Novel compounds

ES 2 389 445 T3

DESCRIPTION

Novel compounds

Pharmaceutical compositions and methods for inducing an immune response against tumor-related antigens are described in this invention. More specifically, polynucleotides, herein referred to as CASB7439 polynucleotides, polypeptides encoded by them (referred to herein as CASB7439 polypeptides), recombinant materials, and methods for their production are described. In another aspect, methods for using such polypeptides and polynucleotides are described, including the treatment of cancer, more particularly colorectal cancer, and autoimmune and other related conditions. In another aspect, pharmaceutical compositions containing CASB7439 polypeptides and polynucleotides, processes for making such compositions, and their use in medicine are described. In a further aspect, methods are described for identifying agonists and antagonists / inhibitors using the materials described herein, and treating conditions associated with CASB7439 polypeptide imbalance with the identified compounds. The invention relates to diagnostic tests for detecting diseases associated with inappropriate activity or levels of the CASB7439 polypeptide.

The polypeptides and polynucleotides described herein are believed to be 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 targeted by specific immune mechanisms. of antigen that lead to the destruction of the tumor cell. They can also be used to diagnose the appearance of tumor cells. Furthermore, its inappropriate expression in some circumstances can cause an 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 described herein. A polypeptide described herein 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.

CASB7439 polypeptides are described herein. 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 at least 97-99% identity, to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 14 throughout the full length of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 14 respectively, provided that said isolated polypeptide is not SEQ ID NO: 2, SEQ ID NO: 12, or SEQ ID NO: 14. Such polypeptides include those that comprise the amino acid of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 11.

Still other peptides 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 at least 97-99% identity, to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID No.: 14, along the full length of SEQ ID No.: 2, SEQ ID No.: 3, SEQ ID No.: 7, SEQ ID No.: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 14 respectively, provided that said polypeptide is not SEQ ID NO: 2, SEQ ID NO: : 12, or SEQ ID NO: 14. Such polypeptides include the polypeptides of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 11 .

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

Additional peptides described herein include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 1.

Also described herein is 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 NO: 3, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, 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 fusion protein) is capable of eliciting an immune response that recognizes the CASB7439 polypeptide. Such an immunogenic fragment can include, for example, the CA polypeptide. SB7439 that lacks an N-terminal leader sequence, a transmembrane domain, or a C-terminal assurance domain. In a preferred aspect the CASB7439 immunogenic fragment according to the invention comprises substantially 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 al less 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: 3, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11,

ES 2 389 445 T3

SEQ ID No.: 12, or SEQ ID No.: 14, along the full length of SEQ ID No.: 2, SEQ ID No.: 3, SEQ ID No.: 7, SEQ ID NO: 10, SEQ ID NO: 11, 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 a CASB7439 epitope will normally 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.

Peptides incorporating these epitopes form a preferred aspect. Mimotopes that have the same characteristics as these epitopes, and immunogens that comprise such mimotopes that elicit an immune response that cross-reacts with an epitope in the context of the CASB7439 molecule,

Isolated peptides encompassing these epitopes themselves, and any mimotopes thereof, are described herein. 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, HM, et al., 1986, Synthetic peptides as antigens. Wiley, Chichester, Ciba foundation symposium 119, p130-149; Gheysen, HM. 1986, Molecular Immunology, 23, 7, 709-715); or they are capable of producing antibodies, when coupled to a suitable carrier, whose antibodies react with the native molecule.

The peptide mimotopes of the above-identified epitopes can be designed for a particular purpose by adding, deleting, or substituting chosen amino acids. Consequently, the peptides can be modified for the 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. Furthermore, it may be desirable for peptides conjugated to a protein carrier to include a hydrophobic end distal of the conjugated end of the peptide, such that the free unconjugated end of the peptide remains associated with the surface of the carrier protein. This reduces the conformational degrees of freedom of the peptide, and thus increases the likelihood that the peptide will present in a conformation that closely resembles that of the peptide as found in the context of the entire molecule. For example, peptides can be altered to have an N-terminal cysteine ​​and a C-terminal hydrophobic nested tail.Alternatively, the addition or substitution of a D-stereoisomer form of one or more of the amino acids can be made 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 fully or partially non-peptide mimotope in which the constituent residues are not necessarily confined to the 20 naturally occurring amino acids. Furthermore, these can be recycled by techniques known in the art to constrain 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.

Furthermore, those skilled in the art will realize that the mimotopes or immunogens of the present invention may be larger than the epitopes identified above and as such may comprise the sequences described herein. Accordingly, the mimotopes of the present invention 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 back sequences of natural sequences, because the sequence orientation is reversed; or alternatively the sequences may fully or at least in part consist of D-stereoisomeric amino acids (reverse sequences). Also, peptide sequences can be retro-reversed in character, because the sequence orientation is reversed and the amino acids are of the D-stereoisomer form. Such retro or retro-inverse peptides have the advantage of being non-independent, and as such they can overcome self-tolerance problems in the immune system.

Alternatively, peptide mimotopes can be identified using antibodies that are themselves capable of binding to the epitopes of the present invention using techniques such as phage display technology (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 to anti-native peptide antibodies, but may not necessarily by themselves share significant sequence homology to native peptide. This approach may have significant advantages in allowing the possibility of identifying a peptide with improved immunogenic properties, or it may overcome any potential antigen self-tolerance problems 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 shared chemical properties among the recognized mimotope sequences.

Covalent coupling of the peptide to the immunogenic carrier can be accomplished 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- [y-maleimidobutyryloxy] succinimide, using common commercially available heterobifunctional linkers such as CDAP and SPDP (using 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.

ES 2 389 445 T3

The types of vehicles used in the Immunogens of the present invention will be readily known to the person skilled in the art. The function of the vehicle is to provide cytokine assistance 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 includes: California keyhole limpet 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 may be directly conjugated to liposome carriers, which may additionally comprise immunogens capable of providing T-cell help. Preferably the ratio of mimotopes to carrier is in the range of 1: 1 to 20: 1, and preferably each carrier it must carry between 3-15 peptides.

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 issued). 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 the N-terminal 100-110 amino acids of protein D (GB 9717953.5 )).

Another preferred method of presenting peptides is in the context of a recombinant fusion 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 may comprise peptides presented in chimeric particles consisting of hepatitis B core antigen. Additionally, recombinant fusion proteins may comprise the mimotopes of the present invention and a carrier protein, such as influenza virus NS1.

Peptides can be readily synthesized by solid phase procedures well known in the art. Suitable syntheses can be carried out using T-boc or F-moc procedures. Cyclic peptides can be synthesized by the solid phase procedure employing the well known F-moc procedure 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. Techniques and procedures for solid phase synthesis are described in 'Solid Phase Peptide Synthesis: A Practical Approach' by E. Atherton and RC Sheppard, published by IRL in Oxford University Press (1989). Alternatively, the peptides can be produced by recombinant procedures, including the expression of nucleic acid molecules encoding the 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, EF, and Sambrook, et al., Molecular cloning, a laboratory manual, 2nd Ed; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

A method of producing a polypeptide is described herein. The process 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 there is provided a process for producing a polypeptide, 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 method 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;

iii) cultivating said transformed host cell under conditions that allow expression of said DNA polymer to produce said protein; and iv) recovering said protein.

The polypeptides or the immunogenic fragment can be in the form of a mature protein or can be part of a larger protein such as a precursor or a fusion protein. It is often 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. Furthermore, addition of the exogenous polypeptide or lipid tail or polynucleotide sequences is also considered to increase the immunogenic potential of the final molecule.

In one aspect, genetically modified soluble fusion proteins comprising a polypeptide as described herein, 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 an 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 Fc part can be eliminated

ES 2 389 445 T3 simply by incorporation of a cleavage sequence that can be cleaved with blood clotting factor Xa. Furthermore, processes are described for the preparation of these fusion proteins by genetic engineering, and the use of the same for drug screening, diagnosis and therapy. A particularly preferred aspect relates to the use of a polypeptide or a polynucleotide in the manufacture of a vaccine to immunotherapeutically treat a patient suffering from or susceptible to carcinoma, especially cancer of the colon or other tumors or diseases associated with the colon. . A further aspect also relates to polynucleotides encoding such fusion proteins. Examples of condensation protein technology can be found in International Patent Application No.^s W094 / 29458 and W094 / 22914.

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

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

Also described herein are xenogenic forms (also called orthologous forms) of the aforementioned polypeptides, said xenogenic forms referring to an antigen that has substantial sequence identity to human antigen (also called autologous antigen) that serves as a reference antigen but which is derived from a different non-human species. In this context substantial identity refers to the match of one amino acid sequence with another amino acid sequence or of a polynucleotide sequence with another polynucleotide sequence when such sequences are arranged in a better alignment in any number of known sequence alignment proteins in The matter. By substantial identity is meant 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 xenogenic 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, a method for inducing an immune response against human CASB7439 having an amino acid sequence as set forth in any of the sequences SEQ ID NO: 2, SEQ ID NO: 3, is also described herein. SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 11 in a human, comprising administering to the patient an effective dose of a composition comprising a xenogenic form of said human CASB7439 as described. described in this document. A preferred embodiment is a method of inducing an immune response against human CASB7439 using xenogeneic CASB7439 isolated from mouse, rat, pig, or rhesus monkey. Another preferred method of inducing an immune response in accordance with the present invention is to use an antigen composition that includes a live viral expression system that expresses said xenogeneic antigen. The preferred xenogeneic CASB7439 polypeptide has the sequence set forth in SEQ ID NO: 12 (mouse) or 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. less 90%, even more preferably an identity of at least 95%, most preferably an identity of at least 97-99%, to SEQ ID NO: 12 or SEQ ID NO: 14, over the full length of SEQ ID NO: 12 or SEQ ID NO: 14. Accordingly, the xenogenic polypeptide will comprise an immunogenic fragment of the polypeptide of SEQ ID NO: : 12 or SEQ ID NO: 14 wherein the immunogenic activity of the immunogenic fragment is substantially equal to the polypeptide of SEQ ID NO: 12 or SEQ ID NO: 14. In addition, the xenogeneic CASB7439 polypeptide may be a fragment of at least about 20 consecutive amino acids, preferably about 30, more preferably about 50, even more preferably about 100, most preferably about 150 contiguous amino acids selected from the sequences of amino acids as shown in SEQ ID NO: 12 or SEQ ID NO: 14. More particularly xenogeneic CASB7439 fragments will retain some functional property, preferably immunological activity, of the larger molecule set forth in SEQ ID NO: 12 or SEQ ID NO: 14, and are useful in the procedures described in the present document (eg, in compositions

ES 2 389 445 T3 pharmaceuticals and vaccines, diagnostics, 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 antologous human form derived from CASB7439 as established in any of SEQ ID NO: 2. In a specific embodiment, the xenogeneic polypeptide can be part of a larger fusion, comprising the xenogeneic CASB7439 polypeptide or fragment thereof and a heterologous protein or part of a protein that acts as a fusion partner as described above.

Also described herein are variants of the aforementioned polypeptides, ie polypeptides that vary from the referents by conservative amino acid substitutions, in which one residue is substituted for another with similar characteristics. Such typical substitutions are found between Ala, Val, Leu, and Ile; between Ser and Thr: between the acidic 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, deleted, or added in any combination.

Polypeptides can be prepared in any suitable way. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. The means of preparing such polypeptides are well understood in the art.

In a further aspect, CASB7439 polynucleotides are described. 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, SEQ ID No.: 3, SEQ ID No.: 7, SEQ ID No.: 10 or SEQ ID No. . °: 11, along the full length of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 11 , 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%. they are the most highly preferred.

Additional polynucleotides 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. less than 95%, to a nucleotide sequence encoding a polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID No .: 11, throughout the entire coding region. 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 an identity of at least 99% are the most highly preferred.

Additional polynucleotides 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. less than 95%, to SEQ ID No.: 1, SEQ ID No.: 4, SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID No.: 8, or SEQ ID No. ID No.: 9, along the full length of said sequences, or to the coding sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 , SEQ ID NO: 8, or SEQ ID NO: 9 over the full length of said coding sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO . °: 5, SEQ ID No.: 6, 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 an identity of at least 99% are the most highly preferred. Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 , or SEQ ID No.: 9, as well as the polynucleotide of SEQ ID No.: 1, SEQ ID No.: 4, SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID NO: 8, SEQ ID NO: 9 or the coding sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9.

Also described in this invention is a nucleic acid encoding the aforementioned xenogenic proteins and its use in medicine. In a preferred embodiment, the xenogeneic CASB7439 polynucleotide for use in pharmaceutical compositions has the sequence set forth in SEQ ID No. 13 (mouse) or SEQ ID No. 15 (rat). Isolated xenogeneic CASB7439 polynucleotides 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 to the sequences described in this invention in SEQ ID No. 13 or SEQ ID No. 15, for example those that comprise sequence identity of at least 70%, preferably of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, Sequence identity compared to a CASB7439 polynucleotide sequence using the procedures described herein (eg, BLAST analysis using standard parameters). In a related embodiment, the isolated xenogeneic polynucleotide will comprise a nucleotide sequence that encodes a polypeptide that has an identity of at least 90%, preferably 95% and higher, to the amino acid sequence of SEQ ID No. 12 or of SEQ ID No. 14, along the full length of SEQ ID No. 12 or SEQ ID No. 14, or a nucleotide sequence complementary to said isolated polynucleotide.

ES 2 389 445 T3

Also described herein are polynucleotides that are complementary to all of the polynucleotides described above.

Such polynucleotides can be inserted into a suitable plasmid, recombinant microorganism vector or a suitable recombinant living microorganism and 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)). According to the foregoing there is provided a recombinant living microorganism or expression vector comprising said polynucleotides as defined above.

Also described in this invention is 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: 4, SEQ ID NO: : 5, SEQ ID No.: 6, SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 13 or SEQ ID No.: 15.

Also described in this invention is a polynucleotide encoding an immunological 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 immunogenic activity level of a polypeptide sequence set forth in SEQ ID No. SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID No. °: 14 or a polypeptide sequence encoded by a polynucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 15.

Polypeptide fragments preferably comprise at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all lengths in between, of a polypeptide composition set forth herein, such as those set forth. in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ iD No.: 14 or those encoded by a polynucleotide sequence set forth in a sequence of SEQ ID No.: 1, SEQ ID No.: 4, SEQ ID No.: 5, SeC ID No.: 6, SEQ ID No. 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 polypeptide encoding sequence (nucleotide 545 to 1126) encoding a 193 amino acid polypeptide, the polypeptide of SEQ ID NO: 2. The nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 may be identical to the sequence encoding polypeptides contained in sEc ID NO: 1 or it may be a different sequence than that contained in SEQ ID NO. . °: 1, which, as a result of the redundancy (degeneracy) 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 is also called human Achaete Scute homolog 2 (HASH2) (accession number NP_005161 and AAB86993).

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

The Ascl2 genes are members of the basic helix-loop-helix (BHLH) family of transcription factors. They activate transcription by binding to the E-box (5'-CANNTG-3 '). Dimerization with other BHLH proteins is required for efficient 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 NO: 6) encodes a 262 amino acid polypeptide, the polypeptide of SEQ ID No. °: 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 sEc ID NO: 11 may be identical to the polypeptide coding sequence contained in SEQ ID NO: 6 or it may be a sequence different from that contained in SEQ ID NO: 6, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptides of SEQ ID NO: 3 and 11. The polypeptide of SEQ ID NO: 3 is structurally related to other proteins of the splice coactivator protein family, having homology and / or structural similarity with the splice coactivator subunit of Homo sapiens srm300 (access to the gene bank AAF21439). The polypeptide of SEQ ID NO: 11 is not related to any known protein. The polypeptide sequences as set forth in SEQ ID NO: 3 and SEQ ID NO: 11, and the polynucleotide sequences as set forth in

ES 2 389 445 T3 SEQ ID NO: 6 are novel.

Preferred polypeptides and polynucleotides are expected to have, among other things, biological functions / properties similar to their homologous polypeptides and polynucleotides. Furthermore, the preferred polypeptides, immunological fragments and polynucleotides of the present invention have at least one activity of either SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO. . °: 11 as appropriate.

Also described in this invention are partial sequences or other incomplete polynucleotide and polypeptide sequences that were first identified prior to the determination of the corresponding complete sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO. . °: 3 and SEQ ID NO: 11.

In accordance with the above, in a further aspect, it is an isolated polynucleotide 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 %, even more preferably at least 97-99% identity, with SEQ ID NO: 4 and 5, over the full length of SEQ ID NO: 4 and 5.

(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 %, even more preferably at least 97-99% identity, with SEQ ID NO: 1 or SEQ ID NO: 6 over the full length of SEQ ID NO: 4 and SEQ ID NO: 5 respectively;

(c) the polynucleotide of SEQ ID NO: 4 and SEQ ID NO: 5; or (d) 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%, even more preferably at least 97-99% identity, with the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 7 respectively, along the full length SEQ ID NO: 2 and 7, as well as polynucleotides of SEQ ID NO: 4 and 5.

Also described in this invention is a polypeptide that:

(a) comprises an amino acid 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 at least 97-99% identity, to that of SEQ ID NO: 2 and 7, over the full length of SEQ ID NO: 2 and 7.

(b) has an amino acid 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 at least 97-99% identity, to the amino acid sequence of SEQ ID NO: 2 or 7 over the full length of SEQ ID NO: 2 or 7;

(c) comprises the amino acid of SEQ ID NO: 2 or 7; and (d) is the polypeptide of SEQ ID NO: 7;

as well as polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 4 and 5.

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

When the polynucleotides are used for the recombinant production of polypeptides, the polynucleotide can include the coding sequence for the mature polypeptide, itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those that encode a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence, or other 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 label from Ha. The polynucleotide may also contain the 5 'and 3' noncoding sequences, such as untranslated, transcribed sequences, splice signals, and

ES 2 389 445 T3 polyadenylation, ribosome binding sites and sequences that stabilize mRNA.

Additional embodiments include polynucleotides encoding polypeptide variants which comprise the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 and in which several amino acid residues are substituted, deleted or added, in any combination, for example, 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1.

Polynucleotides that are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 or 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 invention and to isolate cDNAs and genomic clones from other genes (including genes encoding paralogs derived from human sources and orthologs and paralogs derived from species other than human) that have 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 90% identical, most preferably 95% identical to that of the reference. Probes or primers will generally comprise at least 15 nucleotides, preferably at least 30 nucleotides, and may be 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 elicit a reactive immune response to the human gene.

A polynucleotide encoding a polypeptide, including homologues derived from species other than human, can be obtained by a process comprising the steps of screening an appropriate library under stringent hybridization conditions with a tagged probe having the sequence of SEQ ID N. 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 person. Preferred stringent hybridization conditions include overnight incubation at 42 ° C in a solution comprising: 50% formamide, 5 x SSC (150 mM NaCl from NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate ( pH 7.6), 5x Denhardt's 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. Thus also described herein are polynucleotides obtainable by screening 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.

One of skill in the art will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, because the region coding 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 of cDNA ends (RACE) procedure (see, for example , Forman et al., PNAS Usa 85, 8998-9002, 1988). Recent modifications of the technique, exemplified by Marathon ™ technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for larger cDNAs. In Marathon ™ technology, cDNAs have been prepared from mRNA extracted from selected tissue and an 'adapter' sequence linked to each end. Nucleic acid amplification (PCR) is then carried out to amplify the 5 '"missing" 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 'in the adapter sequence and a gene-specific primer that fuses the 5' additional in the known gene sequence). The products of this reaction can then be analyzed by sequencing constructed full-length DNA and cDNA either by binding the product directly to the existing cDNA to give a complete sequence, or by performing a separate full-length PCR using the new sequence information for the 5 'primer design.

Recombinant polypeptides can be prepared by processes well known in the art from genetically modified host cells comprising expression systems. In accordance with the foregoing, 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 is described in a further aspect. . Cell-free translation systems can also be employed to produce such proteins using RNAs derived from DNA constructs.

For recombinant production, host cells can be genetically modified to incorporate expression systems or parts thereof for polynucleotides.

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, 2^to Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Such preferred procedures include, for example, calcium phosphate transfection, transfection

ES 2 389 445 T3 mediated by DEAE-dextran, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scooping, ballistic introduction or infection.

Preferably the proteins of the invention are co-expressed with thioredoxin in trans (TIT). Coexpression of thioredoxin in trans versus cis is preferred to keep the antigen free from thioredoxin without the need for protease. The co-expression of thioredoxin facilitates the solubilization of the proteins of the invention. The coexpression of thioredoxin also has a significant impact on the production of purified protein, on the solubility and quality of the purified protein.

Representative examples of suitable 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, insert elements, elements yeast chromosomes, from viruses such as baculovirus, papovavirus, such as SV40, vaccinia virus, adenovirus, fowl pox virus, pseudorabies viruses and retroviruses and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. Expression systems can contain control regions that also regulate the expression of spawning. 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 well-known and routine techniques, such as, for example, those set forth in Sambrook et al., Molecular Cloning, A Laboratory Manual (supra). Appropriate secretion signals can be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment. These signals can be endogenous to the polypeptide or they can be heterologous signals.

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

Therefore, in some embodiments, polynucleotides encoding immunogenic polypeptides 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 into 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 {eg, US Patent No. 5,219,740; Miller and Rosman (1989) BioTechniques 7: 980-990; Miller, AD (1990) Human Gene Therapy 1: 5-14; Scarpa et al. (1991) Virology 180: 849852; 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.

In addition, a number of illustrative adenovirus-based systems have also been described. Unlike retroviruses that integrate into the host genome, adenoviruses persist extrachromosomally, consequently minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57: 267274; 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 Rich et al. (1993) Human Gene Therapy 4: 461-476).

Various adeno-associated virus (AAV) vector systems have also been developed for the delivery of polynucleotides. AAV vectors can be easily constructed using techniques well known in the art. See, for example, US Patent Nos. 5,173,414 and 5,139,941; International Publication 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 Harbor Laboratory Press); Crankcase, BJ (1992) Current opinion in Biotechnology 3: 533-539; Muzyczka, N. '(1992) Current Topics in Microbiol. And Immunol. 158: 97-129; Kotin, RM (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 polypeptides by gene transfer include those derived from the smallpox family of viruses, such as vaccinia viruses and fowl pox virus. By way of example, vaccinia virus recombinants expressing novel molecules can be constructed as explained below. 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 thymidine kinase (TK) encoding sequence.

ES 2 389 445 T3

This vector is then used to transfect cells that are simultaneously Infected with the vaccines. Homologous recombination serves to insert the vaccine enhancer plus the gene encoding the polypeptide of interest into the viral genome. The resulting TK.sup. (-) recombinate can be selected by culturing cells in the presence of 5-bromodeoxyuridine and collecting viral plaques 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 vaccinia virus recombinant encoding the T7 RNA polymerase bacteriophage. This polymerase exhibits exquisite specificity because it transcribes only templates that support T7 enhancers. After infection, cells are transfected with the polynucleotide (s) of interest, driven by a T7 enhancer. Polymerase expressed in the cytoplasm from the recombinant vaccinia virus transcribes the transfected DNA into RNA which is then translated into polypeptide by the host translation machinery. The method is provided for the high-level, transient, cytoplasmic production of large amounts 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, fowl pox viruses, such as fowl pox and canary pox viruses, can also be used to deliver the coding sequences of interest. Recombinant fowlpox viruses, which express immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of a fowl pox vector is particularly desirable in human and other mammalian species since members of the fowl pox genus can only replicate productively in susceptible avian species and are therefore not infective in mammalian cells. Procedures for producing recombinant fowlpox viruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, for example, WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a number of alphavirus vectors can be used for delivery of polynucleotide compositions, such as those vectors described in US Patents.^s 5,843,723; 6,015,686; 6,008,035 and 6,015,694. Some vectors based on Venezuelan Equine Encephalitis (VEE) can also be used, Illustrative examples of which can be found in US Patents.^s 5,505,947 and 5,643,576.

Furthermore, molecular conjugated vectors, such as the adenovirus chimeric 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 gene delivery in the invention.

Additional illustrative information on 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. NY Acad. Sci. 569: 86-103, 1989; Flexner et al., Vaccine 8: 17-21, 1990; US Patents °^s 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; US Patent °^s 4,777,127: GB 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 al., Proc. Natl. Acad. Sci. USA 91: 215-219, 1994; Kass-EIsler et al., Proc. Nati. Acad. Sci. USA 90: 1 1498-1 1502, 1993; Guzman et al., Circulation 88: 2838-2848, 1993; and Guzman et al., Cir. Res. 73: 1202-1207, 1993.

The recombinant live microorganisms described above can be virulent, or attenuated in various ways in order to obtain live vaccines.

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 it can be integrated in a random, non-specific position (genetic augmentation). In still further embodiments, the polynucleotide can be stably maintained in the cell as a separate episomal segment of DNA. Such polynucleotide segments or episomes encode sufficient sequences to allow maintenance and replication independent of or in sync with the host cell cycle. How the expression construct is delivered 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 / delivered as pure DNA, eg, as described in Ulmer et al., Science 259: 1745-1749, 1993 and reviewed by Cohen, Science 259: 1691-1692, 1993. Uptake of pure DNA can be increased by coating DNA on biodegradable beads, which are efficiently transported into cells.

In still another embodiment, a composition can be delivered via a particle bombardment approach, much of which has been described. In an Illustrative example, gas-actuated particle acceleration can be achieved with devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines, Inc. (Madison, WI), some examples of which are described in the US Patents °^s 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799. This approach offers a needle-free delivery approach in which a dry powder formulation of microscopic particles, such as

ES 2 389 445 T3 as polynucleotide or polypeptide particles, are accelerated at high speed in a jet of helium gas generated by a portable device, propelling the particles into a target tissue of interest.

In a related embodiment, other devices and methods that may be useful for gas-actuated needleless injection of compositions of the present invention include those provided by Bioject, Inc. (Portland, OR), some examples of which are described in the US Patent °^s 4,790,824; 5,064,413; 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 procedures including ammonium sulfate or ethanol precipitation, acid extraction, anion exchange or cation exchange chromatography, phosphocellulose chromatography, interaction chromatography. hydrophobic, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, ion metal affinity chromatography (IMAC) is employed for purification. Well known techniques can be employed to refold proteins to regenerate the active conformation when the polypeptide is denatured during intracellular synthesis, intracellular isolation, and / or intracellular purification.

This invention 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 reagents specific for the polypeptide of the present invention, as diagnostic reagents.

Identifying genetic or biochemical markers in the blood or tissues that will allow the detection of very early changes in the pathway of carcinogenesis will help determine the best treatment for the patient. Surrogate 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 invention will be useful both in defining the stages of the cancerous disorder and in evaluating the nature of the cancerous tissue. The staging process tracks cancer progression and is determined in the presence or absence of malignant tissue in the biopsied areas. The polynucleotides of the invention can help to refine the staging processes by identifying markers for the aggressiveness of a cancer, for example, the presence in different areas of the body. Cancer screening describes how closely a tumor resembles normal tissue of the same type and is assessed for its cell morphology and other differentiation markers. The polynucleotides of the invention may be useful in determining tumor grade as they aid in determining the different states of cells in a tumor.

Diagnostic assays provide a process for diagnosing or determining a susceptibility to cancers, autoimmune disease, and related conditions by diagnostic procedures comprising determining a sample derived from a subject at an abnormally low or high polypeptide or mRNA level. This diagnostic procedure is known as differential expression. The expression of a particular gene is compared between a diseased tissue and a normal tissue. A difference between the gene related to the polynucleotide, mRNA, or protein in the two tissues is compared, for example, in molecular weight, amino acid or nucleotide sequence, or relative abundance, indicating a change in the gene, or a gene that does it. regulates, in the human tissue that is suspected to be diseased.

Decreased or increased expression can be measured at the RNA level. 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 method. An increased or decreased expression of a given RNA in diseased tissue compared to normal tissue suggests that the 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 relative to the normal level is indicative of the presence of cancer in the patient.

The expression levels of mRNA in a sample can be determined by the generation of a library of expressed sequence tags (ESTs) from the sample. The relative representation of ESTs in the library can be used to assess the relative representation of gene transcription in the starter sample. The EST analysis of the test can then be compared to 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 gene expression methodology (SAGE) (Velculescu et al., Science (1995) 270: 484), differential display methodology (eg US 5,776,683) or analysis hybridization that depends on the specificity of the nucleotide interactions.

Alternatively, the comparison could be made at the protein level. Protein sizes in the two tissues can be compared using antibodies to detect polypeptides in Western banding analysis of protein extracts from the two tissues. Subcellular localization and expression levels can also be detected immunologically using the antibodies to the corresponding protein. Testing techniques

Additional ES 2 389 445 T3 that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well known to those skilled in the art. An increased or decreased 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 disease.

In the assays of the present invention, the diagnosis can be determined by detecting expression levels of gene product encoded by at least one sequence described in SEQ ID No. 1. A comparison of the levels of mRNA or protein in a diseased versus normal tissue to track the progress or remission of a disease.

A large number of polynucleotide sequences can be tested in a sample using polynucleotide bundles. These can be used to examine differential gene expression and to determine gene function. For example, bundles of polynucleotide sequences SEQ ID NO: 1 can be used to determine whether any of the polynucleotides are differentially expressed between a normal and cancer cell. In one embodiment of the invention an oligonucleotide probe array comprising the nucleotide sequence SEQ ID NO: 1 or fragment thereof can be constructed to conduct efficient screening, for example, of genetic mutations. Arranging techniques are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, M. Chee et al., Science , vol 274, pages 610-613 (1996)).

Diagnosis as used herein includes determining the susceptibility of a subject to a disease, determining whether a subject currently has the disease, and also the prognosis of a patient affected by the disease.

The present invention further relates to a diagnostic kit for carrying out a diagnostic test comprising:

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

b) a nucleotide sequence complementary to that of (a), preferably the nucleotide sequence of SEQ ID NO: 6;

c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO: 2 or 3, or a fragment thereof; or

d) an antibody to a polypeptide of the present invention, preferably with the polypeptide of SEQ ID NO: 2 or 3.

The nucleotide sequences described in this invention are also of value for chromosomal localization. The sequence is specifically targeted to, and can be hydrolyzed with, a particular location on an individual human chromosome. The mapping of relevant sequences for chromosomes is an important first step in the correlation of those sequences with the disease associated with the gene. Once a sequence has been mapped to an exact chromosomal location, the physical position of the sequence on the chromosome can be correlated with the genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). The interrelationship between genes and diseases that have been mapped to the same chromosomal region is then identified by linkage analysis (matching of physically adjacent genes). Differences in cDNA or gene sequence between affected and unaffected individuals can also be determined.

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

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

Antibodies raised against the polypeptides described in the present invention can be obtained by administering the polypeptides or epitope support fragments, analogs or cells to an animal, preferably a non-human animal, 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 technique -EBV (Cole et al., Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).

ES 2 389 445 T3

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

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

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

Isolated means altered by the hand of man from its natural state. If a composition or isolated 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 living animal is not isolated, but the same polynucleotide or polypeptide separated from coexisting materials from its natural state is isolated, as the term is used herein.

Polynucleotide generally refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA and DNA or modified RNA or DNA that include single-stranded and double-stranded regions.

"Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but 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 can 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 and variant polypeptide are generally closely similar and, in many regions, identical. A variant and a reference polypeptide can differ in 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 a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur 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 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 polypeptide or polynucleotide sequences, as may be the case, as determined by the inter-strand coupling of such sequences. Identity and similarity can be easily calculated by known procedures, including but not limited to those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; 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, 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. 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 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 polypeptide or polynucleotide sequence comparison using this algorithm include the following:

Gap Penalty: 12

Gap extension penalty: 4

Word size: 2, max. 6

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

ES 2 389 445 T3

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

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

Gap Penalty: 12

Gap Length Penalty: 4

A useful program with these parameters is publicly available as the gap program from Genetics Computer Group, Madison Wl. The above-mentioned 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

Gap Penalty: 50

Gap Length Penalty: 3

A useful program with these parameters is publicly available as the

Genetics Computer Group, Madison Wl. The aforementioned parameters are the default parameters for polynucleotide comparisons.

By way of example, a polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1, that is, 100% identical, or it may include up to a certain integer number of nucleotide alterations in comparison. with 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 wherein said alterations can occur at the 5 'or 3' terminal positions of the sequence of reference nucleotides or anywhere between those terminal positions, interdistributed either individually between 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 percent identity (divided by 100) and subtracting that product from said total number of nucleotides in the SEQ ID NO. 1, or:

nn <Xn - (xn y), where nn is the number of nucleotide alterations, Xn is the total number of nucleotides in SEQ ID NO: 1 and y 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 where 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 missense, missense, or misread mutations in this coding sequence and thus 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, i.e. it may be 100% identical, or it may include up to a certain integer number of amino acid alterations. compared to the reference sequence such that the% identity is less than 100%. Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and in which said alterations may occur at the amino or carboxy terminal positions of the sequence. of reference polypeptide or anywhere between those terminal positions, interdistributed either individually between 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 percentage identity (divided by 100) and then subtracting that product from said number. total 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 y is, for example, 0.70 for the 70%, 0.80 for 80%, 0.85 for 85%, etc., and wherein any non-integer product of Xa and y is rounded down to the nearest integer before subtracting from Xa.

ES 2 389 445 T3

Homologous is a generic term used in the art to indicate a polynucleotide or polypeptide sequence that possesses a high degree of sequence relatedness to a subject sequence. Such a relationship can be quantified by determining the degree of Identity and / or similarity between the sequences being 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 paralog, which means a functionally similar sequence when considered within the same species.

LEGENDS OF THE FIGURES

Figure 1: Shows real-time PCR data using the Taqman probe. The legend is as follows: Adrenal gland: Ad_GI; Bladder: Bl; Bone Marrow: Bo_Ma, Cervix: Ce; Colon: Co; Fallopian tube: Fa_Tu; Ileum: II; Liver: LI; Lung: Lu; Lymph node: Ly_No; Esophagus: Oe, Parathyroid gland: Pa_Thy; Placenta: Pl; Prostate: Pr, Rectum: Re; Skin: Sk; Skeletal muscle; Sk_Mu; Small intestine: Sm_ln; Spleen; Sp; Testicle: You; 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: Sm_ln; Spleen: Sp; Testicle: You; Thyroid gland: Thy; Trachea: Tr, Heart: He.

Figure 3 shows Coomassie blue stained SDS PAGE of the cell extract of the strain expressing CASB7439. Lane 1 shows the molecular markers, lane 2 the Induced cell extract 5 h at 39 ° C; Lane 3 shows Induced cell extract supernatant; and lane 4 shows the Induced cell extract pellet.

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

Figure 5 shows Coomassie blue stained SDS-PAGE of CASB7439 after purification. Lanes 1 and 5 represent molecular weight markers; Lanes 2, 3, 4 are loaded respectively with 2 µl, 4 µΙ, and 6 µΙ of purified protein.

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

Examples

Example 1

Real-time RT-PCR analysis

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

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

Real-time reactions are assembled according to standard PCR protocols using 2 ng of purified mRNA for each reaction. Sybrl stain (Molecular Probes) is added in a final dilution of 1/75000 for real-time detection. Amplification (40 cycles) and real-time detection is performed on a Perkin-Elmer Byosystems PE7700 system using standard instrument settings. Ct values ​​are calculated using PE7700 Sequence Detector software. Several Ct values ​​are obtained for each sample: for the patient samples, the tumor Ct (CtT) and the normal colon coupled Ct (CtN) values ​​in the candidate TAA and for the panel of normal tissue samples, one CtXY for each XY normal tissue. Another Ct (CtA) in the Actin gene is also calculated, as an internal reference, for all samples. Alternatively, real-time PCR amplification can be monitored using a Taqman probe. Amplification (40 cycles) and real-time detection are performed on a Perkin-Elmer Biosystems PE7700 system using standard instrument settings. Ct values ​​are calculated using 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 efficiency of PCR amplification under the prevailing experimental conditions is found

ES 2 389 445 T3 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 Actin transcription level. A value of 1 therefore suggests that the candidate antigen and Actin have the same level of expression.

Real-time PCR reactions were performed first in the tumor colon and by coupling the normal colon derived from biopsies of 12 patients. 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 made 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 previously described. 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; CASB7439 Real Time PCR Expression Results: 12 Patient Data Set.

<td>% of patients with a higher level of mRNA in colon, tumor coupled (positive patients)</td><td> 92 %</td>

<td>% of patients with a mRNA level at least 3 times higher in the coupled tumor colon</td><td> 92 %</td>

<td>% of patients with a mRNA level at least 10 times higher in coupled tumor colon</td><td> 92 %</td>

<td>% of patients with a mRNA level at least 3 times lower in coupled tumor colon.</td><td> 8 %</td>

<td>Average level of normal colon coupled mRNA (standardized actin)</td><td> 0,0026</td>

<td>Average level of mated tumor colon mRNA in positive patients (standardized actin)</td><td> 0,265</td>

<td>Average fold over-expression of mRNA</td><td> 2028</td>

<td>Median times of mRNA overexpression</td><td> 115</td>

<td>Average mRNA level of normal tissues</td><td> 0,0079</td>

<td>Median mRNA level of normal tissues</td><td> 0,0016</td>

<td>Average mRNA level of normal tissues</td><td> 0,0064</td>

<td>Median mRNA level of normal tissues</td><td> 0,0017</td>

<td>% of patients with a mRNA level higher than the average normal tissues</td><td> 92 %</td>

<td>% of patients with a mRNA level greater than 10 times the average of normal tissues</td><td> 75 %</td>

<td>Nondispensable normal tissues above the mean normal tissue mRNA level</td><td>None</td>

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Table 2: CASB743 Real-Time PCR Expression Results - 18 Patient Data Set

<td>% of patients with a higher mRNA level in coupled tumor colon (positive patients)</td><td> 89 %</td>

<td>% of patients with a mRNA level at least 3 times higher in the coupled tumor colon</td><td> 89 %</td>

<td>% of patients with a mRNA level at least 10 times higher in coupled tumor colon</td><td> 78 %</td>

<td>% of patients with a mRNA level at least 3 times lower in coupled tumor colon.</td><td> 5 %</td>

<td>Average level of normal colon coupled mRNA (standardized actin)</td><td> 0,005</td>

<td>Average level of mated tumor colon mRNA in positive patients (standardized actin)</td><td> 0,152</td>

<td>Average fold over-expression of mRNA</td><td> 1100</td>

<td>Median fold of mRNA overexpression</td><td> 60</td>

<td>Average mRNA level of normal tissues</td><td> 0,0065</td>

<td>Median mRNA level of normal tissues</td><td> 0,0015</td>

<td>Average mRNA level of normal tissues</td><td> 0,005</td>

<td>Median mRNA level of normal tissues</td><td> 0,0015</td>

<td>% of patients with a mRNA level higher than the median of average normal tissues</td><td> 94 %</td>

<td>% of patients with a mRNA level greater than 10 times the median of normal tissues</td><td> 94 %</td>

<td>Nondispensable normal tissues above the median normal tissue mRNA level</td><td>None</td>

Table 3: CASB743 Real-Time PCR Expression Results - 24 Patient Data Set

<td>% of patients with a transcription level of CASB7439 superior in tumor colon than adjacent normal colon (positive patients)</td><td> 92 %</td>

<td>% of positive patients with a CASB7439 transcription level at least 10 times higher in the tumor colon than the adjacent normal colon</td><td> 75 %</td>

<td>Average fold over-expression of transcription in tumors from positive patients</td><td> 1289</td>

<td>% of patients with higher CASB7439 transcription level in tumor colon than normal tissue average</td><td> 96 %</td>

<td>% of patients with a mRNA level at least 10 times higher in tumor colon than the average in normal tissue</td><td> 62,5 %</td>

<td>Normal tissues where the expression of transcription of CASB7439 is equivalent to the level of tumor transcription in tumors</td><td>None</td>

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

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

<td>Number of tumor samples from different patients</td><td> 6</td>

<td>% of positive patients with a higher level of CASB7439 transcription in the tumor colon than the adjacent normal colon (positive patients)</td><td> 100 %</td>

<td>% of positive patients with a CASB7439 transcription level at least 10 times higher in the tumor colon than the adjacent normal colon</td><td> 83 %</td>

<td>Average fold over-expression of transcription in tumors from positive patients</td><td> 109</td>

<td>% of patients with higher CASB7439 transcription level in tumor colon than normal tissue average</td><td> 100 %</td>

<td>% of patients with a mRNA level at least 10 times higher in tumor colon than the average in normal tissue</td><td> 100 %</td>

<td>Normal tissues where the expression of transcription of CASB7439 is equivalent to the level of tumor transcription in tumors</td><td>None</td>

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

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

<td>Tissue</td><td>Abbreviation</td>

<td>Adrenal gland</td><td>Ad-Gl</td>

<td>Aorta</td><td>Year</td>

<td>Bladder</td><td>Bl</td>

<td>Bone marrow</td><td>Bo_Ma</td>

<td>Brain</td><td>Bra</td>

<td>Cervix</td><td>EC</td>

<td>Colon</td><td>Co</td>

<td>Fallopian tube</td><td>Fa_You</td>

<td>Heart</td><td>I have</td>

<td>Ileum</td><td>Il</td>

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<td>Kidney</td><td>Ki</td>

<td>Liver</td><td>Li</td>

<td>Lung</td><td>Mon</td>

<td>Lymph node</td><td>Ly_No</td>

<td>Esophagus</td><td>Oe</td>

<td>Parathyroid gland</td><td>Pa_Thy</td>

<td>Right</td><td>Re</td>

<td>Skin</td><td>Sk</td>

<td>Skeletal muscle</td><td>Sk_Mu</td>

<td>Small intestine</td><td>Sm_il</td>

<td>Spleen</td><td>Sp</td>

<td>Stomach</td><td>St</td>

<td>Thyroid gland</td><td>Thyt</td>

<td>Windpipe</td><td>Tra</td>

<td>Ovary</td><td>Ov</td>

<td>Placenta</td><td>Pl</td>

<td>Prostate</td><td>Pr</td>

<td>Testicle</td><td>Tea</td>

Example 2

Differential tracing of cDNA dispositions.

Identification of tumor associated genes in the subtracted cDNA library is done by differential screening.

Total bacterial DNA is extracted from 100 µl in cultures that are allowed to stand overnight. Bacteria are lysed with guanidium isothiocyanate and bacterial DNA is affinity purified using a magnetic beaker (Boehringer). Plasmid inserts 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 tool (Beekman). The transferred cDNA is covalently bound to the membrane by UV irradiation. The first membrane hybridizes with a pooled cDNA probe prepared from a single patient's tumor. The second membrane hybridizes with an equivalent amount of mixed cDNA probe prepared from normal colon from 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 stringent washes are as described in the AlkPhos Direct kit. The hybridized probe is detected by chemiluminescence. Hybridization intensities for each cDNA fragment or for both bands are measured by film densitometry or direct measurement (BioRad Fluor-S Max). The ratio of tumor intensities to normal hybridization (T / N) is calculated for each gene in order to assess the degree of overexpression in the tumor. Genes that are significantly over-expressed 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 (&gt; 18) to calculate the frequency of over-expressing tumors in the patient population.

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

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Example 3

DNA micro-arrangements

Microarrays (microarrays or microarrays) of DNA are used to examine the mRNA expression profiles of large collections of genes in multiple samples. This information is used to complement the data obtained by real-time PCR and provides an independent measurement of gene expression levels in tumors and normal tissues.

Examples of current technologies for the production of DNA microarrays include 1) Affymetrix GeneChip arrays in which oligonucleotides are synthesized on the chip surface by solid phase chemical synthesis using a photolithographic process, 2) staining technology of DNA in which small volumes of a DNA solution are robotically deposited 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 extracted from the tissue of interest (eg, normal tissue, tumor, etc.) and labeled with radioactivity or with a fluorescent reporter molecule. The tagged material is hybridized to the chip and the amount of probe bound to each sequence on the chip is determined using a specialized scanner. The experiment can be set up with a single fluorescent reporter (or with radioactivity) or, alternatively, it can be performed using two fluorescent reporters. In the latter case, each of the two samples is labeled with one of the communicator molecules. The two labeled samples are then fully hybridized to the sequences on the DNA chip. The ratio of the two fluorescent signals is determined for each sequence on the chip. This ratio is used to calculate the relative abundance of the transcript in the two samples. Detailed protocols are available from a number of sources including 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

Northern-Southern band analysis

Limited amounts of mixed tumor and coupled normal colon 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 pg) is electrophoresed on a 1.2% agarose gel and transferred to a nylon membrane. The membrane is hybridized (AlkPhos Direct System) with a probe prepared using a fragment of the candidate TAA cDNA. Northern-Southern analysis provides information on transcript size, presence of splice variants, and transcript abundance in tumor and normal tissues.

Example 6

Northern band analysis

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

Example 7

Experimental identification of the full-length cDNA sequence

Colon tumor cDNA libraries are constructed using the Lambda Zap II system (Stratagene) from 5 pg of polyA + mRNA. The supplied protocol is followed except that Superscriptll (Life Technologies) is used for the reverse transcription step. The oligo dT primed and randomly primed libraries are constructed. Approximately 1.5 x 10® independent phages were plated for each library screen. The phage plates are transferred to nylon filters and hybridized using a cDNA probe labeled with AlkPhos Direct. Positive phage are detected by chemiluminescence. Positive phage are excised from the agrar plate, eluted in 500 µl of SM regulator, and confirmed by gene-specific PCR. Eluted phage are converted into a single M13 strain bacteriophage by in vivo excision. The bacteriophage is then converted to strained double-stranded plasmid DNA by infection with E. coli. Infected bacteria are plated and sent for a second round of cDNA probe screening. Plasmid DNA is purified from positive bacterial clones and sequenced on both strands.

When the full-length gene cannot be obtained directly from the cDNA library, the missing sequence is isolated using RACE technology (Marathon Kit, ClonTech.). This approach relies on reverse transcription of mRNA into double-stranded cDNA, ligating linkers on the ends of the cDNA, and amplifying the desired end of the cDNA using a gene-specific primer and one of the linker oligonucleotides. The Marathon PCR products are cloned into a plasmid (pCRII-TOPO, InVitrogen) and sequenced.

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

Example 8

EST profiles

A complementary approach to characterizing experimental antigen tissue expression is to scan the human EST database. ESTs (Expressed Sequence Tags) are small cDNA fragments 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 tissue cDNA 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 the tissue expression.

CASB7439 EST Distribution:

<td>EST gene bank access number</td><td>EST cDNA tissue library</td>

<td>C00634</td><td>Human adult (K. Okubo)</td>

<td>AA68668</td><td>NCI_CGAP_Co3</td>

<td>AA565752</td><td>NC l_CG AP_Co 11</td>

<td>AA565766</td><td>NC l_CG AP_Co 11</td>

<td>AA565767</td><td>NC l_CG AP_Co 11</td>

<td>AI337239</td><td>NCI_CGAP_Co16</td>

<td>AI337448</td><td>NCI_CGAP_Co16</td>

<td>AI393930</td><td>NCI_CGAP_CLL1</td>

<td>AI473673</td><td>NCI_CGAP_Co14</td>

<td>AI632444</td><td>NCI_CGAP_GC6</td>

<td>A1861937</td><td>NCI_CGAP_Co16</td>

<td>AI825214</td><td>NCI_CGAP_GC6</td>

<td>AW080652</td><td>NCI_CGAP_Co19</td>

<td>AW083899</td><td>NCI_CGAP_Co19</td>

<td>AW206058</td><td>NCI_CGAP_Sub3</td>

<td>AW237006</td><td>NCI_CGAP_GC6</td>

<td>AW364626</td><td>DT0036</td>

<td>AW449612</td><td>NCI_CGAP_Sub5</td>

These ESTs fit perfectly with CASB7439. The list contains 9 ESTs derived from 4 tumor colon libraries, one EST from a normal colon library, 3 ESTs from a tumor germ cell library, one

ESTs from a library of chronic lymphocyte leukemia cells, 2 ESTs from 2 mixed tumor libraries, 2 ESTs from libraries of unknown type. This clearly suggests, as expected, that CASB7439 is over-expressed in tumor tissues, with an emphasis on colorectal tumor tissues, compared to normal tissues.

Example 9

9.1 Expression and purification of tumor-specific antigens

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

ES 2 389 445 T3 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 characteristics for this particular antigen production. In general, the recombinant antigen will be expressed in £ coli and the reagent protein is expressed in yeast.

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

As mentioned above, various builds can undergo benchmarking:

For rapid expression and purification as well as for generation of antibodies against CASB7439, it is proposed to generate in £ coli a full-length CASB7439 protein with NS1 as EFP and a hlstldin 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 hlstldlna tail encoding the cDNA as an AFP (SEQ ID NO: 8). The encoded fusion protein sequence is SEQ ID NO: 10.

Construction 2: Full-length mutated CASB7439 cDNA in condensation with NS1 cDNA as EFP and with a hlstldlna tail encoding cDNA as an AFP (SEQ ID NO: 9). It is proposed in this construct to have the first 50 codons of native CASB7439 cDNA per codon usage specific for £ coli, in order to enhance the expression potential of CASB7439 in its E. coli host. The encoded fusion protein sequence is SEQ ID NO: 10.

The protein layout of CASB7439 is as shown below:

N-terminal end

HIS

C-terminal end

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

The recombinant strain used is AR58: a cryptic λ isogen derived from N99 which is gal E :: Tn 10, Δ-8 ((Μ ° ~ pgl), ΔH1 (cro-cñ / A), N⁺ and cl857 (Proc. Natl. Acad, Sel. USA vol. 82, pp. 88-92, January 1985 Blochemlstry)

When recombinant strains are available, the recombinant product is characterized by 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, total extracts are analyzed by SDS-PAGE. Recombinant proteins are visualized on stained gels and identified by Western banding analysis using specific antibodies.

Plasmid:

name: TCM 281 pRIT .. 15143 replicon: pMB1 selection: Kan promoter: long PL

Insert: NS1-C74-39-Hls

The expression of the recombinant protein derived from construct 1:

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The bacteria were cultured in LB medium + 50 pg / ml Kan at 30 ° C. When the culture reached an OD = 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 complete PBS + regulator ...

Disruption: French press 3X

Centrifugation: 30 min at 14000t

Comment:&gt; 90% in the cell extract supernatant.

The cell extract was subjected to 12.5% ​​SDS PAGE, and subsequently stained with Coomassie blue. Western banding analysis was also performed using a commercial monoclonal antibody against polyhistidine 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 classical approach based on the presence of a His affinity tag 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, Ni⁺⁺Qiagen's NTA) that will specifically retain the recombinant protein. Retained proteins are eluted by a 0-500 mM imidazole gradient (possibly in the presence of a detergent) in a phosphate buffer.

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

Balance regulator: NaH2PO4 100 mM pH 8

10 mM Tris

Urea 6 M

Sample: 6 M urea supernatant, 100 pM NaH2PO4, 10 mM Tris from Tris Wash buffers: 1) 100 mM NaH2PO4 pH 8

Tris 10mM

Urea 6 M

Imidazole 25 mM

Elution buffer:

<td>2) NaH2PO4</td><td>100 mM pH 8</td>

<td>Tris</td><td>10 mM</td>

<td>urea</td><td>6 M</td>

<td>Imidazole 50mM</td><td></td>

<td>NaH2PO4</td><td>100 mM pH 5.5</td>

<td>Tris</td><td>10 mM</td>

<td>Urea</td><td>6 M</td>

<td>Imidazole 500 mM</td><td></td>

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

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

- Same as urea 2 M 2 hrs

- Same as 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 banding analysis, using an anti-polyhistidine monoclonal antibody (Figure 6 ).

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

9.2 Antibody production and immunohistochemistry

Small amounts of relatively purified protein can be used to generate immunological tools in order to

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

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

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

ELISA

96-well microplates (maxisorb Nunc) are coated with 5 pg of protein overnight at 4 ° C. After 1 hour of saturation at 37 ° C with 1% PBS NCS, the serial dilution of the rabbit sera is added for 1:30 hours at 37 ° C (starting at 1/10). After 3 rinses in PBS Tween, biotinylated anti-rabbit antiserum (Amersham) (1/5000) is added. The plates are washed and peroxidase-coupled streptavidin (1/5000) is added for 30 minutes at 37 ° C. After washing, 50 pI of TMB (BioRad) is added for 7 minutes and then the reaction is stopped with 0.2 M H2SO4. OD 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 pg of purified protein. Bleeding is performed 14 days post II and 1 week post 3. The sera are tested by Elisa on purified protein used as coating antigen. Based on these results (intermediate dilution&gt; 10,000) a mouse is selected by condensation.

HAT condensation / selection

Spleen cells are fused 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 / well and resistant clones will be selected in HAT medium. The supernatant from these hybridomas will be tested for 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 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.

Tissue sample preparation

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

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Staining

Tissue sections are dried for 5 minutes at room temperature (RT), fixed in acetone for 10 minutes at RT, dried again, and saturated with PBS, 0.5% BSA, 5% serum. After 30 minutes in RT, either direct staining or indirect staining 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.

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 T-cell and dendritic cell lines are derived from PBMC (peripheral blood mononuclear cells) from healthy donors (preferred HLA-A2 subtype). An HLA-A2.1 / Kb transgenic mouse model is also used to screen for HLA-A2.1 peptides.

CD8 T cell lines are stimulated⁺ newly discovered antigen-specific and maintained by weekly in vitro stimulation. Lytic activity and γ-IFN production of CD8 + lines in response to 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 whole gene-based approach. Both approaches require the newly discovered antigen full-length cDNA 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 efficient 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 be peptide pulsed and used to stimulate sorted CD8 + T cells (by Facs). After several weekly stimulations, CD8 + cell lines will first be tested on autologous peptide-driven BLCL (EBV-B transformed cell lines). To verify proper in vivo processing of the peptide, CD8 + lines will be tested in cDNA transfected tumor cells (LnCaP transfected HLA-A2, Skov3 or CAMA tumor cells). Whole gene approach

CD8 + T cell lines will be primed and stimulated with either gene gun transfected dendritic cells, retrovirally transduced B7.1 transfected fibroblasts, or recombinant pox virus or adenovirus infected dendritic cells. Cells infected with viruses are very efficient at presenting antigenic peptides since the antigen is highly expressed but can only be used once to prevent overgrowth of viral T cell lines.

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

CD4 + T cell response

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

Predicted epitopes (nonamers and decamers) that bind HLA alleles:

HLA Class I binding peptide sequences are predicted either by the Parker algorithm (Parker, KC, MA Bednarek, 20 and JE Coligan. 1994. Scheme for ranking potential 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: 1^st 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.).

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

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

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<td colspan="5">HLA-A 0201: decamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>Parker score *</td><td>SEQ ID:</td>

<td> 1</td><td> 64</td><td>KLVNLGFQAL</td><td> 142,060</td><td>SEQ ID NO: 16</td>

<td colspan="5">*: Estimation of the Average Dissociation Time of a Molecule Containing this subsequence.</td>

<td colspan="5">HLA-A 0201: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>Parker score *</td><td>SEQ ID:</td>

<td> 1</td><td> 182</td><td>ELLDFSSWL</td><td> 507,976</td><td>SEQ ID NO: 17</td>

<td> 2</td><td> 104</td><td>RLLAEHDAV</td><td> 126,098</td><td>SEQ ID NO: 18</td>

<td> 3</td><td> 64</td><td>KLVNLGFQA</td><td> 100,850</td><td>SEQ ID NO: 19</td>

<td colspan="5">*: Estimation of the Average Dissociation Time of a Molecule Containing this subsequence.</td>

<td colspan="5">HLA-A 24: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>Parker score *</td><td>SEQ ID:</td>

<td> 1</td><td> 97</td><td>EYIRALQRL</td><td> 360,000</td><td>SEQ ID NO: 20</td>

<td colspan="5">*: Estimation of the Average Dissociation Time of a Molecule Containing this subsequence.</td>

<td colspan="5">HLA-A 24: decamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>Parker score *</td><td>SEQ ID:</td>

<td> 1</td><td> 97</td><td>EYIRALQRL</td><td> 360,000</td><td>SEQ ID NO: 21</td>

<td colspan="5">*: Estimation of the Average Dissociation Time of a Molecule Containing this subsequence.</td>

<td colspan="5">HLA-B7: decamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>Parker score *</td><td>SEQ ID:</td>

<td> 1</td><td> 111</td><td>AVRNALAGGL</td><td> 600,000</td><td>SEQ ID NO: 22</td>

<td colspan="5">*: Estimation of the Average Dissociation Time of a Molecule Containing this subsequence.</td>

<td colspan="5">HLA-B 4403: decamers</td>

<td>Rank</td><td>Initial position</td><td>List of Waste of</td><td>Score of</td><td>SEQ ID:</td>

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<td></td><td></td><td>Subsequences</td><td>Parker</td><td></td>

<td> 1</td><td> 156</td><td>SEPGSPRSAY</td><td> 600,000</td><td>SEQ ID NO: 23</td>

<td> 2</td><td> 89</td><td>VETLRSAVEY</td><td> 180,000</td><td>SEQ ID NO: 24</td>

<td colspan="5">HLA-DRB1 * 1501: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID:</td>

<td> 1</td><td> 99</td><td>IRALQRLLA</td><td> 5,6</td><td>SEQ ID NO: 25</td>

<td colspan="5">HLA-DRB1 * 1502: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID:</td>

<td> 1</td><td> 99</td><td>IRALQRLLA</td><td> 4,6</td><td>SEQ ID NO: 25</td>

<td colspan="5">HLA-DRB1 * 0402: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID:</td>

<td> 1</td><td> 120</td><td>LRPQAVRPS</td><td> 5,4</td><td>SEQ ID NO: 26</td>

<td colspan="5">HLA-DRB1 * 1101: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID:</td>

<td> 1</td><td> 99</td><td>IRALQRLLA</td><td> 4,8</td><td>SEQ ID NO: 25</td>

<td colspan="5">HLA-DRB1 * 1102: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID:</td>

<td> 1</td><td> 120</td><td>LRPQAVRPS</td><td> 6,2</td><td>SEQ ID NO: 26</td>

<td colspan="5">HLA-DRB1 * 1104: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID:</td>

<td> 1</td><td> 99</td><td>IRALQRLLA</td><td> 5,8</td><td>SEQ ID NO: 25</td>

<td colspan="5">HLA-DRB1 * 1106: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID:</td>

<td> 1</td><td> 99</td><td>IRALQRLLA</td><td> 5,8</td><td>SEQ ID NO: 25</td>

<td colspan="5">HLA-DRB1 * 1301: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID:</td>

<td> 1</td><td> 120</td><td>LRPQAVRPS</td><td> 6,6</td><td>SEQ ID NO: 26</td>

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<td> 2</td><td> 73</td><td>LRQHVPHGG</td><td> 4,9</td><td>SEQ ID NO: 27</td>

<td> 3</td><td> 31</td><td>LLRCSRRRR</td><td> 4,4</td><td>SEQ ID NO: 33</td>

<td colspan="5">HLA-DRB1 * 1302: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID:</td>

<td> 1</td><td> 120</td><td>LRPQAVRPS</td><td> 5,6</td><td>SEQ ID NO: 26</td>

<td colspan="5">HLA-DRB1 * 1304: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID:</td>

<td> 1</td><td> 120</td><td>LRPQAVRPS</td><td> 6,2</td><td>SEQ ID NO: 26</td>

<td> 2</td><td> 73</td><td>LRQHVPHGG</td><td> 4,8</td><td>SEQ ID NO: 27</td>

<td> 3</td><td> 31</td><td>LLRCSRRRR</td><td> 4,6</td><td>SEQ ID NO: 28</td>

<td colspan="5">HLA-DRB1 * 1305: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID:</td>

<td> 1</td><td> 99</td><td>IRALQRLLA</td><td> 4,8</td><td>SEQ ID NO: 25</td>

<td colspan="5">HLA-DRB1 * 0703: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID:</td>

<td> 1</td><td> 112</td><td>VEYIRALQR</td><td> 5,1</td><td>SEQ ID NO: 29</td>

<td> 2</td><td> 98</td><td>YIRALQRLL</td><td> 4,8</td><td>SEQ ID NO: 30</td>

<td> 3</td><td> 65</td><td>LVNLGFQAL</td><td> 4,5</td><td>SEQ ID NO: 31</td>

<td colspan="5">HLA-DRB5 * 0101: nonamers</td>

<td>Rank</td><td>Initial position</td><td>Subsequence Residual List</td><td>User ratings for Tepitope</td><td>SEQ ID NO: 32:</td>

<td> 1</td><td> 96</td><td>VEYIRALQR</td><td> 4,3</td><td></td>

<td colspan="5">*: Estimation of the Average Dissociation Time of a Molecule Containing this subsequence.</td>