenovirus, avian smallpox virus, pseudorabia virus and retrovirus and vectors derived from combinations thereof, such as those derived from genetic elements of plasmids and bacteriophages, such as cosmids and phagemids. Expression systems may contain control regions that also regulate the expression of engendering. Generally, any system or vector that is capable of maintaining, propagating or expressing a polynucleotide can be used to produce a polypeptide in a host. The appropriate nucleotide sequence can be inserted into an expression system by any variety of routine and well-known techniques, such as, for example, those set forth in Sambrook et al., Molecular Cloning, A Laboratory Manual (supra). Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space 20 or the extracellular environment. These signals can be endogenous to the polypeptide or they can be heterologous signals.

The expression system may also be a recombinant living microorganism, such as a virus or bacteria. The gene of interest can be inserted into the genome of a live recombinant virus or bacterium 1. Inoculation and infection in vivo with this live vector will lead to in vivo expression of the antigen and induction of immune responses. 25

Therefore, in some embodiments, polynucleotides encoding immunogenic polypeptides of the present invention are introduced into suitable mammalian host cells for expression using any number of known viral systems. In an illustrated embodiment, retroviruses provide an efficient and convenient platform for genetic delivery systems. A selected nucleotide sequence encoding a polypeptide of the present invention can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can be isolated and then delivered to a patient. A number of illustrative retroviral systems have been described {for example, U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7: 980-990; Miller, A.D. (1990) Human Gene Therapy 1: 5¬14; Scarpa et al. (1991) Virology 180: 849-852; Burns et al. (1993) Proc. Nati Acad. Sci. USA 90: 8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet Develop 3: 102-109. 35

In addition, a number of illustrative adenovirus-based systems have also been described. In contrast to retroviruses that integrate into the host genome, adenoviruses persist extrachromosomically minimizing the risks associated with insertion mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57: 267-274; Bett et al. , (1993) J. Virol. 67: 591 1-5921; 15 Mittereder et al. (1994) Human Gene Therapy 5: 717-729; Seth et al. (1994) J. Virol. 68: 933-940; Barr et al. (1994) Gene Therapy 1: 51-58; Berkner, K. L, (1988) BioTechniques 6: 616-629; and 40 Rich et al. (1993) Human Gene Therapy 4: 461-476).

Various adeno-associated virus (AAV) vector systems have also been developed for polynucleotide delivery. AAV vectors can be easily constructed using techniques well known in the art. See, for example, U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publications Nos. WO 92/01070 and WO 93/03769; Lebkowski et al. (1988) Molec. Cell Biol .. 8: 3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring 45 Harbor Laboratory Press); Carter, B. J. (1992) Current opinion ¡n Biotechnology 3: 533-539; Muzyczka, N. '(1992) Current Topics in Microbiol. And Immunol. 158: 97-129; Kotin, R.M. (1994) Human Gene Therapy 5: 793-801; Shelling and Smith (1994) Gene Therapy 1: 165-169; and Zhou et al. (1994) J. Exp. Med. 179: 1867-1 875.

Additional viral vectors useful for delivering nucleic acid molecules encoding the polypeptides of the present invention by genetic transfer include those derived from the smallpox virus family, such as the 50 vaccine viruses and the smallpox virus. As an example, vaccine virus recombinants expressing novel molecules can be constructed as explained below. The DNA encoding a polypeptide is first inserted into an appropriate vector so that it is adjacent to a vaccine enhancer and flanking vaccine DNA sequences, such as the sequence coding for thymidine kinase (TK). This vector is then used to transfect cells that are simultaneously infected with vaccines. Homologous recombination serves to insert the vaccine enhancer plus the gene encoding the polypeptide of interest in the viral genome. The resulting TK.sup. (-) recombinate can be selected by culturing the cells in the presence of 5-

Bromodeoxyuridine and collecting viral plates resistant to it.

A vaccine-based transfection / infection system can be conveniently used to provide inducible expression or co-expression. transient, of one or more polypeptides described herein in host cells of an organism. In this particular system, cells are first infected in vitro with a vaccine virus recombinant that encodes the T7 RNA polymerase bacteriophage. This polymerase exhibits 5 exquisite specificity because it transcribes only templates that support T7 enhancers. After infection, the cells are transfected with the polynucleotide or polynucleotides of interest, driven by a T7 enhancer. The polymerase expressed in the cytoplasm from the recombinant vaccine virus transcribes the DNA transfected into RNA which is then translated into polypeptide by the host translation machinery. The procedure is provided for high-level cytoplasmic, transient, high-volume production of RNA and its translation products. See, for example, Elroy-Stein and Moss, Proc. Nati Acad. Sci. USA (1990) 87: 6743-6747; Fuerst et al. Proc. Nati Acad. Sci. USA (1986) 83: 8122-8126,

Alternatively, avian smallpox viruses, such as poultry and smallpox canarypox viruses, can also be used to deliver the coding sequences of interest. Recombinant avian pox viruses, which express immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of a bird smallpox vector is particularly desirable in the human species and other mammalian species since members of the bird smallpox genus can only replicate productively in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant birdpox viruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccine viruses. 20 See, for example, WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a certain number of alphavirus vectors can be used for the delivery of polynucleotide compositions of the present invention, such as those vectors described in U.S. Pat. No. 5,843,723; 6,015,686; 6,008,035 and 6,015,694. Some vectors based on Venezuelan Equine Encephalitis (VEE), illustrative examples of which can be found in US Pat. No. 25 5,505,947 and 5,643,576.

In addition, the molecular conjugate vectors, such as the chimeric adenovirus vectors described in Michael et al. J. Biol .. Chem. (1993) 268: 6866-6869 and Wagner et al. Proc. Nati Acad. Sci. USA (1992) 89: 6099-6103, can also be used for genetic delivery under disclosure.

Additional illustrative information about these and other known viral delivery systems can be found, for example, in Fisher-Hoch et al., Proc, Natl. Acad. Sci. USA 86: 317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569: 86-103, 1989; Flexner et al., Vaccine 8: 17-21, 1990; U.S. Patents Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Patent No. 4,777,127: GB documents 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 25 6: 616-627, 1988; Rosenfeld et al., Science 252: 431-434, 1991; Kolls et a!, Proc. Natl Acad. Sci. USA 91: 215-219, 1994; Kass-Eisler et al., Proc. Nati Acad. Sci. USA 90: 1 1498-1 1502, 1993; 35 Guzman et al., Circulation 88: 2838-2848, 1993; and Guzman et al., Cir. Res. 73: 1202-1207, 1993.

The recombinant living microorganisms described above may be virulent, or attenuated in various ways in order to obtain live vaccines. Such live vaccines are also part of the invention.

In some embodiments, a polynucleotide can be integrated into the genome of a target cell. This integration can be in the specific position and orientation by means of homologous recombination (genetic replacement) or can be integrated into a random, non-specific position (genetic augmentation). In still further embodiments, the polynucleotide can be stably maintained in the cell as an episomal segment separated from DNA. Such polynucleotide segments or "episomes" encode sequences sufficient to allow maintenance and replication independent of or in synchronization with the host cell cycle. The manner in which the expression construct is supplied to a cell and where the polynucleotide remains in the cell depends on the type of expression construct employed.

In another embodiment of the invention, a polynucleotide is administered / supplied as "pure" DNA, for example, as described in Ulmer et al., Science 259: 1745-1749, 1993 and reviewed by Cohen, Science 259: 1691- 1692, 1993. The uptake of pure DNA can be increased by coating the DNA in biodegradable beads, which are effectively transported to the cells. fifty

In yet another embodiment, a composition of the present invention can be supplied through a particle bombing approach, of which much has been described. In an illustrative example, gas-driven particle acceleration can be achieved with devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, RU) and Powderject Vaccines, Inc. (Madison, Wl), some examples of which are described in US patents No. 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 55 799. This approach offers a needle-free supply approach in which a dry powder formulation

of microscopic particles, such as polynucleotide or polypeptide particles, are accelerated at high speed in a jet of helium gas generated by a portable device, driving the particles into a target tissue of interest.

In a related embodiment, other devices and methods that may be useful for gas-operated needleless injection of compositions of the present disclosure include those provided by Bioject, Inc. (Portland, OR), some examples of which are described in US Patent No. 4,790,824; 5,064,413; 5 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

The polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well known methods including precipitation of ammonium or ethanol sulfate, acid extraction, anion exchange or cation exchange chromatography, phosphocellulose chromatography, interaction chromatography. hydrophobic, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Very preferably, ion metal affinity chromatography (IMAC) is used for purification. Well-known techniques can be employed to re-fold the proteins in order to regenerate the active conformation when the polypeptide is denatured during intracellular synthesis, intracellular isolation and / or intracellular purification.

Another important aspect of the disclosure relates to a method for inducing, strengthening or modulating an immune response in a mammal that comprises inoculating the mammal with a fragment or all of the polypeptide or polynucleotide of the disclosure, suitable for producing immune response of antibody and / or T cell for immunoprophylaxis or for the therapeutic treatment of cancer, more particularly colorectal cancer, and autoimmune disease and related conditions. Still another aspect of the disclosure relates to a method for inducing, strengthening or modulating the immune response in a mammal that comprises, delivering a polypeptide of the present invention through a vector or cell that directs the expression of the polynucleotide and encoding for the polypeptide in vivo in order to induce such an immune response to produce immune responses for the prophylaxis or treatment of diseases of said mammal.

A further aspect of the invention relates to an immunological / vaccine formulation (composition) and its use in medicine. These compositions, when introduced into a mammalian host, induce, reinforce, or modulate an immune response in that mammal to a polypeptide of the present disclosure in which composition 25 comprises a polypeptide or polynucleotide of the disclosure or an immunological fragment thereof. as defined above in this document. More particularly, the immunogenic composition according to the present invention comprises a safe and effective amount of a CASB7439 polypeptide, or an immunogenic fragment thereof in which the CASB7439 polypeptide is selected from the group comprising SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14. In another embodiment, the immunogenic composition comprises a safe and effective amount of a polynucleotide encoding CASB7439, or fragment thereof in which the CASB7439 coding polynucleotide is selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 15.

The vaccine formulation according to the invention may further comprise a suitable vehicle, that is, pharmaceutically acceptable. Since a polypeptide can be segmented in the stomach, it is preferably administered parenterally (eg, subcutaneous, intramuscular, intravenous, or intradermal injection). Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injection solutions that may contain anti-oxidants, buffers, bacteriostats and solutes that produce the isotonic formulation with the recipient's blood; and sterile aqueous and non-aqueous suspensions that may include suspending agents or thickening agents. The formulations may be presented in single dose or multiple dose containers, for example, sealed ampoules and bottles and may be stored in a lyophilization condition requiring only the addition of the sterile liquid carrier immediately prior to use.

A further aspect of the invention relates to the in vitro induction of immune responses to a fragment or to the entire polypeptide or polynucleotide of the present disclosure or to a molecule comprising the polypeptide or polynucleotide of the present disclosure, using cells from the immune system of a mammal and reimplanting these 45 activated immune cells of the mammal for the treatment of the disease. Activation of cells from the immune system is achieved by incubation in vitro with all the polypeptide or polynucleotide of the present disclosure or with a molecule comprising the polypeptide or polynucleotide of the present disclosure in the presence or absence of various immunomodulatory molecules. A further aspect of the disclosure relates to the immunization of a mammal by the administration of antigen presenting cells modified by in vitro loading with part 50 of or with all the polypeptide of the present disclosure administered in vivo or with a molecule comprising the polypeptide of the present disclosure administered in vivo in an immunogenic manner. Alternatively, the antigen presenting cells can be transfected in vitro with a vector containing a fragment of or all the polynucleotide of the present disclosure or a molecule comprising the polynucleotide of the present disclosure, such as expressing the corresponding polypeptide, and administered in vivo. in an immunogenic way. In accordance with the foregoing, the pharmaceutical compositions of the disclosure will comprise an effective amount of antigen presenting cells, modified by in vitro loading with a CASB7439 polypeptide, or genetically modified in vitro to express a CASB7439 polypeptide and a pharmaceutically effective carrier.

According to another embodiment, the pharmaceutical / immunogenic compositions described herein will comprise one or more immunostimulants in addition to the immunogenic polynucleotide, polypeptide, antibody, T cell and / or antigen presenting cell (APC) compositions of this disclosure. In accordance with the foregoing, a process for the production of said immunogenic composition is provided herein, comprising mixing a CASB7439 polypeptide or a CASB7439 coding polynucleotide with a suitable adjuvant / immunostimulant, a suitable diluent or other carrier pharmaceutically acceptable. An immunostimulant essentially refers to any substance that enhances or potentiates an immune response (antibody mediated and / or cell mediated) to an exogenous antigen. A preferred type of immunostimulant comprises an adjuvant. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil and an immune response stimulator, such as proteins derived from lipid A, Bortadella pertussis or Mycobacterium tuberculosis. Certain adjuvants are commercially available such as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, MI); Adjuvant Merck 65 (Merck and Company Inc., Rahway, NJ); AS-2 (SmithKIine Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; calcium, iron or zinc salts; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derived polysaccharides; polyphosphazenes; biodegradable microspheres; Monophosphoryl and quil A lipid A. Cytokine adjuvants, such as GM-CSF, interleukin-2, -7, -12, and other similar growth factors, can also be used.

In some embodiments of the invention, the adjuvant composition is preferably one that induces a predominantly Th1 type immune response. Elevated levels of Th1-type cytokines (for example, IFN-γ, TNF IL-2 and 20 IL-12) tend to favor the induction of cell-mediated immune responses for a administered antigen. In contrast, elevated levels of Th2-type cytokines (for example, IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. After the application of a vaccine as provided herein, a patient will support an immune response that includes Th1 and Th2 type responses. In a preferred embodiment, in which a response is predominantly of the Th1 type, the level of the Th1 type cytokines will increase to a degree greater than the level of the Th2 type cytokines. The levels of these cytokines can be easily evaluated using standard tests. For a review of the cytokine families, see Mosmann and Coffman, Ann. Rev. Immunol. 7: 145-173, 1989.

Certain preferred adjuvants to produce a predominantly Th1 response include, for example, a combination of monophosphoryl A lipid, preferably 3-de-O-acylated monophosphoryl A lipid, together with an aluminum salt. MPL® adjuvants are available from Corixa Corporation (Seattle, WA; see, for example, U.S. Patents Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induces a predominantly Th1 type response. Such oligonucleotides are well known and are written, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273: 352, 1996. Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 ( Aquila Biopharmaceuticals Inc., Framingham, MA); Spine; Digitonin; or Gypsophila or Chenopodium quinoa saponins. Other preferred formulations include more than one saponin in the combinations of adjuvants of the present invention, for example, combinations of at least two of the following group comprising QS21, QS7, Quil A, β-escina, or digitonin. 40

Alternatively, the saponin formulations can be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide particles and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, polysaccharide or modified polysaccharide composite particles Chemically, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc. Saponins can also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOM. In addition, saponins can be formulated together with a polyoxyethylene ether or ester, either in a non-particulate solution or suspension, or in a particulate structure such as a paucilamellar liposome or ISCOM. Saponins can also be formulated with excipients such as Carbopol® to increase viscosity, or they can be formulated as a dry powder with a powdered excipient such as lactose.

In a preferred embodiment, the adjuvant system includes the combination of a monophosphoryl lipid A and a 50-saponin derivative, such as the combination of QS21 and 3D-MPL® adjuvant, as described in WO 94/00153, or a less reatrogenic composition where QS21 is deactivated with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. Another particularly preferred adjuvant formulation employing QS21, 3D-MPL® adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210. 55

Another improved adjuvant system involves the combination of an oligonucleotide containing CpG and a saponin derivative in particular the combination of CpG and QS21 as described in WO 00/09159. Preferably the formulation further comprises an oil in water emulsion and tocopherol.

Additional illustrative adjuvants for use in the pharmaceutical compositions of the invention include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the adjuvant series SBAS (for example, SBAS-2 or SBAS-4, available from SmithKIine Beecham, Rixensart, Belgium), Detox (Enhanzyn®) (Corixa, Hamilton, MT). RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphates (AGP), such as those described in U.S. Patent Applications Serial No. 08 / 853,826 and 5 09 / 074,720 in process, the disclosures of which are incorporated herein by reference in their entirety, and polyoxyethylene ether adjuvants such as those described in WO 99 / 52549A1.

Other preferred adjuvants include adjuvant molecules of the general formula (I):

 HO (CH2CH2O) n-A-R

wherein, n is 1-50, A is a bond or -C (O) -, R is C1-50 alkyl (1 to 50 carbon atoms) or phenyl with a molecule of 1 to 10 50 carbon atoms.

An embodiment of the present invention consists of a vaccine formulation comprising a polyoxyethylene ether of the general formula (I), in which n is between 1 and 50, preferably 4-24, most preferably 9; The R component is alkyl with a molecule of 1 to 50 carbon atoms, preferably alkyl with a molecule of 4 to 20 carbon atoms and most preferably alkyl with a molecule of 12 carbon atoms and A is a bond. The concentration of the polyoxyethylene ethers would be in the range of 0.1-20%, preferably from 0.1-10% and most preferably in the range of 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35 ether -lauryl and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12th edition: entry 7717). These adjuvant molecules are described in WO 99/52549.

The polyoxyethylene ether according to the general formula (I) set forth above may, if desired, be combined with another adjuvant. For example, a preferred adjuvant combination is preferably with CpG as described in the UK patent application GB 9820956.2 pending.

Preferably a vehicle is also present in the vaccine composition according to the disclosure. The vehicle can be an oil-in-water emulsion, or an aluminum salt, such as aluminum phosphate or aluminum hydroxide.

A preferred oil-in-water emulsion comprises a metabolizable oil, such as squalene, alpha-tocopherol and Tween 80. In a particularly preferred aspect, the antigens in the vaccine composition according to the invention are combined with QS21 and 3D-MPL in such an emulsion. In addition, the oil-in-water emulsion may contain span 85 and / or lecithin and / or tricapriIine.

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

Non-toxic emulsions of oil in water preferably contain a non-toxic oil, for example, squalene or squalene, an emulsifier, for example, Tween 80, in an aqueous vehicle. The aqueous vehicle can be, for example, phosphate regulated saline. 40

A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

The present disclosure also provides a polyvalent vaccine composition comprising a formulation of the revelation vaccine in combination with other antigens, in particular antigens useful for treating cancers, more particularly colorectal cancer, autoimmune diseases and related conditions. Such a polyvalent vaccine composition may include a TH-1 induction adjuvant as described hereinbefore.

In accordance with another embodiment of this invention, an immunogenic composition described herein is supplied to a host through antigen presentation cells (APC), such as dendritic cells, macrophages, B cells, monocytes and other cells that can Designed to be effective APCs. Such cells may, but do not require, be genetically modified to increase the ability to present the antigen, improve activation and / or maintenance of the T-cell response, have antitumor effects per se and / or be immunologically compatible with the receptor (it is say, HLA halotype coupled). APCs can be isolated

generally of any variety of biological fluids and organs, including tumor and peritumoral tissues, and can be autologous, allogenic, syngeneic or xenogeneic.

Certain preferred embodiments of the present disclosure use dendritic or progenitor cells thereof as antigen presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392: 245-251, 1998) and have been shown to be effective as a physiological adjuvant for producing prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med 50: 507-529, 1999), In general, dendritic cells can be identified based on their typical shape (crashed in situ, with marked cytoplasmic processes (dendrites) 5 visible in vitro), their ability to capture, process and present antigens with high efficiency and their ability to activate simple T cell responses. Dendritic cells can, of course, be designed to express specific cell surface receptors or ligands that are not commonly found in dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, dendritic cells loaded with secreted vesicle antigen (called exosomes) can be used in a vaccine (see Zitvogel et al., Nature Med. 4: 594-600, 1998).

Dendritic cells and progenitors can be obtained from peripheral blood, bone marrow, tumor infiltration cells, infiltration cells of peritumoral tissues, lymph nodes, spleen, skin, skin umbilical cord blood, or some other suitable tissue or fluid. For example, dendritic cells can be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and / or TNFα to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood, or bone marrow can be differentiated into dendritic cells by adding combinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, to the culture medium. ligand of flt3 and / or other compound (s) that induce differentiation, maturation and proliferation of dendritic cells.

Dendritic cells are conveniently categorized as "immature" and "mature" cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be interpreted to exclude all possible stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with high expression of the Fcγ receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but by a high expression of cell surface molecules responsible for the activation of T cells such as MHC class I and class II, adhesion molecules (eg, CD54 and CD11) and costimulatory molecules (for example, CD40, CD80, CD86 and 4-1 BB).

APCs can generally be transfected with a polynucleotide of the invention (or portion or other variant thereof) in such a way that the encoded polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection can take place ex vivo and a pharmaceutical composition comprising such transfected cells can then be used for therapeutic purposes, as described herein. Alternatively, a genetic delivery vehicle that targets a dendritic cell or other antigen presenting cell can be administered to a patient, resulting in the transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, can generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al. ., Immunology and cell Biology 75: 456-460, 1997. The antigen load of dendritic cells can be achieved by incubating dendritic cells or progenitor cells with the tumor polypeptide, DNA (pure or in a plasmid vector) or RNA; or with recombinant antigen-expressing bacteria or viruses (eg, vectors of the vaccine virus, of the smallpox, adenovirus or lentivirus). Before loading, the polypeptide can be covalently conjugated to an immune partner that provides T-cell support (for example, a carrier molecule). Alternatively, a dendritic cell with an unconjugated immune partner can be subjected to a pulse, separately or in the presence of the polypeptide.

While any suitable vehicle known to those skilled in the art may be employed in the pharmaceutical compositions of this disclosure, the type of vehicle will typically vary depending on the mode of administration. The compositions of the present invention may be formulated for any appropriate administration, including, for example, topical, oral, nasal, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous and intramuscular administration.

The vehicles for use within such pharmaceutical compositions are biocompatible and can also be biodegradable. In certain embodiments, the formulation preferably provides a relatively constant level of release of active components. However, in other embodiments a faster release rate may be desired immediately after administration. The formulation of such compositions is within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include microparticles of poly (lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative delayed release vehicles include supramolecular biovectors, which comprise a non-liquid hydrophilic core (eg, a degraded polysaccharide or oligasaccharide) and optionally, an outer layer comprising an amphiphilic compound, such as a phospholipid (see, for example , U.S. Patent No.

5,151,254 and PCT applications WO 94/20078, WO / 94/23701 and WO 96/06638). The amount of active compound contained in a sustained release formulation depends on the site of implantation, the rate and expected duration of the release and the nature of the condition to be treated or avoided.

In another illustrative embodiment, biodegradable microspheres (eg, polylactate polyglycolate) are used as vehicles for the compositions of this disclosure. Suitable biodegradable microspheres are described, for example, in U.S. Pat. No. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344; 5,407,609 and 5,942,252. Modified hepatitis B core protein vehicle systems, such as those described in WO99 / 40934, and references cited therein, will also be useful for many applications. Another illustrative delivery / carrier system employs a vehicle comprising particulate-protein complexes, such as those described in U.S. Pat. No. 5,928,647, which are capable of inducing class I restricted cytotoxic T lymphocyte responses in a host. The pharmaceutical compositions of the invention will often additionally comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostatics, chelating agents such as EDTA or glutathione, adjuvants (for example, aluminum hydroxide), solutes that provide the isotonic, hypotonic or weakly hypertonic formulation with the blood of a receptor, suspending agents, thickening agents and / or preservatives. Alternatively, the compositions of the present invention can be formulated as a lyophilisate.

The pharmaceutical compositions described herein may be presented in unit dose or multiple dose containers, such as sealed ampoules or bottles. Such containers are typically sealed in such a way that they preserve the sterility and stability of the formulation until use. In general, the formulations can be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a pharmaceutical composition can be stored in a condition of lyophilization that only requires the addition of a sterile liquid carrier immediately before use.

The development of suitable dosage and treatment regimens to use the particular compositions described herein in a variety of treatment regimens, including, for example, oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation, is well known in the art, some of which are briefly described below for general purposes of illustration.

In some applications, the pharmaceutical compositions described herein may be delivered through oral administration to an animal. As such, these compositions can be formulated with an inert diluent or with an assimilable edible carrier, or they can be included in a hard or soft layer gelatin capsule, or they can be compressed into tablets, or they can be incorporated directly into the diet food.

The active compounds can even be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (see, for example, Mathiowitz et al., Nature 1997 Mar 27; 386 (6623); 410-4; Hwang et al., Crit Rev Ther Drug Carrier Syst. 1998; 1 35 5 (3): 243-84; US Patent 5,641,515; US Patent 5,580,579 and US Patent 5,792,451 ). The tablets, troches, pills, capsules and the like may also contain any of a variety of additional components, for example, a binder, such as gum tragacanth, gum arabic, corn starch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin or a seasoning agent, such as peppermint, gaulteria oil, or cherry flavoring can be added. When the unit dosage form is a capsule, it may contain, in addition to the materials of the above type, a liquid vehicle. Various other materials may be present as coatings or to otherwise modify the physical form of the unit dosage. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, any material used to prepare any unit dosage form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds can be incorporated into the preparation of mamtenide release and formulations.

Typically, these formulations will contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient (s) can, of course, be varied and may conveniently be between about 1 or 1. 2% and about 60% or 70% or more of the weight or volume of the total formulation. Naturally, the amount of the active compound (s) in each therapeutically useful composition can be prepared such that an adequate dose will be obtained at any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product's own life, as well as other pharmacological considerations will be contemplated by those skilled in the art for preparing such pharmaceutical formulations, and as such, a diversity may be desirable. of doses and treatment regimens.

For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, oral tablet, oral atomizer, or orally administered sublingual formulation. Alternatively, the active ingredient may be incorporated into an oral solution such as that containing sodium borate, glycerin and potassium bicarbonate, or it may be dispersed in a dentifrice, or added in a therapeutically effective amount to a composition that may include water. , binders, abrasives, seasoning agents, foaming agents, and humectants. Alternatively, the compositions may be arranged as a tablet or solution that can be placed under the tongue or otherwise dissolved in the mouth.

In some circumstances it will be desirable to deliver the pharmaceutical compositions described herein parenterally, intravenously, intramuscularly, or even intraperitoneally. Such approaches are well known to the skilled worker, some of which are further described, for example, in U.S. Pat. No. 5,543.1 58; in U.S. Pat. No. 5,641,515 and in U.S. Pat. No. 5,399,363. In some embodiments, solutions of the active compounds as a free base or as pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these 15 preparations will generally contain a preservative to prevent the growth of microorganisms.

Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Patent No. 5,466,468). In all cases the form must be sterile and must be fluid to the extent that there is an easy environment for the syringe. It must be stable under the conditions of processing and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Appropriate fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. The prevention of the action of microorganisms may be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, or sodium chloride. Prolonged absorption of the injectable compositions can be caused by the use in the compositions of agents that delay absorption, for example, aluminum monostearate and gelatin. 30

In one embodiment, for parenteral administration in an aqueous solution, the solution should be properly regulated if necessary and first the liquid diluent become isotonic with sufficient saline or glucose solution. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be used will be known to those skilled in the art in light of the present description. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution 35 and added to 1000 ml of hypodermoclysis fluid or injected at the proposed infusion site, (see, for example, "Remington's Pharmaceutical Sciences", 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the patient to be treated. In addition, for human administration, the preparations will of course preferably comply with sterility, pyrogenicity, and general safety and purity standards as required by the FDA Office of Biological Standards. 40

In another embodiment of the invention, the compositions described herein can be formulated in a salt or neutral form. Illustrative pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and those formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric acids. , Mandelic and the like. The salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonia, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. After formulation, the solutions will be administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective.

The vehicles may further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and delayed absorption agents, 50 buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for active pharmaceutical substances is well known in the art. Except to the extent that as any conventional means or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. The complementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce a similar allergic or adverse reaction when administered to a human being.

In some embodiments, the pharmaceutical compositions may be supplied by intranasal sprayers, inhalation and / or other aerosol delivery vehicles. The procedures for supplying genes, nucleic acids and

Peptide compositions directly to the lungs through nasal spray atomizers have been described, for example, in U.S. Pat. No. 5,756,353 and in U.S. Pat. No. 5,804,212. Similarly, the supply of drugs using intranasal microparticulate resins (Takenaga et al., J Controlled Release March 2, 1998; 52 (1-2): 81-7) and lysophosphatidyl glycerol compounds (US Pat. .UN 5,725,871) are also well known in pharmaceutical techniques. Similarly, the delivery of illustrative transmucosal drug 5 in the form of a polytetrafluoroethylene support matrix is described in US Pat. No. 5,780,045.

In some embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles, and the like are used for the introduction of the compositions of the present invention into suitable host cells / organisms. In particular, the compositions of the present disclosure can be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. Alternatively, the compositions of the present disclosure may be covalently or non-covalently bound to the surface of such carrier vehicles.

The formation and use of liposome preparations and liposome-like preparations as potential drug vehicles is generally known to those skilled in the art (see, for example, Lasic, Trends Biotechnol 1998 Jul; 16 (7): 307-21; Takakura , Nippon Rinsho March 1998; 56 (3): 691-5; Chandran et al., Indian J Exp 15 Biol. August 1997; 35 (8): 801-9; Margalit, Crit Rev Ther Drug Carrier Syst .. 1995; 12 (2- 3): 2 33-61; U.S. Patent 5,567,434; U.S. Patent 5,552,157; U.S. Patent 5,565,213; U.S. Patent 5,738,868; and U.S. Patent 5,795,587).

Liposomes have been used successfully with a number of cell types that are normally difficult to transfect by other procedures, including T cell suspensions, primary hepatocyte cultures and 20 PC 12 cells (Renneisen et al., J Biol Chem. 25 de September 1990; 265 (27): 16337-42; Muller et al., DNA Cell Biol. April 1990; 9 (3): 221-9). In addition, liposomes are free of DNA stretch restrictions that are typical of viral delivery systems. Liposomes have been used effectively to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors and the like, into a variety of cultured cell lines and animals. In addition, the use of liposomes does not appear to be associated with autoimmune responses or with unacceptable toxicity after systemic delivery.

In some embodiments, liposomes are formed from phospholipids that are distributed in an aqueous medium and that spontaneously form multilamellar concentric bilayer vesicles (also defined as multilamellar vesicles (MLV)).

Alternatively, in other embodiments, the disclosure is provided for pharmaceutically acceptable nanocapsule formulations of the compositions of the present disclosure. The nanocapsules can generally trap compounds in a stable and reproducible manner (see, for example, Quintanar-Guerrero, et al., Drug Dev Ind Pharm. December 1998; 24 (12): 1113-28). To avoid side effects due to intracellular polymeric overload, such ultrafine particles (about 0.1 µm in size) can be designed using polymers capable of degrading in vivo; such particles can be made as described, for example, by Couvreur et al., Crit Rev 35 Ther Drug Carrier Syst. 1988; 5 (1) 1-20; zur Muhlen et al., Eur J Pharm Biopharm., March 1998; 45 (2): 149-55; Zambaux et al., J Controlled Release, January 2, 1998; 50 (1 -3): 31 -40; and U.S. Pat. No. 5,145,684.

This disclosure also relates to the use of polynucleotides, in the form of primers derived from the polynucleotides of the present invention and of polypeptides, in the form of antibodies or specific reactive agents for the polypeptide of the present disclosure, as diagnostic reactive agents. 40

The identification of genetic or biochemical markers in the blood or tissues that will allow the detection of very early changes in the pathogenesis of carcinogenesis will help determine the best treatment for the patient. Substitute tumor markers, such as polynucleotide expression, can be used to diagnose different forms and states of cancer. The identification of expression levels of the polynucleotides of the disclosure will be useful both in defining stages of the cancerous disorder and in assessing the nature of the cancerous tissue. The stage definition process tracks the progress of cancer and is determined in the presence or absence of malignant tissue in the areas undergoing biopsy. The polynucleotides of the disclosure can help refine the process of defining stages by identifying markers for the aggressiveness of a cancer, for example, the presence in different areas of the body. The cancer evaluation describes how closely a tumor resembles normal tissue of the same type and is evaluated by its cell morphology and other differentiation markers. The 50 polynucleotides of the disclosure can be useful in determining the grade of the tumor since they help in determining the different states of the cells of a tumor.

Decreasing or increasing expression can be measured at the level of RNA. The polyA RNA is first isolated from the two tissues and the detection of mRNA encoded by a gene corresponding to a differentially expressed polynucleotide of the invention can be detected, for example, by in situ hybridization in tissue sections, by reverse transcriptase PCR, using Northern band tests containing poly A + mRNA, or any other direct or indirect RNA detection procedure. An increasing or decreasing expression of a given RNA

in a diseased tissue compared to a normal tissue suggests that transcription and / or the expressed protein has a role in the disease. Consequently, the detection of a higher or lower level of mRNA corresponding to SEQ ID NO: 1 in relation to the normal level is indicative of the presence of cancer in the patient.

The mRNA expression levels in a sample can be determined by the generation of a library of expressed sequence tags (ESTs) from the sample. The relative representation of EST in library 5 can be used to evaluate the relative representation of genetic transcription in the initial sample. The EST analysis of the test can then be compared with the EST analysis of a reference sample to determine the relative expression levels of the polynucleotide of interest.

Other mRNA analyzes can be carried out using serial analysis of the genetic expression methodology (SAGE) (Velculescu et al., Science (1995) 270: 484), differential display methodology (e.g., US 10 5,776,683) or hybridization analysis that depends on the specificity of nucleotide interactions.

Alternatively, the comparison could be made at the protein level. The protein sizes in the two tissues can be compared using antibodies to detect polypeptides in Western band analyzes of the protein extracts of the two tissues. The levels of expression and subcellular localization can also be detected immunologically using the antibodies for the corresponding protein. Additional test techniques that can be used to determine the levels of a protein, such as a polypeptide of the present disclosure, in a sample derived from a host are well known to those skilled in the art. An increasing or decreasing level of polypeptide expression in diseased tissue compared to the same level of protein expression in normal tissue indicates that the expressed protein may be involved in the disease.

The "diagnosis" as used herein includes the determination of the susceptibility of a subject to a disease, determination of whether a subject currently has the disease and also the prognosis of a patient affected by the disease.

The polypeptides of the disclosure or their fragments or analogs thereof, or cells that express them, can also be used as immunogens to produce immunospecific antibodies to polypeptides of the present invention. The term "immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other polypeptides related to the prior art.

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

Antibodies generated against the polypeptides of the present disclosure can be obtained by administering the epitope support polypeptides or fragments, analogs or cells to an animal, preferably an animal that is not a human being, using routine protocols. For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C., Nature (1975) 256: 495-497), the trioma technique, the human B cell hybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) and the hybridoma-EBV technique (Colé and 35 col., Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies, such as those described in US Pat. No. 4,946,778, can also be adapted to produce single chain antibodies to polypeptides of this disclosure. Also, transgenic mice, or other organisms, which include other mammals can be used to express humanized antibodies. 40

The antibodies described above can be used to isolate or identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.

The disclosure antibody can also be used to prevent or treat cancer, particularly colorectal cancer, autoimmune disease and related conditions.

Another aspect of the disclosure relates to a method for inducing or modulating an immune response in a mammal comprising inoculating the mammal with a polypeptide of the present disclosure, suitable for producing immune response of antibodies and / or T cells in order to protect or improve symptoms or disease progression. Yet another aspect of the disclosure relates to a method for inducing or modulating the immune response in a mammal which comprises delivering a polypeptide of the present disclosure by means of a vector that directs the expression of the polynucleotide and encodes the polypeptide in vivo. in order to induce such an immune response to produce antibodies in order to protect said animal from disease.

It will be appreciated that the present disclosure therefore provides a method for treating abnormal conditions such as, for example, cancer and autoimmune diseases, in particular, colorectal cancer, related to either

a presence of either with an excess of, or with a sub-expression of, polypeptide activity of CASB7439. Other abnormal conditions related to the expression of CASB7439 that the invention seeks to treat are chronic lymphocytic leukemia and germ cell tumors.

Genetic therapy can also be used to effect endogenous production of the CASB7439 polypeptide by the relevant cells in the subject. For a general review of gene therapy, see Chapter 20, Genetic Therapy 5 and other Molecular Genetics-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and AP Read, BIOS Scientific Publishers Ltd ( nineteen ninety six).

Vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol. 61 Vaccine Design - the subunit and adyuvant 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 10 liposomes is described, for example, by Fullerton, U.S. Pat. No. 4,235,877. The conjugation of proteins to macromolecules is revealed, for example, by Likhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No. 4,474,757.

The amount of protein in each vaccine dose is selected as an amount that induces an immunoprotective response without significant adverse side effects in typical vaccines. Such amount will vary depending on which specific immunogen is used. Generally, it is expected that each dose will comprise 1-1000 μg of protein, preferably 2-100 μg, most preferably 4-40 μg. An optimal amount for a particular vaccine can be determined by standard studies that involve observation of antibody titres and other responses in subjects. After an initial vaccination, subjects can receive a booster in approximately 4 weeks. twenty

"Isolated" means altered "by the hand of man" from its natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a live animal is not "isolated", but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated," as the term is used herein. document. 25

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

"Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but that maintains its essential properties. A typical variant of a polynucleotide differs from one nucleotide sequence to another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, condensations, and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs from one amino acid sequence to another, reference polypeptide. Generally, the differences are limited so that the sequences of the reference polypeptide and the variant are closely similar in general and, in many regions, identical. A variant and a reference polypeptide may differ in the amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be one that occurs in nature such as an allelic variant, or it may be a variant that is not known if it occurs in nature. Variants of polynucleotides and polypeptides that do not occur in nature can be made by mutagenesis techniques or by direct synthesis.

"Identity" as is known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence affinity between the polypeptide or polynucleotide sequences, as may be the case, as determined by the coupling between chains of such sequences. "Identity" and "similarity" can easily be calculated by known procedures, including but not limited to those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Deveraux, J., eds., M Stockton Press, New York, 50 1991; and Carillo, H., and Lipman D., SIAM J. Applied Math., 48: 1073 (1988). Preferred procedures for determining identity are designed to give the greatest match between the sequences tested. The procedures for determining identity and similarity are encoded in publicly available computer programs. Preferred computer program procedures for determining the identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J et al., Nucleic Acids Research 55 12 (1): 387 (1984) ), BLASTP, BLASTN, and FASTA (Atschul, SF et al., J. Molec. Biol. 215: 403-410 (1990)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM

NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol., 275: 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 comparing the sequence of polypeptides or polynucleotides using this algorithm include the following:

 Hollow Penalty: 12 5

 Hueco extension penalty: 4

 Word size: 2, max. 6

Preferred parameters for polypeptide sequence comparison with other procedures include the following:

 1) Algorithm: Needleman and Wunsch, J. Mol Biol .. 48: 443-453 (1970) 10

 Comparison matrix: BLOSSUM62 by Hentikoff and Hentikoff, Proc. Natl Acad. Sci. USA. 89: 10915-10919 (1992)

 Hollow Penalty: 12

 Gap Length Penalty: 4

A useful program with these parameters is publicly available as the "hollow" program of Genetics 15 Computer Group, Madison Wl, The above parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) .

Preferred parameters for polynucleotide comparison include the following:

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

 Comparison matrix: matches = +10, no matches = 0 20

 Hollow Penalty: 50

 Gap Length Penalty: 3

A useful program with these parameters is publicly available as the "hollow" program of Genetics Computer Group, Madison Wl. The parameters mentioned above are the default parameters for polynucleotide comparisons. 25

By way of example, a polynucleotide sequence of the present disclosure may be identical to the reference sequence of SEQ ID NO: 1, that is, 100% identical, or may include up to a certain integer number of alterations. of nucleotides compared to the reference sequence. Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and in which said alterations can occur at the 5 'or 3' terminal positions of the sequence of 30 reference nucleotides or anywhere between those terminal positions, interdistributed individually between the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the numerical percentage of the respective percentage identity (divided by 100) and subtracting that product from said total number of nucleotides in the SEQ ID NO. 1, or: 35

nn ≤ xn - (xn • y),

where nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO: 1 e and is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, etc., and in which any non-integer product of xn and y is rounded down to the nearest integer before subtracting it from xn. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 can create nonsense mutations, with an erroneous sense or with an erroneous reading, in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.

Similarly, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: 2, that is, it may be 100% identical, or it may include up to a certain integer number of amino acid alterations. compared to the reference sequence in such a way that the% identity is less than 100%. Such alterations are selected from the group consisting of at least one amino acid elimination, substitution, including substitution, or insertion, conservative and non-conservative, and in which said

alterations in the amino or carboxy terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interdistributed either individually between the amino acids in the reference sequence or in one or more contiguous groups in the reference sequence . The number of amino acid alterations for a given identity% is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the numerical percentage of the respective identity percentage (divided by 100) and then subtracting that product from said Total number of amino acids in SEQ ID NO: 2, or:

na ≤ xa - (xa • y),

where na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID NO: 2, and and is, for example, 0.70 for 70%, 0.80 for 80% , 0.85 for 85%, etc., and in which any non-integer product of xa ey is rounded down to the nearest integer before subtracting it from xa. 10

"Homologous" is a generic term used in the art to indicate a polynucleotide or polypeptide sequence that has a high degree of sequence relationship with a subject sequence. Such a relationship can be quantified by determining the degree of identity and / or similarity between the sequences that are compared as described above. Within this generic term are the terms "ortholog", which means a polynucleotide or polypeptide that is the functional equivalent of a polynucleotide or polypeptide in another species and "paralogue" which means a functionally similar sequence when considered within it species.

LEGENDS OF THE FIGURES

Figure 1: shows real-time PCR data using the Taqman probe. The legend is as explained below: Adrenal gland: Ad_GI; Bladder: Bl; Bone Marrow: Bo_Ma, Cervix: Ce; Colon: Co; Fallopian tube: Fa_Tu; Ileon: II; Liver: Li; Lung: Lu; Lymph Node: Ly_No; Esophagus: Oe, Parathyroid gland: Pa_Thy; 20 Placenta: Pl; Prostate: Pr, Straight: Re; Skin: Sk; Skeletal muscle; Sk_Mu; Small intestine: Sm_ln; Spleen; Sp; Testicle: Te; Thyroid gland: Thy; Trachea: Tr.

Figure 2 shows real-time PCR expression using the Sybr protocol. The legend is as follows: Adrenal gland: Ad_GI; Bladder: Bl; Bone Marrow: Bo_Ma; Cervix: Ce; Lymph Node: Ly_No; Esophagus: Oe, Parathyroid gland: Pa_Thy; Placenta: Pl; Prostate; Pr, Straight: Re; Skin: Sk; Skeletal muscle: Sk_Mu; Small intestine 25: Sm_ln; Spleen: Sp; Testicle: Te; Thyroid gland: Thy; Trachea: Tr, Heart: He.

Figure 3 shows SDS PAGE stained with Coomassie blue from the cell extract of the strain expressing CASB7439. Track 1 shows molecular markers, track 2 induced cell extract 5 h at 39 ° C; lane 3 shows the supernatant of induced cell extract; and lane 4 shows the sediment of induced cell extract.

Figure 4 shows an analysis of Western bands of the expressed NS1-CASB7439 protein. The gel is loaded with the cell extract of the strain expressing CASB7439 and developed with anti-NS1 monoclonal antibody.

Figure 5 shows Coomassie blue stained SDS-PAGE of CASB7439 after purification.

Lanes 1 and 5 represent the molecular weight markers; lanes 2, 3, 4 are loaded respectively with 2 μl, 4 μl, and 6 μl of purified protein.

Figure 6 shows a Western band analysis of CASB7439 after purification as revealed by an anti-polyhistidine monoclonal antibody.

Examples

Example 1

RT-PCR analysis in real time

Real-time RT-PCR (U, Gibson. 1996 Genome Research: 6,996) is used to compare the abundance of 40 mRNA transcription of the candidate antigen in normal and matching tumor tissues from multiple patients. In addition, the mRNA levels of the candidate gene in a panel of normal tissues are also evaluated by this approach.

Total RNA derived from the normal and tumor colon is extracted from frozen biopsies using the TriPure reagent (Boehringer). Total RNA derived from normal tissues is acquired from InVitrogen or extracted from frozen biopsies using the TriPure reagent. Poly-A + mRNA is purified from the total mRNA after DNase treatment using oligo-dT (Dynal) magnetic beads. Quantification of the mRNA is carried out by spectrofluorimetry (VersaFluor, BioRad) using Sybrll staining {Molecular Probes). Primers for real-time PCR amplification are designed with Perkin-Elmer Primer Express software using the default options for Taqman amplification conditions. fifty

Real-time reactions are assembled according to conventional PCR protocols using 2 ng of purified mRNA for each reaction. Sybrl (Molecular Probes) staining is added in a final dilution of 1/75000 for real-time detection. Amplification (40 cycles) and real-time detection is carried out in a Perkin-Elmer Byosystems PE7700 system using conventional instrument configurations. Ct values are calculated using the PE7700 Sequence Detector software. Several Ct values are obtained for each sample: 5 for the patient samples, the Ct tumor (CtT) and the values of normal colon coupled Ct (CtN) in the candidate TAA and for the normal tissue sample panel, a CtXY for each normal tissue XY. Another Ct (CtA) in the Actin gene, as an internal reference, is also calculated for all samples. Alternatively, real-time PCR amplification can be monitored using a Taqman probe. Amplification (40 cycles) and real-time detection are carried out in a Perkin-Elmer PE7700 Biosystems system using conventional instrument configurations. Ct values are calculated using the PE7700 Sequence Detector Software. Ct values are obtained from each tissue sample for the target mRNA (CtX) and for the actin mRNA (CtA).

As the efficacy of the PCR amplification in the prevailing experimental conditions is close to the theoretical amplification efficiency, the value 2 (CtN / T / XY-CtA) is a calculation of the relative TAA transcription level of the sample , standardized with respect to the level of transcription of Actin. A value of 1 suggests in consequence that the candidate antigen and Actin have the same level of expression.

Real-time PCR reactions were performed primarily in tumor colon and by coupling the normal colon derived from biopsies of 12 patients. The reactions were carried out on a more complete data set totaling 18 patients (the first 12 patients are included in this data set). Duplicates for 6 of these 18 patients were performed in this data set. Six additional patients were tested, and the results were saved with the previous 18. The statistics in the final saved data set are shown in Table 3, and illustrated in Figure 1.

A series of 48 normal tissue samples, representing 29 different tissues, were also tested by the same procedure (normal tissues analyzed are given in Table 3). TAA transcription levels are calculated as described above. The proportion of patients who overexpress the candidate antigen, as well as the average transcription overexpression against normal tissues is also calculated from this data set. The results are illustrated in Figure 1.

Table 1; Results of real-time PCR expression CASB7439: data set of 12 patients.

 % of patients with a higher level of mRNA in colon, coupled tumor

 92%

 (positive patients)

 % of patients with a mRNA level at least 3 times higher in

 92%

 coupled tumor colon

 % of patients with a mRNA level at least 10 times higher

 92%

 in coupled tumor colon

 % of patients with an mRNA level at least 3 times lower in

 8%

 in coupled tumor colon.

 Average level of normal colon mRNA coupled (Standardized Actin)

 0.0026

 Average mRNA level of coupled tumor colon in positive patients (Actin

 0.265

 standardized)

 Average mRNA overexpression times

 2028

 Median mRNA overexpansion times

 115

 Average mRNA level of normal tissues

 0.0079

 Medium level of normal tissue mRNA

 0.0016

 Average mRNA level of normal tissues

 0.0064

 Medium level of normal tissue mRNA

 0.0017

 % of patients with a mRNA level higher than average normal tissues

 92%

 % of patients with an mRNA level greater than 10 times the average of normal tissues

 75%

 Normal non-dispensable tissues higher than the average mRNA level of normal tissue

 None

Table 2: CASB743 real-time PCR expression results: data set of 18 patients

 % of patients with a higher mRNA level in coupled tumor colon (positive patients)

 89%

 % of patients with an mRNA level at least 3 times higher in coupled tumor colon

 89%

 % of patients with an mRNA level at least 10 times higher in coupled tumor colon

 78%

 % of patients with an mRNA level at least 3 times lower in coupled tumor colon.

 5%

 Average level of normal colon mRNA coupled (Standardized Actin)

 0.005

 Average level of coupled tumor mRNA in positive patients (Standardized Actin)

 0.152

 Average mRNA overexpression times

 1100

 Median times of mRNA overexpression

 60

 Average mRNA level of normal tissues

 0.0065

 Medium level of normal tissue mRNA

 0.0015

 Average mRNA level of normal tissues

 0.005

 Medium level of normal tissue mRNA

 0.0015

 % of patients with a mRNA level higher than average normal tissues

 94%

 % of patients with an mRNA level greater than 10 times the average of normal tissues

 94%

 Normal non-dispensable tissues higher than the average mRNA level of normal tissue

 None

Table 3: CASB743 real-time PCR expression results: data set of 24 patients

 % of patients with a higher CASB7439 transcript level in the tumor colon than the adjacent normal colon (positive patients)

 92%

 % of positive patients with a CASB7439 transcription level at least 10 times higher in the tumor colon than the adjacent normal colon

 75%

 Average times of transcription overexpression in tumors of positive patients

 1289

 % of patients with a transcription level of

 96%

 CASB7439 higher in the tumor colon than the average normal tissue

 % of patients with an mRNA level at least 10 times higher in the tumor colon than the average normal tissue

 62.5%

 Normal tissues where CASB7439 transcription expression is equivalent to the level of tumor transcription in tumors

 None

Real-time PCR reactions were also carried out using the Taqman protocol (as described above) in tumor colon and adjacent normal colon from biopsies of 6 patients. Three duplicate measurements were taken for each one, and the average was used for subsequent calculations. The results are shown in Figure 1. In addition, 36 samples of normal tissue were also tested, representing 28 different tissues (see table 5) by the same procedure. The results are shown in Figure 2.

Table 4: CASB7439 real-time PCR expression results using the Taqman probe

 Number of tumor samples from different patients

 6

 % of positive patients with a higher CASB7439 transcript level in the tumor colon than the adjacent normal colon (positive patients)

 100%

 % of positive patients with a CASB7439 transcription level at least 10 times higher in the tumor colon than the adjacent normal colon

 83%

 Average times of transcription overexpression in tumors of positive patients

 109

 % of patients with a higher CASB7439 transcript level in the tumor colon than the average normal tissue

 100%

 % of patients with an mRNA level at least 10 times higher in the tumor colon than the average normal tissue

 100%

 Normal tissues where CASB7439 transcription expression is equivalent to the level of tumor transcription in tumors

 None

The results clearly suggest that the transcription of CASB7439 is overexpressed in colorectal tumors compared to the adjacent normal colon and with all the normal tissues mentioned above. More than 90% 10 of patients strongly overexpress the transcription of CASB7439 in the tumor, compared to the normal adjacent colon. The average number of times of overexpression in tumors is at least 100. In addition, more than 90% of patients overexpress the transcription of CASB7439 in colorectal tumors compared to other normal tissues, more than 60% of them over- Expressing it at least 10 times.

Table 5: List of normal tissues used for CASB7439 transcription expression analysis 15

 Tissue

 Abbreviation

 Adrenal gland

 Ad_Gl

 Aorta

 Ao

 Bladder

 Bl

 Bone marrow

 Bo_Ma

 Brain

 Bra

 Cervix

 EC

 Colon

 Co

 Fallopian tube

 Fa_Tu

 Heart

 I have

 Ileum

 Il

 Kidney

 Ki

 Liver

 Li

 Lung

 Lu

 Lymph node

 Ly_No

 Esophagus

 Oe

 Parathyroid gland

 Pa_Thy

 Straight

 Re

 Skin

 Sk

 Skeletal muscle

 Sk_mu

 Small intestine

 Sm_il

 Spleen

 Sp

 Stomach

 St

 Thyroid gland

 Thyt

 Windpipe

 Tra

 Ovary

 Ov

 Placenta

 Pl

 Prostate

 Pr

 Testicle

 Tea

Example 2

Differential tracking of cDNA provisions.

The identification of genes associated with tumors in the subtracted cDNA library is performed by differential tracking.

The total bacterial DNA is extracted from 100 μl in cultures that are allowed to stand overnight. The bacteria are lysed with guanidium isothiocyanate and the bacterial DNA is purified by affinity using a magnetic vessel (Boehringer). Plasmid insertions are recovered from bacterial DNA by Advantage PCR amplification (Clontech). The PCR products are transferred to two nylon membranes to produce high-density cDNA arrays using the Biomek 96 HDRT (Beekman) tool. The transferred cDNA is covalently linked to the membrane by UV irradiation. The first membrane is hybridized with a mixed cDNA probe prepared from the tumor of a single patient. The second membrane is hybridized with an equivalent amount of mixed cDNA probe prepared from normal colon of the same patient. The cDNA probe is prepared by PCR amplification as described above and labeled using the AlkPhos Direct System (Amersham). Hybridization conditions and washings under restrictive conditions are as described in the AlkPhos Direct kit. The hybridized probe is detected by chemiluminescence. The hybridization intensities for each cDNA fragment or for both bands are measured by film densitometry or direct measurement (BioRad Fluor-S Max). The proportion of tumor intensities to normal hybridization (T / N) is calculated for each gene in order to assess the degree of overexpression in the tumor. The genes that are significantly overexpressed in colon tumors are followed. Significance is arbitrarily defined as a standard deviation of the T / N frequency distribution. Differential screening experiments are repeated using RNA from multiple patient donors (> 18) to calculate the frequency of 20 overexpression tumors in the patient population.

In addition, DNA arrays are hybridized with mixed cDNA probes from normal tissues other than the colon (see the list above) to determine the level of expression of the candidate gene in these tissues.

Example 3

DNA micro-arrangements

DNA micro-arrangements are used to examine mRNA expression profiles from large collections of genes in multiple samples. This information is used to complement the data obtained by real-time PCR and provides independent measurement of the levels of genetic expression in tumors and normal tissues.

Examples of current technologies for the production of DNA micro-arrangements include 1) Affymetrix's "GeneChip" provisions in which oligonucleotides are synthesized on the surface of the chip by solid phase chemical synthesis using a photolithographic process, 2) DNA staining technology in which small volumes of a DNA solution are deposited robotically and then immobilized on the surface of a solid phase (eg glass). In both cases, the chips are hybridized with cDNA or cRNA that has been removed from the tissue of interest (for example, normal tissue, tumor, etc.) and are labeled with radioactivity or with a fluorescent communicating molecule. The tagged material is hybridized with the chip and the amount of probe attached to each sequence in the chip is determined using a specialized scanner. The experiment can be configured with a single fluorescent communicator (or with radioactivity) or, alternatively, can be performed using two fluorescent communicators. In the latter case, each of the two samples is labeled with one of the communicating molecules. The two labeled samples are then fully hybridized with the DNA chip sequences. The proportion of the two fluorescent signals is determined for each sequence in the chip. This ratio is used to calculate the relative abundance of transcription in the two samples. Detailed protocols are available from a number of sources that include "DNA: Microarrays: A practical approach. Scena M, Oxford University Press 1999" and the World Wide Web (http://cmqm.stanford.edu/pbrown /protocols/index.html), http://arravit.com/DNA-Microarrav-Protocols/) and specialized distributors (for example, Affymetrix).

Example 5 25

Northern-Southern band analysis

Limited amounts of normal-coupled and mixed tumor cDNA are amplified by Advantage PCR (see above). Messenger RNA derived from multiple normal tissues is also amplified using the same procedure. The amplified cDNA (1 μg) is subjected to 1.2% agarose gel electrophoresis and transferred to a nylon membrane. The membrane is hybridized 5 (AlkPhos Direct System) with a probe prepared using a fragment of the candidate TAA cDNA. Northern-Southern analysis provides information on the size of the transcript, presence of ayuste variants and abundance of transcription in tumor and normal tissues.

Example 6

Northern 35 band analysis

Northern bands are produced according to conventional protocols using 1 μg of poly A + mRNA. Radioactive probes are prepared using the Ready-to-Go system (Pharmacia).

Example 7

Experimental identification of the complete stretch cDNA sequence

Colon tumor cDNA libraries are constructed using the Lambda Zap II (Stratagene) system from 40 5 μg of polyA + mRNA. The supplied protocol is followed except that Superscriptll (Life Technologies) is used for the reverse transcription step. Libraries primed with oligo dT and randomly primed are constructed. Approximately 1.5 x 106 independent phages were plated for each library scan. Phage plates are transferred to nylon filters and hybridized using a cDNA probe labeled with AlkPhos Direct. Positive phages are detected by chemiluminescence. Positive phages are excised from the agrar plate, eluted in 500 µL of SM regulator and confirmed by gene specific PCR. Eluted phages become a single bacteriophage of strain M13 by excision in vivo. The bacteriophage is then converted into double stranded plasmid DNA by E. coli infection. Infected bacteria are plaqued and sent to a second round of screening with the cDNA probe. Plasmid DNA is purified from positive bacterial clones and sequenced in both chains.

When the full length gene cannot be obtained directly from the cDNA library, the lost sequence is isolated using RACE technology (Marathon Kit, ClonTech.). This approach is based on the reverse transcription of mRNA in double-stranded cDNA, linking linkers on the ends of the cDNA and amplifying the

desired cDNA tip using a gene specific primer and one of the linker oligonucleotides. Marathon PCR products are cloned into a plasmid (pCRII-TOPO, InVitrogen) and sequenced.

The polynucleotide of SEQ ID NO: 1 was obtained using this procedure.

 Example 8

EST 5 profiles

A complementary approach to the characterization of experimental antigen tissue expression is to explore the human EST database. EST (Expressed Sequence Labels) are small fragments of cDNA made from a collection of mRNA extracted from a particular tissue or cell line. Such a database currently provides a massive amount of human ESTs (2 x 106) from several thousand cDNA tissue libraries, including tumor tissues of various types and disease states. By means of computer tools (Blast), a comparison search of the CASB7439 sequence is carried out in order to have a greater penetration capacity in tissue expression.

EST distribution of CASB7439:

 EST gene bank access number

 EST cDNA tissue library

 C00634

 Human adult (K. Okubo)

 AA68668

 NCI_CGAP_Co3

 AA565752

 NC l_CG AP_Co 11

 AA565766

 NC l_CG AP_Co 11

 AA565767

 NC l_CG AP_Co 11

 AI337239

 NCI_CGAP_Co16

 AI337448

 NCI_CGAP_Co16

 AI393930

 NCI_CGAP_CLL1

 AI473673

 NCI_CGAP_Co14

 AI632444

 NCI_CGAP_GC6

 A1861937

 NCI_CGAP_Co16

 AI825214

 NCI_CGAP_GC6

 AW080652

 NCI_CGAP_Co19

 AW083899

 NCI_CGAP_Co19

 AW206058

 NCI_CGAP_Sub3

 AW237006

 NCI_CGAP_GC6

 AW364626

 DT0036

 AW449612

 NCI_CGAP_Sub5

These ESTs fit perfectly with CASB7439. The list contains 9 ESTs derived from 4 tumor 15 colon libraries, one EST from a normal colon library, 3 EST from a tumor germ cell library, one EST from a chronic lymphocyte leukemia cell library, 2 EST from 2 mixed tumor libraries, 2 ESTs from libraries of unknown type. This clearly suggests, as expected, that CASB7439 is overexpressed in tumor tissues, with an emphasis on colorectal tumor tissues, compared to normal tissues. twenty

Example 9:

9.1 Expression and purification of tumor specific antigens

Expression in microbial hosts, or alternatively in transcription / translation in vitro, is used to produce

the antigen of the invention for vaccine purposes and to produce protein fragments or the whole protein for the purification and rapid generation of antibodies necessary for the characterization of the naturally expressed protein by immunohistochemistry or for the continuation of purification.

Recombinant proteins can be expressed in two microbial hosts, E. coli and in yeast (such as Saccharomyces cerevisiae or Pichia pastoris). This allows the selection of the expression system with the best 5 characteristics for this particular antigen production. In general, the recombinant antigen will be expressed in E. coli and the reactive agent protein is expressed in yeast.

The expression strategy first involves the design of the primary structure of the recombinant antigen. In general, an expression condensation partner (EFP) is placed at the tip of the N-terminal to improve expression levels that could also include a region useful for modulating the immunogenic properties of the antigen, an immune condensation partner ( IFP). In addition, a useful affinity condensation partner (AFP) is included to facilitate further purification at the C-terminal end.

As mentioned earlier, several constructions may undergo benchmarking:

For rapid expression and purification as well as for a generation of antibodies against CASB7439, it is proposed to generate in E. coli a full-length CASB7439 protein with NS1 as EFP and a histidine tail as AFP.

Therefore, two constructions are proposed:

Construction 1: full-length wild type CASB7439 cDNA in condensation with NS1 cDNA as EFP and with a histidine tail encoding the cDNA as an AFP (SEQ ID NO: 8). The encoded condensation protein sequence is SEQ ID NO: 10. 20

Construction 2: full length mutated CASB7439 cDNA in condensation with NS1 cDNA as EFP and with a histidine tail encoding cDNA as an AFP (SEQ ID NO: 9). It is proposed in this construction to have the first 50 cDNA codons of native CASB7439 by codons specific to the use of E. coli codon, in order to improve the expression potential of CASB7439 in its E. coli host. The encoded condensation protein sequence is SEQ ID NO: 10. 25

The CASB7439 protein design is as shown below:

C-terminal end

N-terminal end

"NS1" is the N-terminal fragment (80 amino acids) of the NS1 Influenza protein "HIS" is a polyhistidine tail.

The recombinant strain used is AR58: a cryptic λ linogen derived from N99 which is gal E :: Tn 10, Δ-8 (chlD-pgl), Δ-H1 (cro-chlA), N + and cI857 (Proc. Natl. Acad , Sci. USA vol. 82, pp. 88-92, January 1985 Biochemistry) 30

When recombinant strains are available, the recombinant product is characterized by the evaluation of the level of expression and the prediction of additional solubility of the protein by analysis of the behavior in the crude extract.

After growth in an appropriate culture medium and induction of recombinant protein expression, the total extracts are analyzed by SDS-PAGE. Recombinant proteins are visualized in stained gels identified by Western band analysis using specific antibodies.

Plasmid:

 Name: TCM 281 pRIT .. 15143

 Replicon: pMB1

 selection: Kan 40

 promoter: PL long

 Insert: NS1-C74-39-His

 The expression of the recombinant protein derived from construct 1:

 The bacteria were grown in LB medium + 50 µg / ml of Kan at 30 ° C.

 When the crop reached a D.O. = 0.5 (620 nm), the culture was heated to 39 ° C, after 5 hours of induction, the cells were harvested.

 Extract Preparation:

  Cell concentration: 0.50X .. in full PBS + regulator ... 5

  Disruption: French press 3X

  Centrifugation: 30 min at 14000t

  Comment:> 90% in the cell extract supernatant.

The cell extract was subjected to 12.5% SDS PAGE, and subsequently stained with Coomassie blue. Western band analysis was also performed using a commercial monoclonal antibody against poly-histidine 10 tail (Quiagen). The resulting gels (Figures 3 and 4), show that the protein is expressed, and is visible in the cell extract supernatant.

The purification scheme follows a classic approach based on the presence of a His affinity tail in the recombinant protein. In a typical experiment, the disrupted cells are filtered and the acellular extracts are loaded on an Ion Metal Affinity Chromatography (IMAC, Qiagen Ni ++ NTA) that will specifically retain the recombinant protein. The retained proteins are eluted by 0-500 mM of imidazole gradient (possibly in the presence of a detergent) in a phosphate regulator.

The supernatant from the harvested crop was denatured in 6M urea, 100mM NaH2PO4, 10mM Tris, pH 8, and loaded onto an IMAC Qiagen NTA Ni ++ chromatographic column under the following conditions:

 Balance regulator: 100 mM NaH2PO4 pH 8 20

     10 mM Tris

     Urea 6 M

 Sample: 6 M urea supernatant, 100 µM NaH2PO4, Tris 10 mM Tris

 Wash Buffers: 1) 100 mM NaH2PO4 pH 8

     Tris 10mM 25

     Urea 6 M

     25 mM Imidazole

    2) 100 mM NaH2PO4 pH 8

    10 mM Tris

    urea 6 M 30

    50mM Imidazole

Elution regulator: NaH2PO4 100 mM pH 5.5

    10 mM Tris

    Urea 6 M

    500 mM Imidazole 35

The protein eluted in 500mM imidazole + 6M urea is dialyzed under the following conditions:

- PBS pH 7.2 + 0.5% sarcosyl + 4 M urea

- Idem to urea 2 M 2 hrs

- Idem to urea 0 M 2 hrs

The final material is frozen and stored. Protein content was quantified using a Lowry protein test (0.9 mg / 1.2 ml). Purity was assessed by a 12.5% SDS PAGE stained with Coomassie blue (Figure 5), and the presence of the recombinant protein was verified by Western band analysis, using a monoclonal anti-polyhistidine antibody (Figure 6 ).

A comparative evaluation of the different versions of the expressed antigen will allow the selection of the most promising candidate 5 to be used for further purification and immunological evaluation.

9.2 Production of antibodies and immunohistochemistry

Small amounts of relatively purified protein can be used to generate immunological tools for the purpose of

a) detect expression by immunochemistry in sections of normal or cancerous tissue; 10

b) detect the expression, and follow the protein during the purification process (ELISA / Western Band Analysis); or

 c) characterize / quantify the purified protein (ELISA).

9.2.1 Polyclonal antibodies:

Immunization 15

Rabbits are immunized, intramuscularly (I.M.), 3 times at 3-week intervals with 100 μg of protein, formulated in 3D-MPL / QS21 adjuvant. Three weeks after each immunization a blood sample is taken and the serum antibody titre is estimated by ELISA using the protein as a coating antigen following a conventional protocol.

ELISA 20

96-well microplates (maxisorb Nunc) are coated with 5 μg of protein overnight at 4 ° C. After 1 hour of saturation at 37 ° C with 1% NCS PBS, serial dilution of rabbit sera is added for 1:30 hours at 37 ° C (starting at 1/10). After 3 rinsings in PBS Tween, biotinylated anti-rabbit antiserum (Amersham) (1/5000) is added. The plates are washed and streptavidin coupled with peroxidase (1/5000) is added for 30 minutes at 37 ° C. After washing, 50 μI of TMB (BioRad) is added for 7 minutes and then the reaction with 0.2 M H2SO4 is stopped. It can be measured at 450 nm and intermediate dilutions calculated by SoftmaxPro.

9.2.2. Monoclonal antibodies:

Immunization

5 BALB / c mice are immunized 3 times at 3 week intervals with 5 μg of purified protein. Bleeds are performed 14 days post II and 1 week post 3. The sera are tested by Elisa on purified protein used as a coating antigen. Based on these results (intermediate dilution> 10,000), a mouse is selected by condensation.

HAT condensation / selection

Spleen cells are condensed with SP2 / 0 myeloma according to a standard protocol using 40% PEG and 5% DMSO. The cells are then seeded in 96-well plates 2.5 x 104-105 cells / cavity and the resistant clones will be selected in the HAT medium. The supernatant of these hybridomas will be tested for their specific antibody content and when positive, they will be sent 2 cycles of limited dilution. After 2 rounds of screening, 3 hybridomas will be selected for ascites production.

9.2.3. Immunohistochemistry

When antibodies are available, immunostaining is performed on sections of normal or cancerous tissue, in order to determine:

◊ the level of expression of the antigen of the invention in cancer relative to normal tissue or

 ◊ the proportion of cancer of a certain type that expresses the antigen

◊ if other types of cancer also express the antigen

◊ the proportion of cells that express the antigen in a cancerous tissue. Four. Five

Preparation of tissue sample

After dissection, the tissue sample is installed in a cork disk in OCT compound and quickly frozen in isopentane previously supercooled in liquid nitrogen (-160 ° C). The block will then be stored at -70 ° C until use. Sections of 7-10 μm will be made in a cryostat chamber (-20, -30 ° C).

Staining 5

The tissue sections are dried for 5 minutes at room temperature (RT), fixed in acetone for 10 minutes in RT, dried again, and saturated with PBS, 0.5% BSA, 5% serum. After 30 minutes in RT, direct staining or indirect ticking is performed using antigen specific antibodies. Direct staining leads to better specificity but less intense staining while indirect staining leads to more intense but less specific staining. 10

9.3 Analysis of human cellular immune responses to the antigen of the invention

The immunological relevance of the antigen of the invention can be assessed by in vitro priming of human T cells. All lymphocytic lines of T cells and dendritic cells are derived from PBMC (peripheral blood mononuclear cells) from healthy donors (subtype HLA-A2 preferred). An HLA-A2.1 / Kb transgenic mouse model is also used to track the HLA-A2.1 peptides. fifteen

Recently discovered antigen-specific CD8 + T cell lines are stimulated and maintained by weekly in vitro stimulation. The lytic activity and γ-IFN production of the CD8 + lines in response to the antigen or antigen-derived peptides is tested using standard procedures.

Two strategies are used to stimulate CD8 + T cell lines: a peptide-based approach and a complete gene-based approach. Both approaches require the full length cDNA of the newly discovered antigen 20 in the correct reading frame either to be cloned into an appropriate delivery system or to be used to predict the HLA binding peptide sequence.

Peptide based approach

Briefly, transgenic mice are immunized with HLA-A2 peptide adjuvant, those unable to induce a CD8 + response (as defined by an effective lysis of autologous spleen cells subjected to a peptide pulse) will be further analyzed in the human system .

Human dendritic cells (cultured according to Romani et al.) Will undergo peptide pulse and be used to stimulate classified CD8 + T cells (by Facs). After several weekly stimulations, CD8 + cell lines will first be tested in autologous peptide-driven BLCL (cell lines transformed into EBV-B). To verify the appropriate in vivo processing of the peptide, CD8 + lines will be tested in tumor cells transfected with cDNA (LLA transfected HLA-A2, Skov3 or CAMA tumor cells).

Complete gene based approach

The CD8 + T cell lines will be primed and stimulated with either gene gun transfected dendritic cells, with retrovirally transduced B7.1 transfected fibroblasts, or with dendritic cells infected with recombinant smallpox or adenovirus viruses. Virus-infected cells are very effective in presenting 35 antigenic peptides since the antigen is expressed at a high level but can only be used once to prevent the over-growth of viral T-cell lines.

After alternate stimulations, the CD8 + lines are tested in cDNA transfected tumor cells as indicated above. Peptide specificity and identity is determined to confirm immunological validation. 40

CD4 + T cell response

Similarly, the immune response of CD4 + T cells can also be evaluated. The generation of specific CD4 + T cells is made using dendritic cells loaded with recombinant purified protein or with peptides to stimulate T cells.

Predicted epitopes (nonámeros and decámeros) that unite alleles HLA: 45

Class I HLA binding peptide sequences are predicted either by Parker's algorithm (Parker, KC, MA Bednarek, 20 and JE Coligan. 1994. Scheme for potential ranking HLA-A2 binding peptides based on independent binding of individual peptide side-chains J. Immunol 152: 163 and http: //bimas.dcrt.nih. qov / molbio / h la bind /) or the Rammensee procedure (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). The peptides are then screened in the HLA-A2.1 / Kb transgenic mouse model (Vitiello et al.).

Class I HLA binding peptide sequences are predicted using the Tepitope algorithm, with a score cut set to 6 (Sturniolo, Hammer et al., Nature Biotechnology, 1999, 17; 555-561). 5

The following tables gather the predicted epitope sequences of Class I and II:

 HLA-A 0201: decimers

 Rank

 Starting position Subsequence Residue List Parker score * SEC ID:

 one

 64 KLVNLGFQAL 142,060 SEQ ID NO: 16

 *: Estimation of the Average Dissociation Time of a Molecule Containing this sub-sequence.

 HLA-A 0201: nonámeros

 Rank

 Starting position Subsequence Residue List Parker score * SEC ID:

 one

 182 ELLDFSSWL 507,976 SEQ ID NO: 17

 2

 104 RLLAEHDAV 126,098 SEQ ID NO: 18

 3

 64 KLVNLGFQA 100,850 SEQ ID NO: 19

 *: Estimation of the Average Dissociation Time of a Molecule Containing this sub-sequence.

 HLA-A 24: nonámeros

 Rank

 Starting position Subsequence Residue List Parker score * SEC ID:

 one

 97 EYIRALQRL 360,000 SEQ ID NO: 20

 *: Estimation of the Average Dissociation Time of a Molecule Containing this sub-sequence.

 HLA-A 24: decimers

 Rank

 Starting position Subsequence Residue List Parker score * SEC ID:

 one

 97 EYIRALQRL 360,000 SEQ ID NO: 21

 *: Estimation of the Average Dissociation Time of a Molecule Containing this sub-sequence.

 HLA-B7: decimers

 Rank

 Starting position Subsequence Residue List Parker score * SEC ID:

 one

 111 AVRNALAGGL 600,000 SEQ ID NO: 22

 *: Estimation of the Average Dissociation Time of a Molecule Containing this sub-sequence.

 HLA-B 4403: decimers

 Rank

 Starting position Subsequence Residue List Parker Score SEC ID:

 one

 156 SEPGSPRSAY 600,000 SEQ ID NO: 23

 2

 89 VETLRSAVEY 180,000 SEQ ID NO: 24

 HLA-DRB1 * 1501: nonámeros

 Rank

 Initial position Subsequence Residue List Tepitope Score SEC ID:

 one

 99 IRALQRLLA 5.6 SEQ ID NO: 25

 HLA-DRB1 * 1502: nonámeros

 Rank

 Initial position Subsequence Residue List Tepitope Score SEC ID:

 one

 99 IRALQRLLA 4.6 SEQ ID NO: 25

 HLA-DRB1 * 0402: nonámeros

 Rank

 Initial position Subsequence Residue List Tepitope Score SEC ID:

 one

 120 LRPQAVRPS 5.4 SEQ ID NO: 26

 HLA-DRB1 * 1101: nonámeros

 Rank

 Initial position Subsequence Residue List Tepitope Score SEC ID:

 one

 99 IRALQRLLA 4.8 SEQ ID NO: 25

 HLA-DRB1 * 1102: nonámeros

 Rank

 Initial position Subsequence Residue List Tepitope Score SEC ID:

 one

 120 LRPQAVRPS 6.2 SEQ ID NO: 26

 HLA-DRB1 * 1104: nonámeros

 Rank

 Initial position Subsequence Residue List Tepitope Score SEC ID:

 one

 99 IRALQRLLA 5.8 SEQ ID NO: 25

 HLA-DRB1 * 1106: nonámeros

 Rank

 Initial position Subsequence Residue List Tepitope Score SEC ID:

 one

 99 IRALQRLLA 5.8 SEQ ID NO: 25

 HLA-DRB1 * 1301: nonámeros

 Rank

 Starting position Tepitope Score Residue List SEC ID:

 Subsequences

 one

 120 LRPQAVRPS 6.6 SEQ ID NO: 26

 2

 73 LRQHVPHGG 4.9 SEQ ID NO: 27

 3

 31 LLRCSRRRR 4.4 SEQ ID NO: 33

 HLA-DRB1 * 1302: nonámeros

 Rank

 Initial position Subsequence Residue List Tepitope Score SEC ID:

 one

 120 LRPQAVRPS 5.6 SEQ ID NO: 26

 HLA-DRB1 * 1304: nonámeros

 Rank

 Initial position Subsequence Residue List Tepitope Score SEC ID:

 one

 120 LRPQAVRPS 6.2 SEQ ID NO: 26

 2

 73 LRQHVPHGG 4.8 SEQ ID NO: 27

 3

 31 LLRCSRRRR 4.6 SEQ ID NO: 28

 HLA-DRB1 * 1305: nonámeros

 Rank

 Initial position Subsequence Residue List Tepitope Score SEC ID:

 one

 99 IRALQRLLA 4.8 SEQ ID NO: 32

 HLA-DRB1 * 0703: nonámeros

 Rank

 Initial position Subsequence Residue List Tepitope Score SEC ID:

 one

 112 VEYIRALQR 5.1 SEQ ID NO: 29

 2

 98 YIRALQRLL 4.8 SEQ ID NO: 30

 3

 65 LVNLGFQAL 4.5 SEQ ID NO: 31

 HLA-DRB5 * 0101: nonámeros

 Rank

 Initial position Subsequence Residue List Tepitope Score SEC ID:

 one

 96 VEYIRALQR 4.3 SEQ ID NO: 32

 *: Estimation of the Average Dissociation Time of a Molecule Containing this sub-sequence.

SEQUENCE INFORMATION

SEQ ID NO: 1

SEQ ID NO: 2

SEQ ID NO: 3

SEQ ID NO: 4

SEQ ID NO: 5

SEQ ID NO: 6

SEQ ID NO: 7

SEQ ID NO: 8

SEQ ID NO: 9

SEQ ID NO: 10

SEQ ID NO: 11

SEQ ID NO: 12

SEQ ID NO: 13

SEQ ID NO: 14

SEQ ID NO: 15

SEQ ID NO: 16

KLVNLGFQAL

SEQ ID NO: 17 5

ELLDFSWL

SEQ ID NO: 18

RLLAEHDAV

SEQ ID NO: 19

KLVNLGFQA 10

SEQ ID NO: 20

EYIRALQRL

SEQ ID NO: 21

EYIRALQRLL

SEQ ID NO: 22

AVRNALAGGL

SEQ ID NO: 23

SEPGSPRSAY

SEQ ID NO: 24 5

VETLRSAVEY

SEQ ID NO: 25

IRALQRLLA

SEQ ID NO: 26

LRPQAVRPS 10

SEQ ID NO: 27

LRQHVPHGG

SEQ ID NO: 28

LGFQALRQH

SEQ ID NO: 29 15

VRNALAGGL

SEQ ID NO: 30

YIRALQRLL

SEQ ID NO: 31

LVNLGFQAL 20

SEQ ID NO: 32

VEYIRALQR

SEQ ID NO: 33

LLRCSRRRR

 25

SEQUENCE LIST

<110> SmithKline Beecham Biologicals s.a.

<120> Novel Compounds

<130> BC45300 30

<160> 32

<170> FastSEQ for Windows Version 3.0

<210> 1

<211> 1791 35

<212> DNA

<213> Human being

<400> 1

<210> 2 5

<211> 193

<212> Protein

<213> Human being

<400> 2 10

<210> 3

<211> 262

<212> Protein

<213> Human 5

<400> 3

<210> 4

<211> 1830

<212> DNA

<213> Human being

<400> 4 5

<210> 5

<211> 587

<212> DNA

<213> Human being 10

<400> 5

<210> 6

<211> 1791 15

<212> DNA

<213> Human being

<400> 6

 5

<210> 7

<211> 361

<212> Protein

<213> Human being

 10

<400> 7

<210> 8

<211> 849

<212> DNA

<213> Influenza virus and human being 5

<400> 8

<210> 9

<211> 849

<212> DNA

<213> Influenza virus and human being 5

<400> 9

<210> 10

<211> 282 10

<212> Protein

<213> Influenza virus and human being

<400> 10

<210> 11

<211> 193

<212> Protein

<213> Human 5

<400> 11

<210> 12

<211> 263

<212> Protein

<213> Mouse

 5

<400> 12

<210> 13

<211> 1051

<212> Protein 10

<213> Mouse

<400> 13

<210> 14

<211> 260

<212> Protein

<213> Rat 5

<400> 14

<210> 15

<211> 1526 10

<212> DNA

<213> Rat

<400> 15

 5

<210> 16

<211> 10

<212> Protein

<213> Human being

 10

<400> 16

Lys Leu Val Asn Leu Gly Phe Gln Ala Leu

                                                                 1 5 10

<210> 17

<211> 9 15

<212> Protein

<213> Human being

<400> 17

Glu Leu Leu Asp Phe Be Trp Leu 20

                                                                    fifteen

<210> 18

<211> 9

<212> Protein

<213> Human being

 5

<400> 18

Arg Leu Leu Ala Glu His Asp Ala Val

                                                                     fifteen

<210> 19

<211> 9 10

<212> Protein

<213> Human being

<400> 19

Lys Leu Val Asn Leu Gly Phe Gln Wing 15

                                                                     fifteen

<210> 20

<211> 9

<212> Protein

<213> Human being 20

<400> 20

Glu Tyr Ile Arg Ala Leu Gln Arg Leu

                                                                      fifteen

<210> 21 25

<211> 10

<212> Protein

<213> Human being

<400> 21 30

<210> 22

<211> 10

<212> Protein

<213> Human being 35

<400> 22

Wing Val Arg Asn Wing Leu Wing Gly Gly Leu

                                                                  1 5 10

<210> 23

<211> 10 5

<212> Protein

<213> Human being

<400> 23

Ser Glu Pro Gly Ser Pro Arg Ser Ala Tyr 10

                                                                   1 5 10

<210> 24

<211> 10

<212> Protein

<213> Human being 15

<400> 24

Val Glu Thr Leu Arg Ser Wing Val Glu Tyr

                                                                   1 5 10

<210> 25 20

<211> 9

<212> Protein

<213> Human being

<400> 25 25

Ile Arg Ala Leu Gln Arg Leu Leu Ala

                                                                    fifteen

<210> 26

<211> 9

<212> Protein 30

<213> Human being

<400> 26

Leu Arg Pro Gln Ala Val Arg Pro Ser

                                                                      1 5 35

<210> 27

<211> 9

<212> Protein

<213> Human being

<400> 27 5

Leu Arg Gln His Val Pro His Gly Gly

                                                                      fifteen

<210> 28

<211> 9 10

<212> Protein

<213> Human being

<400> 28

Leu Gly Phe Gln Ala Leu Arg Gln His 15

                                                                     fifteen

<210> 29

<211> 9

<212> Protein

<213> Human being 20

<400> 29

Val Arg Asn Wing Leu Wing Gly Gly Leu

                                                                     fifteen

<210> 30 25

<211> 9

<212> Protein

<213> Human being

<400> 30 30

Tyr Ile Arg Ala Leu Gln Arg Leu Leu

                                                                      fifteen

<210> 31

<211> 9

<212> Protein 35

<213> Human being

<400> 31

Leu Val Asn Leu Gly Phe Gln Ala Leu

                                                                    fifteen

<210> 32 5

<211> 9

<212> Protein

<213> Human being

<400> 32 10

Val Glu Tyr Ile Arg Ala Leu Gln Arg

                                                                       fifteen

<210> 33

<211> 9

<212> Protein 15

<213> Human being

<400> 33

Leu Leu Arg Cys Ser Arg Arg Arg Arg

                                                                    fifteen

Claims (10)

1. An immunogenic composition comprising a CASB7439 polypeptide which comprises an amino acid sequence having at least 70% identity in relation to the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 10 along of the total length of SEQ ID NO: 2 or SEQ ID NO: 10, or an immunogenic fragment of CASB7439 in which the fragment comprises an amino acid sequence of SEQ ID NO: 16 to SEQ ID N .º: 33, and a pharmaceutically acceptable vehicle, for use in medicine. 5 2. An immunogenic composition comprising a polynucleotide encoding CASB7439 or a fragment thereof according to claim 1; wherein the polynucleotide is selected from the group comprising SEQ ID NO: 8 and SEQ ID NO: 9; and a pharmaceutically acceptable vehicle, for use in medicine. 3. An immunogenic composition comprising an effective amount of antigen presenting cells, modified by in vitro loading with a CASB7439 polypeptide according to claim 1, or genetically modified in vitro to express a CASB7439 polypeptide according to claim 1, and a pharmaceutically effective vehicle. 4. A pharmaceutical composition comprising a CASB7439 polypeptide comprising an amino acid sequence having at least 70% identity in relation to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10 along of the total length of SEQ ID NO: 2 or SEQ ID NO: 10, or an immunogenic fragment of CASB7439 in which the fragment comprises an amino acid sequence of SEQ ID NO: 16 to SEQ ID No.: 33, and a pharmaceutically acceptable vehicle, for use in medicine. 5. A pharmaceutical composition comprising a polynucleotide encoding a CASB7439 polypeptide or a fragment thereof according to claim 4; wherein the polynucleotide is selected from the group comprising SEQ ID NO: 8 or SEQ ID NO: 9 and a pharmaceutically acceptable carrier, for use in medicine. twenty 6. A pharmaceutical composition comprising an effective amount of antigen presenting cells, modified by in vitro loading with a CASB7439 polypeptide according to claim 4, or genetically modified in vitro to express a CASB7439 polypeptide according to claim 4 and a pharmaceutically effective vehicle. 7. A pharmaceutical or immunogenic composition as claimed in claim 1 or 4 which additionally comprises an adjuvant that induces TH-1. 8. A pharmaceutical or immunogenic composition as claimed in claim 7 wherein the adjuvant that induces TH-1 is selected from the group of adjuvants comprising: 3D-MPL, QS21, a mixture of QS21 and cholesterol, and a CpG oligonucleotide or a mixture of two or more of said adjuvants. 9. The use of an immunogenic or pharmaceutical composition according to any one of claims 1 to 30 8, in the manufacture of a vaccine to immunotherapeutically treat a patient suffering from or susceptible to, carcinoma in which the carcinoma is cancer of colon, or other conditions associated with the colon or other diseases associated with the colon. 10. A pharmaceutical or immunogenic composition according to any one of claims 1 to 8, for use in the immunotherapeutic treatment of a patient suffering from, or is susceptible to, carcinoma in which the carcinoma is colon or other cancer. conditions associated with the colon or other diseases associated with the colon. Figure 1: Real-time PCR data using the Taqman probe