Tumour-specific animal proteins

The invention relates to a pharmaceutical composition comprising a CASB7439 polypeptide or a polynucleotide encoding said polypeptide for use in the immunotherapeutic treatment of a patient suffering from or susceptible to cancer which is colon cancer, or other colon-related cancers or other colon-related diseases, and a method of diagnosis in a patient with a disease or susceptibility to disease, or in the diagnosis of a patient with colon and rectal cancer, or a colorectal cancer susceptibility associated with the expression or activity of a polypeptide in a subject.

The polypeptides and polynucleotides disclosed 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 as compared to normal cells and thus can be targeted by antigen-specific immune mechanisms. leading to the destruction of the tumor cell. They can also be used to diagnose the presence of cancer cells. Moreover, their inappropriate expression under certain conditions can induce inappropriate autoimmune immune responses which could be corrected by appropriate vaccination with these polypeptides or polynucleotides. In this regard, the most important biological activity of a polypeptide disclosed herein is antigenic and immunogenic activity. The polypeptide disclosed herein may also exhibit at least one other biological activity of the CASB7439 polypeptide that could qualify it as a target for a therapeutic or prophylactic intervention other than that associated with an immune response.

Thus, the invention relates to a pharmaceutical composition comprising a CASB7439 polypeptide that has an amino acid sequence of at least 70% identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10 over the entire length of SEQ ID NO: 2 or SEQ ID NO: 10, respectively. or an immunogenic fragment of CASB7439, wherein said fragment comprises the amino acid sequence of one or more of SEQ ID NO: 16 to SEQ ID NO: 33 and a pharmaceutically acceptable carrier, for use in the immunotherapeutic treatment of a patient suffering from or susceptible to cancer which is colon cancer, or other colon related cancers or other colon related diseases.

The invention also relates to a pharmaceutical composition comprising a polynucleotide encoding a CASB7439 polypeptide or fragment thereof as defined above, wherein said polynucleotide is selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9, and a pharmaceutically acceptable carrier for use in the immunotherapeutic treatment of a patient. suffering from or susceptible to cancer, which cancer is colon cancer, or other colon related cancers, or other colon related diseases.

The pharmaceutical compositions preferably additionally comprise a TH-1 inducing adjuvant, said agents having as a TH-1 inducing adjuvant an adjuvant, preferably selected from the group of adjuvants consisting of 3D-MPL, QS21, a mixture of QS21 and cholesterol, and a CpG oligonucleotide or mixtures of two or more of these. adjuvants.

The invention also relates to a pharmaceutical composition comprising antigen presenting cells, modified by loading in vitro with the CASB7439 polypeptide or fragment thereof, as defined above, or genetically modified in vitro so as to express the above-defined CASB7439 polypeptide and a pharmaceutically effective carrier.

Furthermore, the invention relates to a method of diagnosing a disease or susceptibility to a disease in a patient, or diagnosing a patient for the presence of colorectal cancer, or a susceptibility to colorectal cancer, associated with the expression or activity of a polynucleotide in a patient characterized by or the amount of said polynucleotide in a sample from said patient, said polynucleotide being selected from the group consisting of:

(a) a polynucleotide that encodes an amino acid sequence of at least 70% identity to SEQ ID NO: 2 over the entire length of SEQ ID NO: 2;

(b) a polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2; and (c) a polynucleotide or coding region for said polynucleotide of SEQ ID NO: 1.

The invention also relates to a method of diagnosing a disease or susceptibility to a disease in a patient, or diagnosing a patient for the presence of colorectal cancer, or a susceptibility to colorectal cancer, associated with the expression or activity of a polypeptide in a patient characterized by the fact that the presence or amount of said polypeptide in a sample taken from said patient is analyzed, said polypeptide being selected from the group consisting of:

(a) a polypeptide having an amino acid sequence that has at least 70% identity to SEQ

ID NO: 2 for the entire length of SEQ ID NO: 2;

(b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2;

(c) a polypeptide comprising an immunogenic fragment of the polypeptide of SEQ ID NO: 2, wherein the immunogenic activity of the immunogenic fragment is substantially the same as that of the polypeptide of SEQ ID NO: 2;

(d) an immunogenic fragment of SEQ ID NO: 2, which fragment comprises the sequence of one or more from SEQ ID NO: 16 to SEQ ID NO: 33.

In a first aspect, the disclosure relates to CASB7439 polypeptides. Such peptides include isolated polypeptides having an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97-99% identical. with 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 for the entire length, respectively 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, with the proviso that said isolated polypeptide is not SEQ ID NO: 2, SEQ ID NO: 12 or SEQ ID NO: 14. Such polypeptides include those comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 7, NO: 10 and SEQ ID NO: 11.

Further peptides disclosed herein include isolated polypeptides wherein said amino acid sequence is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97-99. % identical 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 in full length, respectively 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, with the proviso that said isolated polypeptide is not SEQ ID NO: 2, SEQ ID NO: 12 or SEQ ID NO: 14. Such polypeptides include those comprising the amino acid sequence of SEQ ID NO: 14. 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 disclosed herein are purified and are substantially free of any other protein or contaminating host material.

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

The disclosure also provides an immunogenic fragment of a CASB7439 polypeptide that is a contiguous portion of a CASB7439 polypeptide having the same or similar immunogenic properties as a 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. This means that this fragment (linked to a carrier if necessary or as part of a larger fusion protein) is capable of eliciting an immune response that recognizes the CASB7439 polypeptide. Such an immunogenic fragment may include, e.g., a CASB7439 polypeptide lacking an N-terminal leader sequence, a transmembrane domain, or a C-terminal anchor domain. In a preferred aspect, the immunogenic fragment of CASB7439 disclosed herein comprises substantially the entire extracellular domain of the polypeptide which is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97-99% identical to 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 for full length , respectively, 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.

Preferably, an immunogenic fragment disclosed herein comprises at least one epitope.

Peptide fragments containing the CASB7439 epitope will typically contain at least 7, and preferably 9 or 10, contiguous amino acids of SEQ ID NO: 2. Preferred epitopes are shown in SEQ ID NO: 16 to SEQ ID NO: 33.

Preferably, the peptides disclosed herein contain these epitopes. Mimotopes that have the same characteristics as those epitopes and immunogens containing such immune-generating mimotopes that cross-react with an epitope in the context of the CASB7439 molecule also form part of the disclosure.

The disclosure thus encompasses isolated peptides comprising these epitopes as such and any mimotope thereof. A mimotope is defined as an entity that is similar to a native epitope

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CASB7439 enough to be recognized by antibodies that recognize the native molecule (Gheysen, HM et al., 1986, Syntethetic peptides as antigens. Wiley, Chichester, Ciba foundation symposium 119, pp. 130-149; Gheysen, HM, 1986, Molecular Immunology, 23, 7, 709-715) or, when conjugated to an appropriate carrier, is capable of generating antibodies that cross-react with a native molecule.

Peptide mimotopes of the above-identified epitopes can be designed for a given target by adding, deleting, or substituting selected amino acids. Thus, the peptides disclosed herein may be modified to facilitate conjugation to a protein carrier. For example, for certain chemical conjugation methods it may be preferable that the epitope includes a terminal cysteine. Furthermore, it may be preferred that the peptides coupled to the protein carrier contain a hydrophobic end opposite the peptide's coupled end such that the free, non-conjugated 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 be presented in a conformation that is as close as possible to that of the peptide as a whole in the context of the molecule. For example, peptides can be altered to have an N-terminal cysteine ​​and a hydrophobic amidated C-terminus. Alternatively, the addition or substitution of the D-stereoisomer of one or more amino acids may be performed to generate a derivative of interest, e.g. to increase the stability of the peptide. It will be appreciated by those skilled in the art that such modified peptides or mimotopes may be a wholly or partially non-peptide mimotope in which the constituent moieties are not necessarily limited to the 20 naturally occurring amino acids. Additionally, they can be cyclized using known techniques that limit the peptide to a conformation that is very close to its shape when the sequence of the peptide is in the context of the entire molecule. A preferred method of cyclizing the peptide involves the addition of a pair of cysteine ​​residues, which allows the formation of a disulfide bridge.

Moreover, those skilled in the art will recognize that the mimotopes or immunogens disclosed herein may be larger than the epitopes identified above and as such may include the sequences disclosed herein. Thus, the mimotopes disclosed herein may have N- and / or C-terminal extensions added from a number of other natural moieties at one or both ends. The peptide mimotopes can also be retro-natural sequences with reversed sequence orientation, or the sequences can be wholly or partially composed of D-stereoisomeric amino acids (inverse sequences). Peptide sequences can also be retro-inverted sequences when the sequence orientation is reversed and the amino acids are D-stereoisomeric. Such retro or retro-inverse peptides have the advantage of being non-self and as such can overcome self-tolerance problems in the immune system.

Alternatively, peptide mimotopes can be identified using antibodies that are themselves capable of binding epitopes disclosed herein, such as by 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 the native peptides, and thus are capable of binding antibodies against the native peptide, but do not necessarily exhibit significant sequence homology with the native peptide in themselves. This approach can have significant advantages in allowing the identification of a peptide with enhanced immunogenic properties, or can overcome any potential antigen self-tolerance problems that may be associated with the use of native peptide sequences. Additionally, this technique allows the identification of a recognition pattern for each native peptide in terms of its common chemical properties among the recognized mimotope sequences.

Covalent linkage of the peptide to an immunogenic carrier can be accomplished by known methods. For example, a carbodiimide, glutaraldehyde, or (N- [Y-maleimidobutyryloxy] succinimide) ester may be used to obtain a direct covalent linkage using commercially available heterodifunctional linkers such as CDAP and SPDP (using manufacturer's instructions). Following the conjugation reaction, the immunogen can be easily isolated and purified by dialysis, gel filtration, fractionation, etc.

The types of carriers used in the immunogens disclosed herein will be well known to those skilled in the art. The function of the carrier is to provide the assistance of cytokines to help induce an immune response against the peptide. A non-exhaustive list of carriers that may be used according to the disclosure includes: keyhole limpet hemocyanin (KLH), serum albumin such as bovine serum albumin (BSA), inactivated bacterial toxins such as tetanus or diphtheria toxins (TT and DT), or thereto. recombinant fragments (e.g. domain 1 of fragment C from TT or the translocation domain DT),

PL 209 127 B1 or purified tuberculin protein derivative (PPD). Alternatively, mimotopes or epitopes may be directly coupled to liposome carriers, which may additionally contain immunogens capable of delivering T-cell help.

Preferably, the ratio of mimotopes to carrier is from 1: 1 to 20: 1, with each carrier preferably carrying 3-15 peptides.

In one embodiment, a preferred carrier is Protein D from Haemophilus influenzae (EP 0 594 610 B1). Protein D is an IgD binding protein from Haemophilus influenzae and has been patented by Forsgren (WO 91/18926, EP 0 594 610 B1). Under certain conditions, e.g. in recombinant immunogen expression systems, it may be advantageous to use protein D fragments, e.g. protein D 1/3 (containing the N-terminal 100-110 amino acids of protein D (GB 9 717 953.5)).

Another preferred method of displaying the peptides disclosed herein uses a recombinant fusion molecule. For example, EP 0 421 635 B describes the use of chimeric hepadna virus core antigen particles to display foreign peptide sequences in a virus-like particle. As such, the immunogens disclosed herein may include peptides presented in chimeric particles composed of the hepatitis B core antigen. In addition, recombinant fusion proteins may include the mimotopes and carrier protein disclosed herein, such as influenza NS1. For any recombinantly expressed protein forming part of the disclosure, a nucleic acid that encodes that immunogen also forms an aspect of this disclosure.

The peptides used herein can be readily synthesized using well known solid phase procedures. Relevant syntheses can be carried out using the "T-boc" or "F-moc" procedures. Cyclic peptides can be synthesized by the solid-phase method using the well-known "F-moc" procedure and a polyamide resin in a fully automated device.

Alternatively, those skilled in the art will know the necessary laboratory procedures to perform 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, publ. IRL in Oxford University Press (1989).

Alternatively, peptides may be produced by recombinant methods, including by expressing nucleic acid molecules encoding mimotopes in a bacterial or mammalian cell line, and then purifying the expressed mimotope.

Techniques for expressing recombinant peptides and proteins are known and are described in Maniatis, T., Fritsch, EF and Sambrook et al. Molecular cloning: a laboratory manual, ed. 2, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

Another embodiment is a method of producing a polypeptide as described herein. The method disclosed herein may be performed using known recombinant techniques such as those described in Maniatis et al. Molecular Cloning - A Laboratory Manual; Cold Spring Harbor, 1982-1989. Thus, the method of producing a polypeptide disclosed herein comprises culturing the host cells under conditions sufficient to produce the polypeptide, and isolating the polypeptide from the culture medium. In particular, the method disclosed herein may advantageously include the steps of:

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

ii) transforming host cells with this vector;

iii) culturing said transformed host cells under conditions allowing expression of said DNA polymer to produce said protein, and iv) isolating said protein.

The polypeptides or immunogenic fragment disclosed herein may be in the form of a "mature" protein or may be part of a larger protein, such as a precursor or fusion protein. It is often preferable to include an additional amino acid sequence that includes secretory or leader sequences, pro-sequences, purification aid sequences, such as multiple histidine residues, or an additional sequence for stability during recombinant production. In addition, the addition of an exogenous polypeptide or lipid tail or polynucleotide sequence may also be considered to increase the immunogenic potential of the final molecule.

In one of its aspects, the disclosure relates to genetically engineered soluble fusion proteins comprising a polypeptide or fragment thereof as disclosed herein and various portions of the constant regions of heavy or light chains of immunoglobulins of various subgroups (IgG, IgM, IgA, IgE).

PL 209 127 B1

Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, especially IgG1, where the fusion occurs at the hinge region. In a particular embodiment, the Fc portion can be removed simply by including a cleavage sequence that can be cleaved by blood coagulation factor Xa. Furthermore, methods for producing these fusion proteins by genetic engineering and their use in drug discovery, diagnosis and therapy are disclosed. A particularly preferred aspect relates to the use of a polypeptide or polynucleotide in the manufacture of a vaccine for the immunotherapeutic treatment of a patient suffering from or susceptible to cancer, especially colon cancer or other cancers or diseases related to the colon. A further aspect relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Applications Publication Nos. WO 94/29458 and WO 94/22914.

The proteins can be chemically coupled or expressed as recombinant fusion proteins, which allows for the production of larger amounts of the product in an expression system as compared to the production of non-fusion proteins. The fusion partner may help to deliver T helper epitopes (immunological fusion partner), preferably T helper epitopes recognized in human organisms, or help to express the proteins (expression enhancer) with greater efficiency than that obtained with native recombinant proteins.

Preferably, the fusion partner will be both an immunological fusion partner and an expression enhancer.

The fusion partners include the Haemophilus influenzae B protein D and the nonstructural influenza virus protein, NS1 (haemagglutinin).

Another immunological fusion partner is the protein known as LYTA. Preferably the C-terminal part of the molecule is used. Lyta is derived from Streptococcus pneumoniae, which synthesizes N-acetyl-L-alanine amidase, LYTA amidase (encoded by the lytA gene) (Gene, 43 (1986) pp. 265-272), an autolysin that specifically breaks down certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity for choline or for certain choline analogs such as DEAE. This property has been used to develop E. coli C-LYTA expressing plasmids useful for the expression of fusion proteins. Purification of hybrid proteins containing a C-LYTA fragment at their N-terminus has been described (Biotechnology: 10 (1992) pp. 795-798). It is possible to use a repeat portion of a Lyta molecule occurring at the C-terminus, starting at residue 178, e.g. residues 188-305.

Also disclosed herein are xenogeneous forms (also called orthologous forms) of the above-mentioned polypeptides, which xenogeneic forms refer to an antigen having a significant degree of sequence identity with a human antigen (also called autologous antigen), which serves as a reference antigen but comes from a species other than human. In this context, "appreciable degree of identity" refers to the correspondence of an amino acid sequence with another amino acid sequence or a polynucleotide sequence with another polynucleotide sequence when such sequences match as closely as possible to any of a variety of known sequence comparison proteins. By a significant degree of identity is meant at least 70-95%, preferably at least 85-95% and most preferably at least 90-95% identity of the compared sequences. Thus, in accordance with the disclosure, the xenogeneic CASB7439 polypeptide will be CASB7439 which is xenogeneic to human CASB7439, i.e., is isolated from a non-human species. In a preferred embodiment, the polypeptide will be isolated from a mouse, rat, pig or rhesus monkey, most preferably a mouse or a rat. Thus, a method of inducing an immune response in a human against a human CASB7439 having the amino acid sequence as shown in any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 11 consists in administering to said patient an effective dose of an agent containing the xenogeneic form of said human CASB7439, as described herein. A preferred embodiment is a method of inducing an immune response against human CASB7439 using xenogeneic CASB7439 isolated from a mouse, rat, pig or rhesus monkey. Another preferred method of inducing an immune response is by using an antigen composition comprising a live viral expression system that expresses the xenogeneic antigen.

A preferred CASB7439 xenogeneic polypeptide has the sequence shown in SEQ ID NO: 12 (mouse) or in SEQ ID NO: 14 (rat).

In general, an isolated CASB7439 xenogeneic polypeptide will have significant sequence similarity and will contain isolated polypeptides having an amino acid sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least

PL 209 127 B1

90% identical, even more preferably at least 95% identical, and most preferably at least 97-99% identical to SEQ ID NO: 12 or SEQ ID NO: 14 over the entire length of SEQ ID NO: 12 or SEQ ID NO: 14. Thus, the xenogeneic polypeptide will comprise an immunogenic fragment of a polypeptide of SEQ ID NO: 12 or SEQ ID NO: 14, wherein the immunogenic activity of the immunogenic fragment is substantially the same as that of the polypeptide of SEQ ID NO: 12 or SEQ ID NO: 14. Furthermore, the xenogeneic CASB7439 polypeptide may be a fragment of at least about 20 contiguous amino acids, preferably about 30, more preferably about 50, even more preferably about 100, and most preferably about 150 contiguous amino acids selected from the amino acid sequences set forth in SEQ ID NO: 12 or SEQ ID NO: 14. In particular, the CASB7439 xenogeneic fragments will retain certain functional properties, preferably immunological activity, of the larger molecule shown in SEQ ID NO: 12 or SEQ ID NO: 14 and are useful in the methods described herein (e.g., pharmaceuticals and vaccines, diagnostics, etc. .). In particular, these fragments will be able to generate an immune response against the human counterpart, such as generating cross-reacting antibodies that react with the autologous human CASB7439 shown in any of SEQ ID NO: 2. In a particular embodiment, the xenogeneic polypeptide disclosed herein may be part of a larger fusion comprising the xenogeneic CASB7439 polypeptide or fragment thereof and a heterologous protein or protein portion acting as a fusion partner as described herein above.

Also disclosed herein are variants of the aforementioned polypeptides, that is, polypeptides that differ from the reference polypeptides by conservative amino acid substitution, where a given residue is substituted for another with similar characteristics. Typical substitutions are made 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 with aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted or added in any combination.

The polypeptides disclosed herein may be produced by any suitable means. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Methods for producing such polypeptides are well known.

In another aspect, the disclosure relates to CASB7439 polynucleotides. Such polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide that is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, and even more preferably at least 95% identical 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 for the entire length, respectively SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 11. In this regard, encoded polypeptides that are at least 97% identical are highly preferred, while those 98-99% identical are even more preferred and those that are at least 99% identical are most preferred.

Further polynucleotides disclosed herein include isolated polynucleotides having a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, and even more preferably at least 95% identical to the nucleotide sequence encoded by c A polypeptide with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 11 over the entire coding region. In this regard, polynucleotides that are at least 97% identical are highly preferred, 98-99% identical are more preferred, and those that are at least 99% identical are most preferred.

Further polynucleotides disclosed herein include isolated polynucleotides having a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, and even more preferably at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or 5 SEQ ID NO: 9 over the entire length of these sequences or with the coding sequence 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 entire length of this coding sequence 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. relative to polynucleotides that are at least 97% identical are highly preferred, those 98-99% identical are more preferred, and those that are at least 99% identical are most preferred. Such polynucleotides include a polynucleotide comprising the polynucleotide 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

PL 209 127 B1

ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9 or coding region 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 disclosed herein are nucleic acid encoding the aforementioned xenogeneic proteins and their uses in medicine. In a preferred embodiment, the xenogeneic CASB7439 polynucleotide for use in pharmaceuticals has the sequence shown in SEQ ID NO: 13 (mouse) or in SEQ ID NO: 15 (rat). The isolated CASB7439 xenogeneic polynucleotides disclosed herein may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. Additional coding or non-coding sequences may or may not be present in a polynucleotide disclosed herein. Other related embodiments provide polynucleotide variants having a significant degree of identity to the sequences disclosed herein in SEQ ID NO: 13 or in SEQ ID NO: 15, e.g. having at least 70% sequence identity, and preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity as compared to sequence of the polynucleotides disclosed herein, according to the methods described herein (e.g., BLAST analysis with standard parameters). In a related embodiment, an isolated xenogeneic polynucleotide disclosed herein will contain a nucleotide sequence encoding a polypeptide that is at least 90%, preferably 95% or greater identical to the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 14 over the entire length of SEQ. ID NO: 12 or SEQ ID NO: 14, or a nucleotide sequence complementary to that isolated polynucleotide.

Further disclosed herein are polynucleotides that are complementary to all of the above-described polynucleotides.

These polynucleotides can be inserted into a suitable plasmid, recombinant microorganism vector or recombinant live microorganism and used for immunization (see, e.g., 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)). Thus, an expression vector or a recombinant living microorganism containing these polynucleotides as defined above is provided.

Further provided herein is a fragment of a CASB7439 polynucleotide which, when administered to a patient, has the same immunogenic properties as the polynucleotide 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.

Further provided herein is a polynucleotide encoding an immunological fragment of the CASB7439 polypeptide as defined above.

These 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 the polypeptide sequence shown in SEQ ID NO: 2, 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 the polypeptide sequence encoded by the polynucleotide sequence shown in 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.

These polypeptide fragments of the invention preferably contain at least about 5, 10, 15, 20, 25, 50, or 100 or more contiguous amino acids, including all intermediate lengths of the 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 the polynucleotide sequence shown in 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.

The nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence which comprises a polypeptide coding sequence (nucleotides 545 to 1126) coding for a 193 amino acid polypeptide, i.e., SEQ ID NO: 2 polypeptide. The nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 may be identical. with the sequence encoding the polypeptide in SEQ ID NO: 1 or may be a sequence other than that contained in SEQ ID NO: 1, which, due to the redundancy (degeneracy) of the genetic code, also encodes a polypeptide with 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 homologue 2 (HASH2) gene, officially named human ASCL2 (from

Achaete Scute complex like 2) is a chronologist of the Achaete and Scute Drosophila genes. Human ASCL2 is only expressed on the extracellular trophoblasts of the developing placenta and maps to chromosome 11p15 near IGF2 and H19. The murine achaete scute homolog 2 (MASH2) gene encodes a transcription factor involved in trophoblast development. The Mash2 gene is paternally stigmatized

In mice, and the lack of expression of human ASCL2 in human benign acinar (androgenetic) molars indicates that human ASCL2 is also stigmatized in humans.

ASCL2 genes are members of the basic helix-loop-helix (BHLH) family of transcription factors. They activate transcription by binding to the E sequence (5'-CANNTG-3 '). Dimerization with other BHLH proteins is required for efficient DNA binding. They are also involved in the determination of neuronal precursors in the peripheral nervous system and the axis of 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 includes two other sequences encoding the polypeptides. The first sequence encoding a polypeptide (nucleotides 1184 to 399 in SEQ ID NO: 1, nucleotides 608 to 1393 in SEQ ID NO: 6) encodes a polypeptide with 262 amino acids, that is, the polypeptide of SEQ ID NO: 3. The second sequence encoding the polypeptide (nucleotides 840 to 262 in SEQ ID NO: 1, nucleotides 952 to 1530 in SEQ ID NO: 6) encodes a polypeptide with 193 amino acids, that is, the polypeptide of SEQ ID NO: 11. The nucleotide sequences encoding the SEQ ID NO: 3 and SEQ ID NO: 11 polypeptides may be identical to the polypeptides encoding the sequence contained in SEQ ID NO: 6, or it may be a sequence other than that contained in SEQ ID NO: 6, which as a result of redundancy (degeneration) ) of the genetic code also encodes SEQ ID NOS: 3 and 11 polypeptides. The SEQ ID NO: 3 polypeptide is structurally related to other proteins of the splicing coactivator family, having homology and / or structural similarity with the homo sapiens srm300 splicing coactivator subunit (gene bank accession number AAF21439). The polypeptide of SEQ ID NO: 11 is not related to any known protein. The polypeptide sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 11 and the polynucleotide sequences set forth in SEQ ID NO: 6 are novel and also form part of the disclosure.

Preferred polypeptides and polynucleotides disclosed herein are expected to have, inter alia, biological functions / properties similar to those of homologous polypeptides and polynucleotides. In addition, the preferred polypeptides, immunological fragments, and polynucleotides disclosed herein have at least one activity, respectively, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 11.

Further disclosed herein are partial or other incomplete polynucleotide and polypeptide sequences which were identified prior to the determination of the corresponding full length sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 11. Further provided herein is a polypeptide, which:

(a) has an amino acid sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical, and most preferably at least 97-99% identical identical to SEQ ID NO: 2 over the entire length of SEQ ID NO: 2;

(b) has an amino acid sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical, and most preferably at least 97-99% identical. identical to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2; and (c) comprises the amino acid sequence of SEQ ID NO: 2.

The polynucleotides disclosed herein can be prepared using standard cloning and screening techniques from a cDNA library derived from mRNA from human colon cancer cells (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The polynucleotides disclosed herein may also be obtained from natural sources, such as genomic DNA libraries, or synthesized using techniques known and commercially available.

When the polynucleotides disclosed herein are used to recombinantly produce the polypeptides disclosed herein, the polynucleotide may include the mature polypeptide coding sequence as such or the mature polypeptide coding sequence in frame with other coding sequences, such as those encoding a leader or a secretory sequence, a pre-sequence , pro or prepro proteins, or other portions of the fusion peptides. For example, one can encode a marker sequence that facilitates purification of the fusion polypeptide.

In some preferred embodiments of this aspect, the marker sequence is a hexahistidine peptide, such as provided in the pQE vector (Quiagen, Inc.) and described in Gent et al., Proc. Natl. Acad. Sci., USA (1989) 86: 821-824 or HA tag. The polynucleotide may also contain 5 'and 3' non-coding sequences, such as transcribed, untranslated sequences, splicing and polyadenylation signals, ribosome binding sites, and mRNA stabilizing sequences.

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Further embodiments include polynucleotides encoding variant polypeptides that 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, e.g., 5 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 are substituted, deleted, or added, in any combination.

Polynucleotides that are identical or sufficiently identical to the nucleotide sequence contained in SEQ ID NO: 1 or SEQ ID NO: 6, can be used as probes to hybridize to cDNA and genomic DNA, or as primers for nucleic acid amplification (PCR) reactions, to isolate full-length cDNA and genomic clones encoding the polypeptides disclosed herein, and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from non-human species) which 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, and most preferably 95% identical to the reference sequence. Probes or primers will generally be at least 15 nucleotides, and preferably at least 30 nucleotides, and may be at least 50 nucleotides. Particularly preferred probes will contain 30-50 nucleotides. Particularly preferred primers will contain 20-25 nucleotides. In particular, polypeptides and polynucleotides derived from sequences derived from a homologous animal could be used as immunogens leading to a cross immune response to the human gene.

A polynucleotide encoding a polypeptide as disclosed herein, including homologues from non-human species, can be produced by a method comprising the steps of screening a suitable 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 and isolating the clones. full-length and genomic cDNAs containing this polynucleotide sequence. Such hybridization techniques are well known to those skilled in the art. Preferred stringent hybridization conditions include overnight incubation at 42 ° C in a solution containing: 50% formamide, 5 x SSC (150 mM NaCl, 15 mM sodium tricitrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate and 20 µg / ml denatured fragmented DNA from salmon sperm; followed by rinsing the filters in 0.1 x SSC at about 65 ° C. Thus, the disclosure further includes polynucleotides obtainable by screening a suitable 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 skilled in the art will appreciate that in many instances the isolated cDNA sequence will be incomplete and that the polypeptide coding region is truncated at the 5 'end of the cDNA.

There are several methods available and well known to those skilled in the art to obtain full-length cDNAs or to extend short cDNAs, e.g., methods based on the rapid amplification of cDNA ends (RACE) (see, e.g., Frohman et al., PNAS USA 85, 8998-9002, 1988). Recent modifications to this technique, such as the Marathon ™ technology (Clontech Laboratories Inc.), have greatly simplified the search for longer cDNAs. In the Marathon ™ technology, cDNA is obtained from mRNA extracted from the selected tissue and an "adapter" sequence is ligated to each end. Nucleic acid amplification (PCR) is then performed to amplify the "missing" end of the cDNA using a combination of gene and adapter specific oligonucleotide primers. The PCR reaction is then repeated using "nested" primers, that is, primers designed to hybridize to the amplified product (usually an adapter-specific primer that hybridizes further 3 'in the adapter sequence and a gene-specific primer that hybridizes further to the amplified product). 5 'side in a known gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA can be constructed either by attaching the product directly to an existing cDNA to form the complete sequence, or by performing a separate full-length PCR process using the new sequence information to design the 5 'primer.

The recombinant polypeptides disclosed herein can be produced by well-known methods from genetically engineered host cells containing expression systems. Thus, in a further aspect, the disclosure relates to an expression system that comprises a polynucleotide as disclosed herein, engineered host cells with such expression systems, and a method of producing the polypeptides disclosed herein using recombinant techniques. Cell-free translation systems can also be used to produce such proteins, using RNA obtained from the DNA constructs disclosed herein.

To carry out the production process by recombinant techniques, host cells can be obtained by genetic engineering techniques which contain the expression systems of the polynucleotides or portions thereof disclosed herein. Introduction of polynucleotides into host cells can be accomplished by methods 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, 5th ed. 2, Cold Spring Harber Laboratory Press, Cold Spring Harber, NY (1989). Preferred methods include, eg, calcium phosphate transfection, DEAE-dextran transfection, transvection, microinjection, cationic lipid transfection, electroporation, transduction, scrape loading, ballistic introduction, and infection.

Preferably, the proteins disclosed herein are co-expressed with trioredoxin in trans (TTT). Co-expression of thioredoxin in trans as opposed to cis is beneficial in keeping the antigen free of thioredoxin without the need for a protease. Co-expression of thioredoxin facilitates solubilization of the proteins disclosed herein. The co-expression of thioredoxin also has a significant influence on the protein purification efficiency, solubility of the purified protein and quality.

Representative examples of suitable hosts include bacterial cells such as Streptococcus, Staphylococcus, E. coli, Streptomyces, and Bacillus subtilis cells; fungal cells such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells and plant cells.

A wide variety of expression systems can be used, e.g., chromosomal, episomal, and viral-derived systems, e.g. vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, pap viruses such as SV40, vaccinia virus, adenoviruses, avipox virus, pseudorabies virus and retrovirs, and vectors derived from combinations thereof, such as those derived from the genetic elements of plasmids and bacteriophages, such as cosmids and phagemids. Expression systems can contain control regions that regulate as well as trigger expression. In general, any system or vector that is capable of maintaining, propagating, or expressing a polynucleotide to produce a polypeptide in a host may be used. The appropriate nucleotide sequence can be inserted into the expression system using any of a variety of well-known and routine techniques, such as e.g. the techniques presented in Sambrook et al., Molecular Cloning, A Laboratory Manual (supra). Appropriate secretion signals can be incorporated into the desired polypeptide, which allows the secreted protein to be secreted 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 recombinant living microorganism such as a virus or bacteria. The gene of interest can be inserted into the genome of a live recombinant virus or bacteria. Inoculation and in vivo infection with this live vector will lead to in vivo expression of the antigen and induction of immune responses.

Thus, in certain embodiments, polynucleotides encoding the disclosed immunogenic polypeptides are introduced into appropriate mammalian hosts for expression using any of a variety of known virus-based systems. For example, retroviruses provide a convenient and effective basis for gene delivery systems. The selected nucleotide sequence encoding the disclosed polypeptide can be inserted into the vector and packaged into retroviral particles using known techniques. The recombinant virus can then be isolated and administered to the patient. A number of exemplary retroviral systems have been described (e.g., US 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: 849-852; Burns et al (1993) Proc. Natl. Acad. Sci. USA 90: 8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3: 102-109 .

Additionally, a number of exemplary adenovirus-based systems have been described. Unlike retroviruses which integrate into the host genome, adenoviruses remain outside the chromosome, which minimizes the risks of insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57: 267-274; Bett et al. (1993) J Virol. 67: 5911-5921; 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, KL (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. AAV vectors are readily constructed using known techniques, see, e.g., US No. 5,173,414 and UD No. 5,139,941; 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); Carter, BJ (1992) Current

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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: 1867-1875.

Additional vectors useful for the production of nucleic acid molecules encoding the polypeptides disclosed herein by gene transfer include those derived from the pox virus family such as vaccinia virus and the avipox virus family. For example, vaccinia virus recombinants expressing new molecules can be constructed as follows. The DNA encoding the polypeptide is first inserted into an appropriate vector to adhere to the vaccinia promoter and flanking vaccinia DNA sequences, such as the thymidine kinase (TK) encoding sequence. This vector is then used to transfect cells that are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter and the gene encoding the polypeptide of interest into the viral genome. The resulting recombinant TK. sup (-) can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and selecting virus-resistant plaques.

A vaccinia-based infection / transfection system can conveniently be used to provide inducible transient expression or co-expression of one or more of the polypeptides described herein in cells of a host organism. In this particular system, cells are first infected in vitro with a recombinant vaccinia virus that encodes bacteriophage T7 RNA polymerase. This polymerase exhibits exceptional specificity as it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide or polynucleotides of interest, driven by the T7 promoter. The cytoplasmic polymerase from recombinant vaccinia virus transcribes the transfected DNA into RNA, which is then translated into a polypeptide by the host's translational machinery. The method enables high levels of transient cytoplasmic production of large amounts of RNA and its translation products, see, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87: 6763-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986) 83: 8122-8126.

Alternatively, avipox viruses such as fowlpox and canarypox viruses can also be used to provide coding sequences of interest. Recombinant avipox expressing immunogens from mammalian pathogens is known to confer protective immunity when administered to non-avian species. The use of the avipox vector is particularly advantageous in humans and other mammalian species as members of the genus Avipox can only reproduce productively in susceptible bird species and are therefore not infectious in mammalian cells. Methods for producing recombinant avipox viruses are known and use the genetic recomination described above for the production of vaccinia viruses, see e.g. WO 91/12882; WO 89/03429 and WO 92/03545.

Any of a variety of alphavirus vectors may also be used to prepare the polynucleotide compositions disclosed herein, e.g., the vectors described in US No. 5,843,723, US No. 6,015,686, US No. 6,008,035, and US No. 6,015,694. for Venezuelan equine meningitis virus (VEE), see, e.g., US No. 5,505,947 and US No. 5,643,576.

In addition, molecular conjugate vectors may also be used to deliver the genes of the disclosure, such as the chimeric adenoviral vectors described in Michael et al. J. Biol. Chem. (1993) 268: 6866-6869 and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89: 6099-6103.

Additional information on these and other virus-based 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; U.S. No. 4,603,112, U.S. No. 4,769,330 and U.S. No. 5,017,487; WO 89/01973; No. 4,777,127; GB No. 2,200,651; EP 0 345 242; WO 91/02805; Berkner, Biotechniques 6: 616-627, 1988; Resenfeld et al., Science 252: 431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91: 215-219,1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90: 11498-11502,1993; 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 to obtain live vaccines. Such live vaccines also form part of the disclosure.

In some embodiments of the invention, the polynucleotide may be integrated into the genome of the target cell. This integration can be performed at a specific location and orientation by homologous recombination (gene replacement) or it can be performed at a random, non-specific site (gene addition). In still other embodiments of the invention, the polynucleotide may be stably maintained in the cell as a discrete episomal stretch of DNA. Such polynucleotide segments or "episomes" encode sequences sufficient to maintain and replicate independent of the host cell cycle or synchronized with the host cell cycle. The method of delivering an expression construct into a cell and the location in the cell where the polynucleotide will remain are dependent on the type of expression construct used.

In another embodiment of the invention, the polynucleotide is administered / delivered as "naked" DNA, eg, as described in Ulmer et al., Science 259: 1745-1749, 1993 and reviewed by Cohen, Science 259: 1691-1692, 1993. The DNA can be increased by coating the DNA with biodegradable beads that are efficiently transported into the cells.

In yet another embodiment of the invention, the agent of the invention may be provided by particle bombardment techniques, many of which have already been described. For example, gas driven particle acceleration can be achieved using equipment such as those manufactured by Powderjet Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc. (Madison, WI), and some examples are described in US No. 5,846,796, US No. 6,010,478, US No. 5,865,796, US No. 5,584,807 and EP 0 500 799. This allows for syringe-free administration as a microscopic particle size dry powder formulation, such as polynucleotide or polypeptide particles, is accelerated to high velocity by the helium gas stream generated by the hand held device introducing the particles into the target tissue.

In a related embodiment of the invention, other devices and methods are employed which are useful for the gas-guided needleless injection of an agent of the invention, e.g., provided by Bioject, Inc. (Portland, OR), and some examples are described in U.S. No. 4,790,824, U.S. No. 5,064,413, U.S. No. 5,312,335, U.S. No. 5,383,851, U.S. No. 5,399,163, U.S. No. 5 520,639 and U.S. No. 5 993 412.

The polypeptides disclosed herein can be isolated from recombinant cell cultures and purified by known methods, e.g. . Most preferably, metal ion affinity chromatography (IMAG) is used for purification. Well known protein refolding techniques can be used to regenerate the active conformation when the polypeptide is denatured during intracellular synthesis, isolation, or purification.

Another important aspect of the disclosure relates to a method of inducing, enhancing, or modulating an immune response in a mammal, which comprises vaccinating the mammal with a fragment or all of a polypeptide or polynucleotide disclosed herein sufficient to generate an antibody and / or T cell immune response for the immunoprophylaxis or therapeutic treatment of cancer, and in particular, colon and rectal cancer, autoimmune diseases and related conditions. Yet another aspect relates to a method of inducing, enhancing, or modulating an immune response in a mammal which comprises delivering a polypeptide as disclosed herein through a vector or cell controlling the expression of the polynucleotide and encoding the polypeptide in vivo to elicit such an immune response to generate immune responses for prophylaxis or therapy diseases in such a mammal.

A further aspect of the invention relates to an immunological / vaccine preparation (composition) and its use in medicine. When introduced into a mammalian host, these agents induce, enhance, or modulate an immune response in that mammal to a polypeptide disclosed herein, wherein the agent comprises a predefined polypeptide or polynucleotide or an immunological fragment thereof. In particular, the immunogenic agent of the invention comprises a safe and effective amount of a CASB7439 polypeptide or an immunogenic fragment thereof, wherein the CASB7439 polypeptide is selected from the group consisting 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. In another embodiment of the invention, the immunogenic agent comprises a safe and effective amount of a CASB7439 encoding polynucleotide or fragment thereof, wherein the CASB7439 encoding polynucleotide is selected from the group consisting 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.

The vaccine formulation according to the invention may furthermore contain a suitable, i.e., pharmaceutically acceptable carrier. Since the polypeptide can be degraded in the stomach, it is preferably administered parenterally (e.g., by subcutaneous, intramuscular, intravenous or intradermal injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, bacteriostats, and dissolved compounds that render the formulation isotonic with the patient's blood, as well as aqueous and non-aqueous sterile suspensions which may include suspending or thickening agents. Preparations can be placed in containers,

In single or multiple dose forms, for example, sealed ampoules and vials, and can be stored in a lyophilized form requiring only the addition of a sterile liquid carrier immediately prior to use.

A further aspect of the disclosure relates to inducing in vitro immune responses to a fragment or all of a polypeptide or polynucleotide or molecule comprising a polypeptide or polynucleotide disclosed herein, using cells from the immune system of a mammal and reintroducing these activated mammalian immune cells to treat a disease. Activation of cells from the immune system is achieved by in vitro incubation with the entire polypeptide or polynucleotide disclosed herein, or with a molecule containing a polypeptide or polynucleotide as disclosed herein, in the presence or absence of various immunomodulatory molecules. A further aspect relates to immunizing a mammal by administering antigen presenting cells modified by in vitro loading with some or all of a polypeptide or molecule containing a polypeptide disclosed herein and administered in vivo in an immunogenic manner.

Alternatively, antigen presenting cells may be transfected in vitro with a vector containing a fragment or all of a polynucleotide disclosed herein, or a molecule containing a polynucleotide disclosed herein so as to express the appropriate polypeptide, and administered in vivo in an immunogenic manner. Thus, the pharmaceutical compositions of the invention will contain an effective amount of antigen presenting cells modified by in vitro loading with CASB7439 polypeptide or genetically modified in vitro to express CASB7439 polypeptide and a pharmaceutically effective carrier.

In accordance with another embodiment of the invention, the pharmaceutical / immunogenic agents described herein will contain one or more immunostimulants in addition to the immunogenic polynucleotide, polypeptide, antibody, T-cells, and / or antigen presenting cells (APCs) of the invention. Thus, the method of producing this immunogenic agent comprises mixing the CASB7439 polypeptide or the CASB7439 encoding polynucleotide with a suitable adjuvant / immunostimulant, diluent or other pharmaceutically acceptable carrier. An immunostimulant is essentially any substance that enhances or enables an immune response (mediated by antibodies and / or cells) to an exogenous antigen.

A preferred type of immunostimulant includes an adjuvant. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulant of immune responses, such as lipid A, proteins derived from Bortadella pertussis or Mycobacterium tuberculosis. Certain adjuvants are commercially available, e.g. Incomplete Freund's Adjuvant and Complete Freund's Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ), AS-2 (SmithKline Beecham, Philadelphia, PA), aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate, calcium, iron or zinc salts, acylated tyrosine insoluble suspension, acylated sugars, cationic or anionic substituted polysaccharides, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A and quil A. Cytokines such as GM-CSF, interleukin-2, -7, -12, and other growth factors can also be used as adjuvants.

According to some embodiments, it is preferable to use an adjuvant composition that induces an immune response, primarily of the Th1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TN-Fa, IL-2 and IL-12) may promote the induction of cell mediated immune responses to an administered antigen. Conversely, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) may favor the induction of humoral immune responses. After vaccination, the patient will maintain an immune response that includes both Th1 and Th2 responses. According to a preferred embodiment, the response is primarily a Th1 type response and the level of Th1 type cytokines will increase to a greater extent than the level of Th2 type cytokines. The levels of these cytokines can be readily determined using standard assays. For a review of the cytokine families, see Mosmann and Coffman, Ann. Rev. Immunol. 7: 145-173,1989.

Certain adjuvants suitable for inducing a Th1-type response primarily include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, with an aluminum salt. MPL adjuvants^® are available from Corixa Corporation (Seattle, WA; see, e.g., US No. 4,436,727, US No. 4,877,611, US No. 4,866,034 and US No. 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described e.g. in WG 96/02555, WG 99/33488, US No. 6,008,200 and US No. 5,856,462. Immunostimulatory DNA sequences are described e.g. in Sato et al., Science 273: 352.

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1996. Another preferred adjuvant includes a saponin such as Quil A or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Farmingham, MA), escin, digitonin, or Gypsophila saponins or

Chenopodium guinoa. Other preferred formulations include more than one saponin in the adjuvant combinations disclosed herein, e.g., combinations of at least two saponins selected from the group consisting of QS21, QS7, Quil A, β-escin, and digitonin.

Alternatively, saponin preparations can be combined with vaccine carriers composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, a polymer matrix based on poly-N-acetyl-glucosamine, particles composed of polysaccharides or chemically modified polysaccharides and liposomal particles, lipid-based particles composed of glycerol monoesters etc. Saponins can also be formulated in the presence of cholesterol to form partial structures such as liposomes or ISCOMs. Furthermore, saponins may be formulated with a polyoxyethylene ether or ester, in solution or suspension with or without particles, or in a partial structure such as a multilayer liposome or an ISCOM. Saponins can also be formulated with excipients such as Carbopol^R, to increase the viscosity, or in the form of a dry powder with a powder excipient such as lactose.

In one preferred embodiment, the adjuvant system comprises a combination of monophosphoryl lipid A and a saponin derivative such as the composition QS21 and a 3D-MPL adjuvant.^® described in WO 94/00153 or less of a reactogenic composition wherein QS21 is quenched with cholesterol as described in WO 96/33739. Other preferred formulations include an oil in water emulsion and tocopherol. Another particularly preferred adjuvant formulation comprising QS21, a 3D-MPL adjuvant^® and tocopherol in the form of an oil in water emulsion is described in WO 95/17210.

Another enhanced adjuvant system comprises a combination of a CpG containing oligonucleotide and a saponin derivative, especially the combination of CpG and QS21 disclosed in WO 00/09159. A preferred formulation further comprises an oil in water emulsion and tocopherol.

Additional exemplary adjuvants for use in the pharmaceutical compositions of the invention include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, USA), ISCOMS (CSL), MF-59 (Chiron), a series of SBAS adjuvants (e.g. -2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Enhazyn^®) (Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkylglucosaminide-4-phosphates (AGP) such as those described in U.S. Patent Applications Serial Numbers 08/853826 and 09/074720 , the disclosures of which are incorporated in their entirety herein as references and polyoxyethylene ether type adjuvants such as those described in WO 99 / 52549A1.

Other preferred adjuvants include adjuvant molecules of general formula (I): HO (CH2CH2O) nAR where n is 1-50, A is a bond or -C (O) - and R is C1-50-alkyl or phenyl-C1- 50-alkyl.

One embodiment of the invention is a vaccine formulation comprising a polyoxyethylene ether of general formula (I), wherein n is 1-50, preferably 4-24, and most preferably 9; R is C1-50-alkyl, preferably C4-20-alkyl, most preferably C12-alkyl, and A is a bond. The concentration of polyoxyethylene ethers should be 0.1-20%, preferably 0.1-10% and most preferably 0.1-1%. Preferred polyoxyethylene ethers are selected from the group consisting of polyoxyethylene 9-lauryl ether, polyoxetylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene 35-lauryl ether and polyoxyethylene 23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Mercx Index (12th Ed., Entry 7717). These adjuvant molecules are described in WO 99/52549.

The polyoxyethylene ether of the above general formula (I) may, if desired, be combined with another adjuvant. A preferred combination of adjuvants comprises CpG as described in pending British Application No. GB ​​9 820 956.2.

Preferably, a carrier is also present in the vaccine formulation of the invention. The carrier can be an oil-in-water emulsion or an aluminum salt such as aluminum phosphate or aluminum hydroxide.

A preferred oil-in-water emulsion comprises a metabolizable oil such as squalene, α-tocopherol and Tween 80. Particularly preferably in such an emulsion, the vaccine antigens of the invention are combined with QS21 and 3D-MPL. In addition, the oil-in-water emulsion may contain Span 85 and / or lecithin and / or tricapryline.

Typically, when administered to humans, QS21 and 3D-MPL will be present in the vaccine in an amount of 1-200 µg, eg 10-100 µg, and preferably 10-50 µg per dose. Typically oil in water will contain 2-10% squaly16

% Α-tocopherol, and 0.3-3% Tween 80. Preferably, the ratio of squalene: α-tocopherol is 1 or less than 1, resulting in a more stable emulsion. Span 85 may also be present at a concentration of 1%. In some cases it may be advantageous that the vaccines of the invention also contain a stabilizer.

Non-toxic oil-in-water emulsions preferably contain a non-toxic oil, e.g. squalane or squalene, and an emulsifier, e.g. Tween 80, in an aqueous carrier. The aqueous carrier can be, for example, a phosphate buffered saline solution.

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

Further provided is a polyvalent vaccine composition comprising the vaccine formulation according to the invention in combination with other antigens, especially antigens useful in the treatment of cancer, especially colorectal cancer, autoimmune diseases and conditions associated therewith. Such a polyvalent vaccine composition may include a TH-1 inducing adjuvant as previously described.

According to another embodiment of the invention, the immunogenic agent described herein is administered to the host by antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes, and other cells that can be engineered to be effective APCs. Such cells may, but need not be, genetically modified to increase antigen-presenting capacity, to improve activation and / or maintenance of T cell responses, to have anti-tumor activity as such and / or to be immunologically compatible with the recipient (HLA matched haplotype). APCs can generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumor tissues, and can be autologous, allogeneic, syngeneic, or xenogeneic cells.

In some preferred embodiments, dendritic cells or their precursors are used as antigen presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392: 245-251, 1998) and have been shown to be effective as a physiological adjuvant for inducing prophylactic or therapeutic immunity against cancer (see Timmerman and Levy, Ann. Rev. Med. 50: 507-529,1999). In general, dendritic cells can be identified by their typical shape (stellate in situ, with prominent protrusions (dendrites) visible in vitro), their ability to pick up, process and present antigens with high efficiency, and their ability to activate a response. naive T cells Of course, dendritic cells can be engineered to express cell-specific cell surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are within the scope of the disclosure. As an alternative to dendritic cells, isolated vesicles from antigen-loaded dendritic cells (called exosomes) can be used in the vaccine (see Zitvogel et al., Nature Med. 4: 594-600, 1998).

Dendritic cells and precursors can be obtained from peripheral blood, bone marrow, tumor infiltrating cells, cells infiltrating tissues surrounding the tumor, lymph nodes, spleen, skin, umbilical cord blood, or any other suitable tissue or fluid. For example, dendritic cells can be differentiated ex vivo by adding combinations of cytokines such as GM-CSF, IL-4, IL-13, and / or TNF? To the culture of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood, or bone marrow can be differentiated into dendritic cells by adding a combination of GM-CSF, IL-3, TNF?, CD40 ligand, LPS, fit3 ligand and / or other compound to the culture medium ( other compounds) that induces the differentiation, maturation and proliferation of dendritic cells.

Dendritic cells are classified into the categories of "immature" and "mature" cells, which allows a simple distinction between two well-characterized phenotypes. However, this nomenclature should not be considered as excluding all possible intermediate states of differentiation. Immature dendritic cells are characterized as APCs with high antigen uptake and processing capacity, which correlates with high expression of the Fcy receptor and the mannose receptor. The mature phenotype is typically characterized by lower expression of these markers, but high expression of cell surface molecules responsible for T cell activation such as MHC class I and II, adhesion molecules (e.g. CD54 and CD11) and co-stimulatory molecules (e.g. CD40, CD80 , CD86 and 4-1BB).

The APCs can generally be transfected with a polynucleotide (or a portion or other variant thereof) disclosed herein such that the encoded polypeptide, or immunogenic portion thereof, is expressed on

Cell surface. Such transfection may occur ex vivo, and the pharmaceutical agent containing such transfected cells may then be used for the therapeutic purposes described herein. Alternatively, a gene delivery vehicle that targets a dendritic cell or other antigen-presenting cell can be administered to a patient to effect in vivo transfection. For example, in vivo and ex vivo transfection of dendritic cells can be performed using any known methods such as those described in WO 97/24447 or the gene gun method described by Mahvi et al., Immunology and Cell Biology 75: 456-460, 1997. Dendritic cell antigen loading may be achieved by incubating dendritic cells or progenitor cells with a tumor polypeptide, DNA (naked or within a plasmid vector) or RNA, or with recombinant bacteria or viruses expressing the antigen (e.g. vaccinia, avipox, adenoviral or lentiviral vectors) . Prior to loading, the polypeptide may be convalently coupled to an immune partner that provides T-cell help (e.g. carrier molecule). Alternatively, the dendritic cell may be pulsed with an unconjugated immune partner, either alone or in the presence of the polypeptide.

Any carrier known to those skilled in the art can be used in the pharmaceutical compositions of the invention, however, typically the type of carrier will vary with the mode of administration. The agents of the invention may be formulated for any suitable mode of administration, including, for example, topical, oral, intranasal, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous, and intramuscular administration.

Carriers for use in such pharmaceuticals are biocompatible and may also be biodegradable. In some embodiments, the formulation preferably provides a relatively constant level of active ingredient release. In other embodiments, however, a faster release rate immediately after administration may be desirable. It is within the ordinary skill to formulate such agents and can be carried out using known techniques. Examples of useful carriers include microparticles of poly (lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran, and the like. Other exemplary delayed release carriers include supramolecular biovectors that contain a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide). ) and optionally an outer layer containing an amphiphile such as a phospholipid (see e.g. US No. 5,151,254, WO 94/20078, WO 94/23701 and WO 96/06638). The amount of active ingredient contained in the sustained release formulation depends on the site of the implant, the rate and duration of release expected, and the nature of the condition to be treated or prevented.

In another exemplary embodiment, biodegradable microspheres (e.g., polylactate or polyglycolate) are used as carriers for the agent of the invention. Suitable biodegradable microspheres are disclosed e.g. in U.S. No. 4,897,268, U.S. No. 5,075,109, U.S. No. 5,928,647, U.S. No. 5,811,128, U.S. No. 5,820,883, U.S. No. 5,853,763, U.S. No. 5,814,344, U.S. No. 5,407,609 and US No. 5,942,252. Modified hepatitis B core protein carrier systems described in WO / 99 4 0934 and the literature cited therein can be useful in many applications. Other exemplary delivery / delivery systems are in the form of particulate protein complexes such as those described in US 5,928,647, which are capable of causing a class I restricted cytotoxic T cell response in the host.

The pharmaceutical compositions of the invention will often further contain one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g. glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g. aluminum hydroxide), dissolved compounds that render the preparation isotonic, hypotonic or slightly hypertonic to the blood of the recipient, suspending agents, thickening agents and / or preservatives. Alternatively, the agents of the invention may be formulated as a lyophilisate.

The pharmaceuticals described herein can be presented in single-dose or multi-dose containers such as sealed ampoules or vials. Such containers are typically sealed to maintain the sterility and shelf life of the formulation until use. Generally, formulations can be stored as suspensions, solutions, or emulsions in oily or aqueous vehicles. Alternatively, the pharmaceuticals may be stored in a lyophilized state requiring the addition of a sterile liquid carrier just prior to use.

Methods of developing appropriate dosing and therapy regimens for the use of the particular agents described herein, including oral, parenteral, intravenous,

Intranasal and intramuscular formulation and formulation methods are well known, some of which are discussed briefly below for illustration.

In some applications, the pharmaceuticals disclosed herein can be administered orally to animals. As such, these compositions may be formulated with an inert diluent or an digestible, edible carrier, or enclosed in a hard or soft gelatin capsule, or compressed into tablets, or incorporated directly into foodstuffs.

Active substances can be combined with excipients and used in the form of edible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers etc. (see e.g. Mathiowitz et al., Nature 1997 Mar 27; 386 (6623): 410-4 ; Hwang et al., Crit. Rev. Ther. Drug Carrier Syst. 1998; 15 (3): 243-84; US No. 5,641,515; US No. 5,580,579 and US No. 5,792,451). The tablets, troches, pills, capsules and the like may also contain any of a variety of additional ingredients, e.g. a binder such as gum tragacanth, acacia, corn starch or gelatin, excipients such as dicalcium phosphate, a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate, and a sweetening agent such as sucrose, lactose, or saccharin, or a flavoring agent such as mint, wintergreen oil or cherry flavor. When the dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials can be used as coatings or to otherwise modify the physical form of the dosage form. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, any materials used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, the active compounds can be incorporated into sustained release preparations.

Typically, these formulations will contain at least about 0.1% or more active ingredient, although the percentage of active ingredient (s) may, of course, vary from about 1-2% to about 60% or 70% or more of the active ingredient (s). on the total weight or volume of the preparation. Of course, the amount of active ingredient in any given therapeutically useful agent may be selected such that a suitable dosage form is contained in a given dosage form. One skilled in the art of preparing pharmaceutical formulations will take into account factors such as solubility, bioavailability, biological half-life, route of administration, product shelf-life, and other factors of importance in pharmacy, and various dosages and modes of administration may be desirable.

The compositions of the invention for oral administration may contain one or more excipients and be in the form of a mouth rinse, dentifrice, buccal tablet, mouth spray or sublingual preparation. Alternatively, the active ingredient may be incorporated into an oral solution, such as a solution containing sodium borate, glycerin, and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically effective amount to a composition which may contain water, binders, abrasives, flavors, foaming agents, and moisturizers. Alternatively, the agent may be formulated into a tablet or solution to be placed under the tongue or otherwise dissolve in the mouth.

Under certain conditions, it will be desirable to administer the pharmaceuticals described herein parenterally, intravenously, intramuscularly, or even intraperitoneally. Such approaches are well known to those skilled in the art, some of which are described, for example, in US No. 5,543,158, US No. 5,641,515 and US No. 5,399,363. In some embodiments, a solution of the active ingredient in the form of free bases or pharmaceutically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be made in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations will generally contain a preservative to prevent the growth of microorganisms.

Exemplary injectable pharmaceutical forms include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions (see, e.g., US No. 5,466,468). In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must contain preservatives against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Adequate fluidity can be maintained, e.g. by the use of a coating such as lecithin, by the maintenance of the desired particle size in the case of dispersion and / or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by the use of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, isotonic agents, e.g., sugars or sodium chloride, are preferably added. Prolonged absorption of injectable agents may be brought about by the use of agents delaying absorption, e.g., aluminum monostearate and gelatin.

In one embodiment, the aqueous solutions for parenteral administration should be suitably buffered if necessary and the liquid diluent made isotonic with sufficient saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Suitable sterile aqueous media are known to those skilled in the art. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml subcutaneous infusion fluid or injected at the proposed infusion site (see, eg, "Remington's Pharmaceutical Sciences" 15th edition, pp. 1035-1038 and 1570- 1580). Certain dose adjustments will necessarily be made depending on the condition of the patient being treated. Furthermore, formulations for human administration will, of course, meet the standards of sterility, pyrogenicity, and general safety and purity required by the FDA Office of Biological Standards.

In another embodiment, the agents disclosed herein may be formulated as neutral or salt forms. Examples of pharmaceutically acceptable salts are acid addition salts (formed with the free amino groups of the protein) obtained from inorganic acids such as e.g. hydrochloric acid or phosphoric acid, or from organic acids such as acetic, oxalic, tartaric, mandelic and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as e.g. sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. Once formulated, solutions will be administered in a consistent manner in the form administered and in a therapeutically effective amount.

Carriers can also be any and all solvents, dispersion media, bases, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents with pharmaceutically active substances is generally known. Any media and means that are compatible with the active ingredient can be used in pharmaceuticals. Additional active ingredients can also be incorporated into the compositions. The term "pharmaceutically acceptable" refers to molecules and agents that do not produce an allergic or other untoward reaction when administered to a human.

In some embodiments, the pharmaceutical compositions can be administered by nasal spray, inhalation, and / or other aerosol carriers. Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via intranasal aerosol sprays are described, for example, in US 5,756,353 and US 5,804,212. Similarly, drug delivery using microparticle nasal resins (Takenaga et al. , J. Controlled Release 1998 Mar 2: 52 (1-2): 81-7) and lysophosphatidyl glycerol compounds (US 5,725,871) is also well known in the pharmaceutical field. An example of transmucosal drug delivery using a polytetrafluoroethylene carrier matrix is ​​described in US 5,780,045.

In some embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles, and the like are used to introduce an agent of the invention into appropriate host cells / organisms. In particular, the agents of the invention may be formulated by encapsulation in lipid particles, liposomes, vesicles, nanospheres or nanoparticles, etc. Alternatively, the agents of the invention may be bonded, covalently or non-covalently, to the surface of such carrier substrates.

The preparation and use of liposomes and liposome-like preparations as potential drug carriers is known to those skilled in the art (see, e.g., Lasic, Trends Biotechnol., Jul 1998; 16 (7): 307-21; Takakura, Nippon Rinsho, March 1998; 56 ( 3): 691-5; Chandran et al., Indian J. Exp. Biol., Aug 1997; 35 (8): 801-9; Margalit, Crit. Rev. Ther. Drug Carrier Syst. 1995; 12 ( 2-3); 233-61; U.S. No. 5,567,434; U.S. No. 5,552,157; ​​U.S. No. 5,565,213; U.S. No. 5,738,868 and U.S. No. 5,795,587.

PL 209 127 B1

Liposomes have been used successfully with many cell types that are normally difficult to transfect using other procedures, including T-cell suspensions, primary hepatocyte cultures, and PC12 cells (Renneisen et al., J. Biol. Chem., Sept. 25, 1990). r., 265 (27): 16337-42; Muller et al., DNA Cell Biol. Apr. 1990; 9 (3): 221-9). Moreover, liposomes are free of the DNA length limitations that are typical of viral vector based delivery systems. Liposomes have been successfully used to deliver genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors, etc. to a variety of cultured cell lines and animals. Moreover, the use of liposomes does not appear to be associated with autoimmune responses or unacceptable toxicity after systemic delivery.

In some embodiments, liposomes are made of phospholipids that disperse in an aqueous medium where they spontaneously form multilayer concentric bilayer vesicles (also called multilamellar vesicles (MLVs)).

Alternatively, in other embodiments, the disclosure provides pharmaceutically acceptable nanocapsule formulations of the agent of the invention. In general, compounds can be stably and reproducibly encapsulated in nanocapsules (see, e.g., Quintanar-Guerrero et al., Drug

Dev. Indium. Pharm. 1998 Dec 24 (12): 1113-28). To avoid side effects due to excessive amounts of polymer inside the cell, such ultrafine particles (size about 0.1 µm) can be made from polymers capable of being degraded in vivo. Such particles can be produced as described in, for example, Couvreur et al., Crit. Rev. Ther. Drug Carrier Syst. 1988; 5 (1): 1-20; zur Muhlen et al., Eur. J. Pharm. Biopharm. 1998 Mar, 45 (2): 149-55; Zambaux et al., J. Controlled Release Jan 1998, 2:50 (1-3): 31-40 and in US No. 5,145,684.

Polynucleotides in the form of primers derived from the polynucleotides disclosed herein and polypeptides in the form of antibodies or reagents specific for a polypeptide disclosed herein may also be used as diagnostic reagents.

The identification of genetic or biochemical markers in blood or tissues, which will allow the detection of very early stages in the carcinogenesis pathway, will determine the best therapy for the patient. Surrogate tumor markers, such as polynucleotide expression, can be used to diagnose various forms and conditions of cancer. Identification of the expression levels of the polynucleotides disclosed herein will be useful in both establishing the stage of a neoplastic disorder and classifying the nature of the cancerous tissue. Staging monitors the stage of cancer and determines the presence or absence of malignant tissue in the biopsy areas. The polynucleotides disclosed herein can improve the staging method by identifying markers of aggressiveness in cancer, e.g., presence in various regions of the body. Cancer classification helps to determine how similar a tumor is to normal tissue of the same type and is assessed by its cell morphology and other markers of differentiation. The polynucleotides disclosed herein may be useful in determining the class of cancer as they may assist in determining the differentiation status of cancer cells.

Diagnostic tests are a method of diagnosing or determining susceptibility to cancers, autoimmune diseases, and related conditions through diagnostics that determine an abnormally decreased or increased level of a polypeptide or mRNA in a patient sample. This way of diagnosing is called differential expression. The expression of a given gene is compared in the diseased tissue and the normal tissue. The difference between a polynucleotide-related gene, mRNA, or protein in two tissues, compared, e.g., by comparing the molecular weight, amino acid or nucleotide sequence, or relative amount, is a measure of changes in that gene or gene that regulates it in the human tissue that he was suspected of being sick.

Reduced or increased expression can be measured at the RNA level. First, polyA RNA is isolated from two tissues, and detection of mRNA encoded by a gene corresponding to a differentially expressed polynucleotide disclosed herein may be performed, e.g., by in situ hybridization in tissue sections, by reverse transcriptase PCR, using Northern blots containing polyA + mRNA or any other another direct or indirect method of RNA detection. Increased or decreased expression of a given RNA in diseased tissue compared to normal tissue suggests that the transcript and / or the protein expressed play a role in the disease. Thus, the detection of a higher or lower level of mRNA corresponding to SEQ ID NO: 1 relative to the normal level is an indication of the presence of cancer in the patient.

MRNA expression levels in a sample can be determined by generating a library of expressed sequence labels (ESTs) from the sample. A relative representation of the EST in the library may be used

To evaluate the relative representation of the gene transcript in the original sample. The EST analysis of the test sample may then be compared to the EST analysis of the reference sample to determine the relative expression levels of the polynucleotide of interest.

Other mRNA analyzes can be performed using serial gene expression analysis (SAGE) methodology (Velculescu et al., Science (1995) 270: 484), differential exposure methodology (e.g. US No. 5,776,638) or hybridization analysis which is based on specificity. nucleotide interactions.

Alternatively, the comparison can be made at the protein level. The sizes of proteins in two tissues can be compared using antibodies to detect polypeptides in Western blots of protein extracts from the two tissues. Expression levels and subcellular localization can also be detected immunologically using antibodies to the appropriate protein. Further assay techniques that can be used to determine the levels of a protein, such as a polypeptide disclosed herein, in a host-derived sample are well known to those of skill in the art. An elevated or lowered level of polypeptide expression in diseased tissue compared to the level of expression of the same protein in normal tissue indicates that the expressed protein may be involved in the disease.

In assays according to the invention, the diagnosis can be established by detecting the expression levels of the gene products encoded by at least one sequence shown in SEQ ID NO: 1. Comparing mRNA or protein levels in diseased tissue versus normal tissue can also be used to track progress or remission. diseases.

A significant amount of polynucleotide sequences in a sample can be determined using an array of polynucleotides. They can be used to determine the differential expression of genes and to determine the function of the genes. For example, the arrays of the SEQ ID NO: 1 polynucleotide sequences can be used to determine whether any of the polynucleotides are differentially expressed between a normal and a cancer cell. In one embodiment of the invention, an array of oligonucleotide probes comprising the nucleotide sequences of SEQ ID NO: 1 or fragments thereof can be constructed to perform an efficient screening of e.g. genetic mutations. The methods of matrix technology are well known and widely applicable and can be used to explain a wide variety of problems in the field of molecular genetics, including gene expression, genetic linkage, and genetic variation (see, e.g., M. Chee et al., Science, vol. 274, pp. 610-613 (1996)).

"Diagnosis" as used herein means determining the susceptibility of a patient to a disease, determining whether the patient is sick at any given time, and making prognosis for an afflicted patient.

The polypeptides disclosed herein, or fragments or analogs thereof, or cells expressing them, can also be used as immunogens to generate immunospecific antibodies for the polypeptides disclosed herein. The term "immunospecific" means that the antibodies have a significantly higher affinity for the polypeptides disclosed herein than for other related polypeptides known in the art.

In a further aspect, the disclosure provides an antibody immunospecific for a polypeptide disclosed herein or an immunological fragment thereof as defined above.

Preferably, the antibody is a monoclonal antibody.

Antibodies generated against a polypeptide disclosed herein can be obtained by administering epitope-bearing polypeptides or fragments, analogs or cells to an animal, preferably a non-human, using routine protocols. Any technique that provides antibodies produced by continuous cell line cultures can be used to obtain monoclonal antibodies. 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 EBV hybridoma technique (Cole et al. , Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R., Liss Inc., 1985).

Techniques for producing single chain antibodies, such as those described in US 4,946,778, may also be adapted to produce single chain antibodies to the polypeptides disclosed herein. Also, transgenic mice or other organisms, including other mammals, can be used to express the humanized antibodies.

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

The antibody disclosed herein can also be used to prevent or treat cancer, particularly colorectal cancer, autoimmune diseases, and related conditions.

A method of inducing or modulating an immune response in a mammal comprises vaccinating the mammal with a polypeptide as disclosed herein that is suitable for producing an antibody and / or a response.

T cell immune function, for protection against or alleviation of symptoms or disease progression. Yet another aspect of the disclosure relates to a method of inducing or modulating an immune response in a mammal which comprises delivering a polypeptide as disclosed herein through a vector controlling the expression of the polynucleotide and encoding the polypeptide in vivo to induce an immune response and cause the production of an antibody to protect an animal from disease.

It should be appreciated that the disclosure provides a method of treating abnormal conditions, such as, e.g., cancer and autoimmune diseases, in particular colorectal cancer, associated with either an excess or deficiency in the expression of CASB7439 polypeptide activity. Other abnormal conditions associated with the expression of CASB7439 that can be treated by the invention are chronic lymphocytic leukemias and germ cell tumors.

Gene therapy can also be used to endogenously produce the CASB7439 polypeptide using appropriate cells in a patient. For a review of gene therapy, see Chapter 20, Gene Therapy and Other Molecular Genetic-based Therapeutic Approaches (and references cited therein) in Human Molecular Genetics, T. Strachan and AP Read, BIOS Scientific Publishers Ltd. (1996).

Vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol. 61, Vaccine Design - the subunit and adjuvant approach, eds. Powell and Newman, Plenum Press, 1995, New Trends and Developments in Vaccines, ed. Voller et al., University Park Press, Baltimore , Maryland, USA, 1978. Liposome encapsulation is described, for example, by Fullerton in US No. 4,235,877. Protein-macromolecule conjugation is described, for example, in US No. 4,372,945 (Likhite) and in US No. 4,474,757 (Armor et al.) .

The amount of protein in each vaccine dose is selected to induce an immunoprotective response without the significant adverse side effects associated with conventional vaccines. Such amount will vary depending upon the specific immunogen used. Generally, it is expected that each dose will contain 1-1000 µg of protein, preferably 2-100 µg and most preferably 4-40 µg. The optimal amount for a given vaccine can be determined by standard testing of antibody titers and other responses in patients. Following initial vaccination, patients may receive a booster dose after approximately 4 weeks.

"Isolated" means changed "by human hand" from its natural state. If the "isolated" agent or substance occurs in nature, it has been altered or removed from its environment, or changed and removed at the same time. For example, a polynucleotide or polypeptide that occurs naturally in living animals is not "isolated", but the same polynucleotide or polypeptide separated from coexisting materials in its natural state is "isolated" as used herein.

A "polynucleotide" is generally a polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA, including single stranded and double stranded regions.

A "variant" refers to a polynucleotide or polypeptide that differs from the template polynucleotide or polypeptide but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from a template polynucleotide. Variations in the nucleotide sequence of the variant may or may not alter the amino acid sequence of the polypeptide encoded by the template polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in a polypeptide encoded by the template sequence, as discussed below. A typical polypeptide variant differs in amino acid sequence from another, template polypeptide. Overall, the differences are limited, so that the template and variant polypeptide sequences are generally very similar and, in many regions, identical. The variant and template polypeptide may differ in their amino acid sequence by one or more substitutions, additions, and deletions in any combination. The substituted or inserted amino acid residue may or may not be encoded by the genetic code. The variant of the polynucleotide or polypeptide may occur in nature, such as an allelic variant, or may be a variant that is known not to occur naturally. Non-naturally occurring variant polynucleotides and polypeptides can be produced by mutagenesis techniques or by direct synthesis.

As is known, "identity" is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. It is known that "identity" also means the degree of relatedness between the sequences of a polypeptide or polynucleotide as determined by the alignment of stretches of such sequences. "Identity" and "similarity" can be readily calculated by known methods including, but not limited to, those described in Computational Molecular Biology, Lesk, AM, eds. 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, Ed., Humana Press New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G. Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J. Ed. M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).

Preferred methods to determine identity are designed to give the greatest match between the sequences tested. The methods of determining identity and similarity are codified in publicly available computer programs. Preferred programs for computerized determination of two sequence identity and similarity include, but are not limited to, the GCG program suite (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. 215: 403-410 (1990) The well-defined Smith Waterman algorithm can also be used to determine identity.

The preferred algorithm used is FASTA.

The preferred parameters for comparing the sequence of a polypeptide or polynucleotide using this algorithm are as follows:

Gap penalty: 12.

Penalty for extending the break: 4.

Word size: 2, max 6.

Preferred parameters for comparing polypeptide sequences by other methods are as follows:

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.

Break Length Penalty: 4.

A program using these parameters is publicly available as a "gap" program from Genetics Computer Group, Madison, WI. The aforementioned parameters are the default parameters for polypeptide comparisons (with no penalty for end gaps).

The preferred parameters for polynucleotide comparisons are as follows:

1) Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-353 (1970).

Comparison matrix: matches = +10, does not match = 0.

Gap penalty: 50.

Break Length Penalty: 3.

A program using these parameters is publicly available as a "gap" program from Genetics Computer Group, Madison, WI. The aforementioned parameters are the default parameters for polynucleotide comparisons.

For example, the sequence of a polynucleotide disclosed herein may be identical to the template sequence of SEQ ID NO: 1, that is, it may be 100% identical, or it may contain a number of nucleotide changes as compared to the template sequence. Such changes are selected from the group consisting of at least one deletion, substitution, including nucleotide transitions and transversions, and insertions, which changes may occur at the 5 'or 3' ends of the nucleotide template sequence or anywhere between these terminal positions, and are individually between the nucleotides of a template sequence or in one or more contiguous groups within the template sequence. The number of nucleotide changes is defined as the difference between the total number of nucleotides in SEQ ID NO: 1 and the product of the total number of nucleotides in SEQ ID NO: 1 and the number percent of the corresponding percent identity (divided by 100), or:

nn <Xn - (Xn y), where nn is the number of nucleotide changes, xn is the total number of nucleotides in SEQ ID NO: 1, and y is e.g. 0.70 for 70%, 0.80 for 80%, 0, 85 for 85% and 0.90 for 90%, 0.95 for 95%, etc., with each non-integer product of xn and y being rounded to the nearest integer before subtracting xn. Alterations to the sequence of the polynucleotides encoding the polypeptide of SEQ ID NO: 2 may occur

Create nonsense, missense, or frame alteration mutations in this coding sequence and thus alter the polypeptide encoded by the polynucleotide following such changes.

Similarly, the sequence of a polypeptide disclosed herein may be identical to the template sequence SEQ ID NO: 2, that is, be 100% identical, or it may contain an integer number of amino acid changes from the template sequence such that the percentage identity is less than 100%. Such changes are selected from the group consisting of at least one deletion, substitution, including conservative and non-conservative, and amino acid insertion, wherein such changes may occur at the amino or carboxyl termini of the template polypeptide sequence or anywhere between these terminal positions and are found individually among the amino acids in a template sequence or in one or more contiguous groups within a template sequence. The number of amino acid changes for a given percent identity is defined as the difference between the total number of amino acids in SEQ ID NO: 2 and the product of the total number of amino acids in SEQ ID NO: 2 and the number percent of the respective percent identity (divided by 100), respectively:

n_and <Xa - (Xa y), where na is the number of amino acid changes, xa is the total number of amino acids in SEQ ID NO: 2, and y is e.g. 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, etc., with each non-integer product of Xa and y being rounded to the nearest integer before subtracting from xa.

A "homologue" is a generic term used to indicate a polynucleotide or polypeptide sequence having a high degree of relatedness to a target sequence. Such relatedness can be quantified by determining the degree of identity and / or similarity between the sequences being compared, as previously described. The term includes the term "ortholog" meaning a polynucleotide or polypeptide that is functionally equivalent to a polynucleotide or polypeptide in another species and the term "paralog" meaning a functionally similar sequence within the same species.

Legend for figures

Figure 1 shows real-time PCR data using a Taqman probe. Legend: Adrenal gland: Ad_Gl; Bladder: Bl; Bone marrow: Bo_Ma; Cervix: Ce; Colon: What; Oviduct: Fa_Tu; Stole: II; Liver: Li; Lung: Lu; Lymph Node: Ly_No; Esophagus: Oe; Parathyroid gland: Pa_Thy; Bearing: Pl; Prostate: Pr; Rectum: Re; Leather: Sk; Skeletal muscles: Sk_Mu; Small intestine: Sm_In; Spleiona: Sp; Kernel: Te; Thyroid: Thy; Trachea: Tr.

Figure 2 shows real-time PCR expression using the Sybr protocol. Legend: Adrenal gland: Ad_Gl; Bladder: Bl; Bone marrow: Bo_Ma; Cervix: Ce; Colon: What; Lymph Node: Ly_No; Esophagus: Oe; Parathyroid gland: Pa_Thy; Bearing: PI; Prostate: Pr; Rectum: Re; Leather: Sk; Skeletal muscles: Sk_Mu; Small intestine: Sm_In; Spleiona: Sp; Kernel: Te; Thyroid: Thy; Trachea: Tr; Heart: He.

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

Figure 4 shows Western analysis of the expressed NS1-CASB7439 protein. The cell extract of the CASB7439 expressing strain was applied to the gel and detected with an anti-NS1 monoclonal antibody.

Figure 5 shows Coomassie Blue Stained SDS PAGE CASB7439 after purification. Lanes 1 and 5 are molecular weight markers; lanes 2, 3, 4 are loaded with 2 µl, 4 µl and 6 µl of purified protein, respectively.

Figure 6 shows a CASB7439 Western blot after purification detected with an anti-histidine monoclonal antibody.

Examples

Example 1. Real-time RT-PCR analysis

Real-time RT-PCR (U. Gibson, 1996. Genome Research: 6, 996) was used to compare the amount of candidate antigen mRNA transcript in matched colon tumor tissues and normal colon tissue from multiple patients. In this method, the candidate gene mRNA levels in a panel of normal tissues were additionally assessed.

PL 209 127 B1

Total RNA from normal colon tissue and colon tumor tissue was extracted from flash-frozen biopsies using TriPure reagent (Boehringer). Total RNA from normal tissues was purchased from InVitrogen or extracted from flash-frozen biopsies using TriPure reagent. PolyA + mRNA was purified from total RNA after treatment with DNAse using oligo-dT magnetic beads (Dynal). Quantification of mRNA was performed by spectrofluorimetry (VersaFluor, BioRad) using SybrII dye (Molecular Probes). The real-time PCR amplification primers were designed using Perkin-Elmer Primer Express software using the default options for TaqMan amplification conditions.

The real-time reactions were assembled according to standard PCR protocols using 2 ng of purified mRNA for each reaction. Sybrl dye (Molecular Probes) was added at a final dilution of 1/75000 for real-time detection. Amplification (40 cycles) and real-time detection were performed on a Perkin-Elmer Biosystems PE7700 system using standard instrument settings. Ct values ​​were calculated using Sequence Detector PE7700 software. Several Ct values ​​were obtained for each sample: for patient samples, tumor Ct (CtT) and CtN matched normal colon tissue (CtN) on TAA candidate, and for a normal tissue sample panel, CtXY for each normal XY tissue. Other Ct (CtA) values ​​were calculated for all samples against the actin gene as internal standard. Alternatively, you can monitor real-time PCR amplification using a Taqman probe. Amplification (40 cycles) and real-time detection were performed on a Perkin-Elmer Biosystems PE7700 system using standard instrument settings. Ct values ​​were calculated using Sequence Detector PE7700 software. Ct values ​​for each tissue sample were calculated for the target mRNA (CtX) and for the actin mRNA (CtA).

Since the efficiency of PCR amplification under the experimental conditions used is close to the theoretical amplification efficiency, the value of 2^{(CtN / T / XY-CtA)} is an estimate of the relative TAA transcript level of a sample standardized against the actin transcript level. A value of 1 thus suggests that the candidate antigen and the actin have the same expression level.

Real-time PCR reactions were first performed on colon tumor tissues and matched normal colon tissue biopsies from 12 patients. The reactions were then run on a more complete data set of 18 patients (this data set included the data set for the first 12 patients). In this dataset, replicates were used for 6 patients out of 18. Six more patients were tested and the results combined with the previous 18 patients. The statistics for the final pool are presented in Table 3 and illustrated in Figure 1.

A series of 48 normal tissue samples representing 29 different tissues was tested using the same procedure (the normal tissues analyzed are listed in Table 3). TAA transcript levels were calculated as described above. The proportion of patients overexpressing the candidate antigen as well as the mean overexpression of the transcript relative to normal tissues was also calculated in this data set. The results are shown in Fig. 1.

Table 1

CASB7439 Expression Results in Real-Time PCR: Data Set for 12 Patients

<td> 1</td><td> 2</td>

<td>% of patients with higher mRNA levels in matching colon tumor tissues (positive patients)</td><td> 92%</td>

<td>% of patients with mRNA levels at least 3 times higher in matching colon tumor tissues</td><td> 92%</td>

<td>% of patients with mRNA levels at least 10 times higher in matching colon tumor tissues</td><td> 92%</td>

<td>% of patients with mRNA levels at least 3 times lower in matching colon tumor tissues</td><td> 8%</td>

<td>Average mRNA level in matched normal colon tissues (actin standardized)</td><td> 0,0026</td>

<td>Mean mRNA level in matching colon tumor tissues in positive patients (standardized to actin)</td><td> 0,265</td>

<td>Mean fold mRNA overexpression</td><td> 2028</td>

<td>Median fold mRNA overexpression</td><td> 115</td>

PL 209 127 B1 cont. table 1

<td> 1</td><td> 2</td>

<td>Average mRNA levels in normal tissues</td><td> 0,0079</td>

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

<td>Average mRNA levels in normal tissues</td><td> 0,0064</td>

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

<td>% of patients with mRNA levels higher than average for normal tissues</td><td> 92%</td>

<td>% of patients with mRNA levels higher than ten times the mean for normal tissues</td><td> 75%</td>

<td>Normal unnecessary tissues with mRNA levels higher than the median for normal tissues</td><td>lack</td>

Table 2

CASB7439 Expression Results in Real Time PCR: Data Set for 18 Patients

<td>% of patients with higher mRNA levels in matching colon tumor tissues (positive patients)</td><td> 89%</td>

<td>% of patients with mRNA levels at least three times higher in matching colon tumor tissues</td><td> 89%</td>

<td>% of patients with mRNA levels at least ten times higher in matching colon tumor tissues</td><td> 78%</td>

<td>% of patients with mRNA levels at least three times lower in matching colon tumor tissues</td><td> 5%</td>

<td>Average mRNA level in matched normal colon tissues (actin standardized)</td><td> 0,005</td>

<td>Average mRNA level in matched normal colon tissues (actin standardized)</td><td> 0,152</td>

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

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

<td>Average mRNA levels in normal tissues</td><td> 0,0065</td>

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

<td>Average mRNA levels in normal tissues</td><td> 0,005</td>

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

<td>% of patients with mRNA levels higher than average for normal tissues</td><td> 94%</td>

<td>% of patients with mRNA levels greater than ten times the mean level for normal tissues</td><td> 94%</td>

<td>Normal unnecessary tissues with mRNA levels higher than the median for normal tissues</td><td>lack</td>

Table 3

CASB7439 Expression Results in Real Time PCR: Data Set for 24 Patients

<td>% of patients with CASB7439 transcript levels higher in colon tumor tissues than in adjacent normal colon tissues (positive patients)</td><td> 92%</td>

<td>% positive patients with CASB7439 transcript levels at least ten times higher in colon tumor tissues than in adjacent normal colon tissues</td><td> 75%</td>

<td>Mean fold transcript overexpression in tumors of positive patients</td><td> 1289</td>

<td>% of patients with CASB7439 transcript levels higher in colon cancer than average in normal tissue</td><td> 96%</td>

<td>% of patients with mRNA levels at least ten times higher in colon cancer than average in normal tissue</td><td> 62,5%</td>

<td>Normal tissues where CASB7439 transcript expression corresponds to tumor transcript expression in tumors</td><td>lack</td>

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Real-time PCR reactions were also performed using the Taqman protocol (as described above) on colorectal tumor tissues and adjacent normal colon tissue biopsied from 6 patients. 3 replicates were taken for each and the mean was used for further calculations. The results are shown in Figure 1. In addition, 36 normal tissue samples representing 28 different tissues were also tested using the same procedure (see Table 5). The results are shown in Fig. 2.

Table 4

Results of CASB7439 expression in real-time PCR using the Taqman probe

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

<td>% of patients with CASB7439 transcript levels higher in colon tumor tissues than in adjacent normal colon tissues (positive patients)</td><td> 100%</td>

<td>% positive patients with CASB7439 transcript levels at least ten times higher in colon tumor tissues than in adjacent normal colon tissues</td><td> 83%</td>

<td>Mean fold transcript overexpression in tumors of positive patients</td><td> 109</td>

<td>% of patients with CASB7439 transcript levels higher in colon cancer than average in normal tissue</td><td> 100%</td>

<td>% of patients with mRNA levels at least ten times higher in colon cancer than average in normal tissue</td><td> 100%</td>

<td>Normal tissues where CASB7439 transcript expression corresponds to tumor transcript expression in tumors</td><td>lack</td>

The results clearly suggest that the CASB7439 transcript is overexpressed in colon and rectal tumors as compared to adjacent normal colon tissues and all the above-mentioned normal tissues. More than 90% of patients strongly overexpress CASB7439 transcript in the tumor, compared to adjacent normal colon tissues. The average overexpression in tumors is at least a hundred fold. Furthermore, more than 90% of patients overexpress CASB7439 transcript in colon and rectal tumors compared to other normal tissues, and more than 60% of them overexpress it at least tenfold.

Table 5

List of normal tissues used for analysis of CASB7439 transcript expression

<td>Tissue</td><td>Shortcut</td>

<td> 1</td><td> 2</td>

<td>The adrenal glands</td><td>Ad_Gl</td>

<td>Aorta</td><td>Ao</td>

<td>Bladder</td><td>BI</td>

<td>Marrow</td><td>Because he has</td>

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

<td>Cervix</td><td>Ce</td>

<td>Colon</td><td>What</td>

<td>Fallopian tube</td><td>Fa_Tu</td>

<td>Heart</td><td>He</td>

<td>The swirling gland</td><td>Loam</td>

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

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

<td>Lung</td><td>Lu</td>

PL 209 127 B1 cont. table 5

<td> 1</td><td> 2</td>

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

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

<td>Parathyroid glands</td><td>Pa_Thy</td>

<td>Anus</td><td>Re</td>

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

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

<td>Small intestine</td><td>Sm_In</td>

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

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

<td>Thyroid</td><td>Thy</td>

<td>Trachea</td><td>Tra</td>

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

<td>Bearing</td><td>PI</td>

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

<td>Kernel</td><td>These</td>

Example 2. Differential screening of cDNA templates

Identification of tumor associated genes in the subtracted cDNA library is performed by a differential screening method.

Total bacterial DNA was extracted from 100 µl of the overnight cultures. The bacteria were lysed with guanidine isothiocyanate and the bacterial DNA was affinity purified using magnetic glass (Boehringer).

Plasmid inserts were recovered from bacterial DNA by Advantage PCR amplification (Clontech). The PCR products were spotted on two nylon membranes to produce high-density cDNA templates using the Biomek 96 HDRT tool (Beekman). The spotted cDNA was covalently bound to the membrane by means of UV irradiation. The first membrane was hybridized with a mixed cDNA probe prepared from a single patient tumor. The second membrane was hybridized with an equivalent amount of a mixed cDNA probe prepared from normal colon tissue of the same patient. The probe cDNA was prepared by PCR amplification as described above using the AlkPhos Direct system (Amersham).

The hybridization conditions and the stringent washing conditions are as described in the AlkPhos Direct kit. The hybridized probe is detected by chemiluminescent analysis. The hybridization intensities for each cDNA fragment in both blots are measured by membrane densitometry or directly (BioRad Fluor-S Max). The ratio of the intensity of hybridization to tumor tissues to the intensity of hybridization to normal tissues (Y / N) is calculated for each gene to assess the amount of overexpression in the tumor.

Genes that are significantly overexpressed in colon tumors are further investigated. Significance is arbitrarily defined as one standard deviation of the Y / N frequency distribution. Differential screening experiments were repeated using RNA from multiple patient donors (&gt; 18) to assess the incidence of overexpressing tumors in the patient population. Additionally, the DNA arrays were hybridized with mixed cDNA probes from normal tissues other than the colon (see list above) to determine the level of candidate gene expression in these tissues.

Example 3. DNA microarrays

DNA microarrays are used to study mRNA expression profiles of large collections of genes in multiple samples. This information is used to complete the data obtained by the route

Real-time PCR and provides an independent measure of gene expression levels in tumors and normal tissues.

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Examples of contemporary 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 photolithography process; 2) DNA spotting technology, where small volumes of DNA solution are applied by a robot and then immobilized on the surface of a solid phase (e.g. glass). In both cases, the chips are hybridized with cDNA or cRNA extracted from the tissue of interest (e.g., normal tissue, tumor, etc.) and radiolabeled or with a fluorescent reporter molecule.

The labeled material is hybridized to the chip and the amount of probe associated with each sequence on the chip is determined using a specialized scanner. The experiment can be performed with a single fluorescent (or radioactivity) reporter or alternatively with two fluorescent reporters. In the latter case, each of the two samples is labeled with one of the reporter molecules. The two labeled samples are then hybridized in competition 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 amount of transcript in the two samples. Detailed protocols are available from a number of sources including DNA Microarrays: A practical approach. Schena M., Oxford University Press 1999 "and on the Internet (http://cmgm.Stanford.edu/pbrown/protocols/index.html), http://arrayit.com/DNA-Microarray-Protocols/) and at specialized distributors ( e.g. Affymetrix).

Example 5. Northern-Southern blot analysis

Limited amounts of mixed cDNA from tumor tissues and matched normal colon tissues were amplified by Advantage PCR (see above). Template RNA from many normal tissues was also amplified using the same procedure. The amplified cDNA (1 µg) was subjected to 1.2% agarose gel electrophoresis and transferred to a nylon membrane. The membrane was hybridized (AlkPhos Direct System) with a probe prepared with the candidate TAA cDNA fragment. Northern-Southern analysis provides information on the size of the transcript, the presence of splice variants, and the amount of transcripts in the tumor and normal tissues.

Example 6. Northern blot analysis

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

Example 7. Experimental Identification of Full Length cDNA Sequences

Colon cancer cDNA libraries were constructed using the Lambda Zap II system (Stratagene) with 5 µg polyA + mRNA. The provided protocol was used, however SuperscriptII (Life Technologies) was used for the reverse transcription step. Oligo dT primer and random primer libraries were constructed.

For each screening, about 1.5 x 10 were plated in libraries⁶ independent phages. Phage plaques were transferred to nylon filters and hybridized with an AlkPhos Direct labeled cDNA probe. Positive phage were detected by chemiluminescence. Positive phages were excised from the agar plate, eluted into 500 µl of SM buffer and confirmed by gene specific PCR. The eluted phages were transformed into single-stranded M13 bacteriophages by in vivo excision. Bacteriophages were transformed into double-stranded plasmid DNA by E. coli infection. Infected bacteria were plated and subjected to a second round of cDNA probe screening. Plasmid DNA was purified from positive bacterial clones and sequenced on both strands.

When the full length genome cannot be obtained directly from the cDNA library, the missing sequence is isolated using RACE technology (Marathon Kit, ClonTech). This approach is based on the reverse transcription of mRNA into double-stranded cDNA, ligation of linkers at the ends of the cDNA, and amplification of the desired cDNA ends using a gene-specific primer and one of the linker oligonucleotides. Marathon PCR products cloned into a plasmid (pCRII-TOPO, InVitrogen) and sequenced. Using this procedure, the polynucleotide of SEQ ID NO: 1 was obtained.

Example 8. EST profiles

The characterization of experimental antigen expression in tissues is complemented by the study of the human EST database. ESTs (expressed sequence labels) are small cDNA fragments obtained from a collection of mRNA extracted from a given tissue or cell line. Such a database now provides large amounts of human ESTs (2 x 10⁶) from several thousand cDNA tissue libraries, including tumor tissues from various disease types and states. An exploratory sequence comparison of CASB7439 was performed using information technology tools (Blast) to gain further insight into tissue expression.

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Distribution of EST CASB7439

<td>EST GenBank Accession Number</td><td>EST cDNA tissue library</td>

<td>C00634</td><td>Adult (K. Okubo)</td>

<td>AA468668</td><td>NCI_CGAP_Co3</td>

<td>AA565752</td><td>NCI_CGAP_Co11</td>

<td>AA565766</td><td>NCI_CGAP_Co11</td>

<td>AA565767</td><td>NCI_CGAP_Co11</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>AI861937</td><td>NCI_CGAP_Co16</td>

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

<td>AW080652</td><td>NCl_CGAP_Co19</td>

<td>AW083899</td><td>NCl_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 the CASB7439 perfectly. The list includes 9 ESTs from 4 different colon cancer libraries, one EST from a normal colon tissue library, 3 ESTs from one germ cell tumor library, one EST from a chronic lymphocytic leukemia cell library, 2 ESTs from 2 mixed tumor libraries, 2 ESTs from libraries of unknown type. This clearly suggests, as expected, that CASB7439 is overexpressed in tumor tissues, especially colon and rectal tumor tissues, as compared to normal tissues.

Example 9

9.1 Expression and purification of tumor specific antigens

Expression in microbial hosts or alternatively in vitro transcription / translation is used to produce the antigen disclosed herein to obtain vaccines and to generate protein fragments or whole protein for rapid purification and generation of antibodies needed for characterization of a naturally expressed protein by immunohistochemistry or for purification tracking.

Recombinant proteins can be expressed in two host microorganisms, E. coli and yeast (such as Saccharomyces cerevisiae or Pichia pastoris). This allows for the selection of the expression system with the best characteristics for the production of a given antigen. Generally, the recombinant antigen will be expressed in E. coli and the protein reagent will be expressed in yeast.

The expression strategy involves first designing the primary structure of the recombinant antigen. In general, to improve expression levels, an expression fusion partner (EFP) is placed at the N-terminus, which may also include a region useful for modulating the immunogenic properties of the antigen, the immune fusion partner (IFP). Additionally, one fusion affinity partner (AFP) is incorporated at the C-terminus to facilitate further purification.

As mentioned above, several constructs can be compared. For the rapid expression and purification and generation of antibodies against CASB7439, protein generation in E. coli has been proposed

CASB7439 full length with NS1 as EFP and Histidine tail as AFP. Thus, two constructs are proposed:

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Construct 1: CASB7439 wild-type full length cDNA fused to the NS1 cDNA as EFP and a histidine tail encoding the cDNA as AFP (SEQ ID NO: 8). The sequence of the encoded fusion protein is SEQ ID NO: 10.

Construct 2: Mutant CASB7439 cDNA full length fused to NS1 cDNA as EFP and a histidine tail encoding the cDNA as AFP (SEQ ID NO: 9).

It is proposed that in this construct the first 50 codons of the native CASB7439 cDNA be replaced with codons specific to the E. coli codon usage to enhance the potential for expression of CASB7439 in an E. coli host. The sequence of the encoded fusion protein is SEQ ID NO: 10.

The project of the CASB7439 protein is shown below:

<img file="PL209127B1_D0001.tif" />

"NS1" is the N-terminal fragment (80 amino acids) of the influenza NS1 protein.

"HIS" is a poly-histidine tail.

The recombinant strain used is AR58: cryptic λ lysogen derived from N99, gal E :: Tn 10, Δ-8 (ch1D-pg1), Δ-H (ero-ch1A), N + and cI857 (Proc. Natl. Acad. Sci. USA vol. 82, pp. 88-92, Jan. 1985 Biochemistry).

When recombinant strains are available, the recombinant product is characterized by assessing the expression level and predicting further protein solubility by analyzing the behavior in the crude extract.

After growing in a suitable culture medium and inducing expression of recombinant protein, the total extracts were analyzed on SDS-PAGE.

Recombinant proteins are visualized in stained gels and identified by Western blot analysis using specific antibodies.

Plasmid name: TCM 281 pRIT..15143 replicon: pMB1 selection: Kan promoter: PL long insert: NSl-GT74-39-His

Expression of recombinant protein from construct 1

The bacteria were grown in LB + 50 µg / ml Kan medium at 30 ° C. When the culture reached OD = 0.5 (620 nm), the culture was heated to 39 ° C and the cells were harvested after 5 hours of induction.

Preparation of the extract

Cell concentration: 50 x ... in PBS + total ...

Breaking: French press 3 x

Spinning: 30 min. at 14,000 t

Note:&gt; 90% in cell extract supernatant

The cell extract was run through 12.5% ​​SDS PAGE and then stained with Coomassie blue.

A Western blot was also prepared using a commercial monoclonal anti-polyhistidine tail antibody (Quiagen).

The resulting gels (Figures 3 and 4) show that the protein is expressed and is visible in the supernatant of the cell extract.

The purification scheme was carried out according to the classical approach based on the presence of a His tail in the recombinant protein.

In a typical experiment, the broken cells were drained and the cell extracts subjected to metal ion affinity chromatography (IMAC; Ni ** NTA from Quiagen) which specifically retained the recombinant protein. Retained proteins were eluted with a 0-500 mM imidazole gradient (optionally in the presence of detergent) in phosphate buffer.

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The supernatant from the harvested culture was denatured in 6M urea, 100 mM NaH2PO4, 10 mM Tris, pH 8 and loaded onto an IMAG Quiagen NTA Ni ** chromatography column under the following conditions:

<td>Balancing buffer:</td><td>NaH2PO4 Tris Urea</td><td>100 mM pH 8 10 mm 6M</td>

<td>Sample: supernatant in 6M urea:</td><td>100 mM NaH2PO4</td><td>10 mM Tris</td>

<td>Rinsing buffers:</td><td>1) NaH2PO4</td><td>100 mM pH 8</td>

<td></td><td>Tris</td><td>10 mm</td>

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

<td></td><td>Imidazole</td><td>25 mm</td>

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

<td></td><td>Tris</td><td>10 mm</td>

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

<td></td><td>Imidazole</td><td>50 mm</td>

<td>Elution buffer:</td><td>NaH2PO4</td><td>100 mM pH 5.5</td>

<td></td><td>Tris</td><td>10 mm</td>

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

<td></td><td>Imidazole</td><td>500 mm</td>

Eluted protein in 500 mM imidazole + 6M urea was dialyzed under the following conditions:

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

- the same with the use of 2M urea for 2 hours,

- the same with the use of 0M urea for 2 hours.

The final material was frozen and stored. Protein content was quantified using the Lowry protein assay (0.9 mg / 1.2 ml). Purity was assessed by 12.5% ​​SDS PAGE stained with Coomassie blue (Figure 5) and the presence of recombinant protein was checked by Western blot using an anti-polyhistidine monoclonal antibody (Figure 6).

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

9.2 Antibody production and immunohistochemistry

Small amounts of relatively pure protein can be used to generate immunological tools to:

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

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

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

9.2.1 Polyclonal antibodies

Immunization

Rabbits were immunized intramuscularly (IM) 3 times at 3-week intervals with 100 µg of protein formulated in 3D-MPL / QS21 adjuvant. Three weeks after each immunization, a blood sample was taken and serum antibody titer was assessed by ELISA, using protein as antigen coating according to standard protocol.

ELISA

96 well plates (Nunc maxisorb) were coated with 5 µg of protein overnight at 4 ° C. After 1 hour of saturation at 37 ° C with PBS NCS 1%, serial dilutions of rabbit sera for 1 hour were added. 30 minutes. at 37 ° C (starting at 1/10). After 3 washes in PBS Tween, anti-rabbit biotinylated antiserum (Amersham) (1/5000) was added. The plates were washed and peroxidase combined streptavidin (1/5000) was added for 30 min at 37 ° C. After washing, 50 µl of TMB (BioRad) was added for 7 min. and the reaction was then stopped with 0.2M H2SO4. OD can be measured at 450 nm and middle dilutions calculated using SoftmaxPro.

9.2.2 Monoclonal antibodies

Immunization BALB / c mice were immunized 3 times at 3-week intervals with 5 µg of purified protein. Bleeding was performed 14 days post II and 1 week post 3. Sera were tested with Elisa na

Purified protein used as coating antigen. Based on these results (middle dilution&gt; 10000) one mouse was selected for fusion.

HAT fusion / selection

Spleen cells were fused to SP2 / 0 myeloma according to standard protocol, using 40% PEG and 5% DMSO. Cells were then seeded into 2.5 x 10 96 well plates⁴-10⁵cells / well and resistant clones in HAT medium were selected. The supernatant of these hybridomas were tested for specific antibody content and, when positive, subjected to 2 cycles of limited dilution. After 2 rounds of screening, 3 hybridomas for ascitic fluid production were selected.

9.2.3 Immunohistochemistry

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

- expression level of an antigen disclosed herein in cancer relative to normal tissue or

- the proportion of cancer of a certain type expressing an antigen,

- do other types of cancer also express the antigen?

- the proportion of cells expressing the antigen in the cancer tissue.

Preparation of tissue samples

After excision, the tissue sample was mounted on a stopper disc in OCT compound and rapidly frozen in isopentane previously supercooled in liquid nitrogen (-160 ° C). The block was then stored at -70 ° C until use. 7-10 µm sections were prepared in a cryostat chamber (-20, -30 ° C).

Coloring

Tissue sections were dried for 5 min at room temperature (RT), fixed in acetone for 10 min at RT, dried again and saturated with PBS serum 0.5% BSA 5%. After 30 min at room temperature, direct or indirect staining was performed with antigen-specific antibodies. Direct staining leads to better specificity but less intense staining, while indirect staining produces more intense but less specific staining.

9.3. Analysis of human cellular immune responses to the antigen disclosed herein

The immunological significance of the antigen disclosed herein can be assessed by in vitro loading of human T cells. All T and dendritic lymphocyte lines are derived from PBMCs (peripheral blood mononuclear cells) of healthy donors (preferably the HLA-A2 subtype). An HLA-A2.1 / Kb transgenic mouse model is also used to screen HLA-A2.1 peptides.

CD8 T cell lines were grown⁺ newly discovered antigen and maintained by weekly in vitro stimulation. Lytic activity and IFNγ production of the CD8 + line in response to antigen or antigen-derived peptides were tested using standard procedures.

Two strategies were used to obtain the CD8 T cell line⁺: the peptide-based approach and the whole-gene-based approach. Both approaches require either the cloning of the full-length cDNA of the newly discovered antigen in the correct reading frame in an appropriate delivery system, or using it to predict the sequence of HLA binding peptides.

Peptide-based approach

Briefly, transgenic mice were immunized with adjuvanted HLA-A2 peptides, followed by those unable to induce a CD8 response.⁺ (defined by the efficient lysis of protein pulsed autologous spleen cells) was further analyzed in the human system. Human dendritic cells (cultured according to Romani et al.) Were pulsed with peptides and used to stimulate CD8 sorted⁺ T cells (using FACS). After several weeks of stimulation, CD8 lines⁺first they were tested on peptide pulsed autologous BLCL (EBV-B transformed cell lines). To verify proper in vivo processing of the peptide lines CD8⁺ are tested on cDNA transfected tumor cells (LnCaP, Skov3 or CAMA tumor cells transfected with HLA-A2).

Whole gene approach

CD8 T cell lines⁺ initiated and stimulated with dendritic cells transfected with a gene shotgun, fibroblasts transduced by retroviruses and transfected with

B7.1, dendritic cells infected with recombinant pox or adenovirus.

Virus infected cells more efficiently present the antigenic peptides because the antigen is highly expressed but can only be used once to avoid overgrowth

Of viral T lines. After alternating stimulations, CD8 lines⁺ were tested on tumor cells transfected with cDNA as indicated above. The specificity and identity of the peptide was determined to confirm immunological validity.

CD4 T cell response⁺

The immune response of CD4 T cells can also be assessed similarly⁺. Generation of specific CD4 T cells⁺ is performed using dendritic cells loaded with purified recombinant protein or peptides to stimulate T cells.

Predicted epitopes (nonamers and decamers) binding to HLA alleles

HLA class I binding peptide sequences are predicted either using the Parker algorithm (Parker, KC, MA Bednarek 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.ni.gov/molbio/hla_bind/), or by the Rammensee method (Rammensee, Friede, Stevanovic, MHC ligands and peptide motifs: 1st listing, Immunogenetics 41, 178-228, 1995; Rammensee, Bachmann, Stevanovic: MHG ligands and peptide motifs. Landes Bioscience 1997 and http://134.2.96.221/scripts/hlaserver.dll/home.htm).

The peptides can then be 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 cutoff value of 6 (Sturniolo, Hammer et al., Nature Biotechnology. 1999, 17; 555-561).

The following tables summarize the predicted sequences of class I and II epitopes.

<td colspan="5">HLA-A0201: decamers</td>

<td>Rank</td><td>Take-off position</td><td>List of residual subsets</td><td>Parker points °</td><td>SEQ ID:</td>

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

°: Evaluation of the dissociation half-life of a molecule containing this subsection

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

<td>Rank</td><td>Take-off position</td><td>List of residual subsets</td><td>Parker points °</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>

°: Evaluation of the dissociation half-life of a molecule containing this subsection

<td colspan="5">HLA-A24: nonamery</td>

<td>Rank</td><td>Take-off position</td><td>List of residual subsets</td><td>Parker points °</td><td>SEQ ID:</td>

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

°: Evaluation of the dissociation half-life of a molecule containing this subsection

<td colspan="5">HLA-A 24: set-top boxes</td>

<td>Rank</td><td>Take-off position</td><td>List of residual subsets</td><td>Parker points °</td><td>SEQ ID:</td>

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

°: Evaluation of the dissociation half-life of a molecule containing this subsection

<td colspan="5">HLA-B7: set-top boxes</td>

<td>Rank</td><td>Take-off position</td><td>List of residual subsets</td><td>Parker points °</td><td>SEQ ID:</td>

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

°: Evaluation of the dissociation half-life of a molecule containing this subsection

PL 209 127 B1

<td colspan="5">HLA-B 4403: set-top boxes</td>

<td>Rank</td><td>Take-off position</td><td>List of residual subsets</td><td>Parker points °</td><td>SEQ ID:</td>

<td> 1</td><td> 156</td><td>SEPGSPRSAY</td><td> 360,000</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>

°: Evaluation of the dissociation half-life of a molecule containing this subsection

HLA-DRB1 * 1501: nonamers

<td>Rank</td><td>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</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"></td>

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

<td>Rank</td><td>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</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-DRB 1 * 0402: nonamers</td>

<td>Rank</td><td>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</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>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</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>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</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>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</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>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</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>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</td><td>SEQ ID:</td>

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

<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>

PL 209 127 B1

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

<td>Rank</td><td>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</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>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</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>LGFQALRQH</td><td> 4,6</td><td>SEQ ID NO: 28</td>

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

<td>Rank</td><td>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</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-DRB 1 * 0703: nonamers</td>

<td>Rank</td><td>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</td><td>SEQ ID:</td>

<td> 1</td><td> 112</td><td>VRNALAGGL</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>Take-off position</td><td>List of residual subsets</td><td>Tepitope points</td><td>SEQ ID:</td>

<td> 1</td><td> 96</td><td>VEYIRALQR</td><td> 4,3</td><td>SEQ ID NO: 32</td>

PL 209 127 B1

INFORMATION ABOUT SEQUENCES

SEQ LD NO: 1 gtaccttgctttgggggcgcactaagtacctgccgggagcagggggcgcaccgggaactcgcagatttcgcc 5 agttgggcgcactggggatctgtggactgcgtccggggggatggacgcttggggcccgcttggggcccgctttgggg

TTACAGAATGTGATCGCGCGAGGGGGAGGGCGAAGCGTGGCGGGAGGGCGAGGCGAAGGAAGGAGGGCGTGA

GAAAGGCGACGGCGGCGGCGCGGAGGAGGGTTATCTATACATTTAAAAACCAGCCGCCTGCGCCGCGCCTGC

GGAGACCTGGGAGAGTCCGGCCGCACGCGCGGGACACGAGCGTCCCACGCTCCCTGGCGCGTACGGCCTGCC

ACCACTAGGCCTCCTATCCCCGGGCTCCAGACGACCTAGGACGCGTGCCCTGGGGAGTTGCCTGGCGGCGCC

GTGCCAGAAGCCCCCTTGGGGCGCCACAGTTTTCCCCGTCGCCTCCGGTTCCTCTGCCTGCACCTTCCTGCG GCGCGCCGGGACCTGGAGCGGGCGGGTGGATGCAGGCGCGatggacggcggcacactgcccaggtccgcgcc ccctgcgccccccgtccctgtcggctgcgctgcccggcggagacccgcgtccccggaaccgttgcgctgcag ccggcggcggcgaccggccaccgcagagaccggaggcggcgcagcggccgtagcgcggcgcaatgagcgcga gcgcaaccgcgtgaagctggtgaacttgggcttccaggogctgcggcagcacgtgccgcacggcggcgccag caagaagctgagcaaggtggagacgctgcgctcagccgtggagtacatccgcgcgcrgcagcgcctgctggc cgagcacgacgccgtgcgcaacgcgctggcgggagggcrgaggccgcaggccgtgcggccgtctgcgccccg cgggccgccagggaccaccccggtcgccgcctcgccctcccgcgcttcttcgtccccgggccgcgggggcag ctcggagcccggctccccgcgttccgcctactcgtcggacgacagcggctgcgaaggcgcgctgagtcctgc ggagcgcgagctactcgacttctccagctggttagggggctactgaGCGCCCTCGACCTATGAGCCTCAGCC

CCGGAAGCCGAGCGAGCGGCCGGCGCGCTCATCGCCGGGGAGCCCGCCAGGTGGACCGGCCCGCGCTCCGCC CCCAGCGAGCCGGGGACCCACCCACCACCCCCCGCACCGCCGACGCCGCCTCGTTCGTCCGGCCCAGCCTGA CCAATGCCGCGGTGGAAACGGGCTTGGAGCTGGCCCCATAAGGGCTGGCGGCTTCCTCCGACGCCGCCCCTC CCCACAGCTTCTCGACTGCAGTGGGGCGGGGGGCACCAACACTTGGAGATTTTTCCGGAGGGGAGAGGATTT TCTAAGGGCACAGAGAATCCATTTTCTACACATTAACTTGAGCTGCTGGAGGGACACTGCTGC-CAAACGGAG acctatttttgtacaaagaacccttgacctggggcgtaataaagatgacctggacccctgcccccactatct ggagttttccatgctggccaagatctggacacgagcagtccctgaggggcgggggtccctggcgtgaggcccc cgtgacagcccaccctggggtgggtttgtgggcactgctgctactactactactactactactactactactactactactactactactactactactactactactactactactaactactactactactaactactactacta

SEQIDNO: 2 mdggtlprsappappvpvgcaarrrpaspellrcsrrrrpataetgggaaavarrnerernrvklvnlgfqa lrqhvphggaskklskvetlrsaveyiralqrllaehdavrnalagglrpqavrttpaprgppg

RASSSPGRGGSSEPGSPRSAYSSDDSGCEGALSPAERELLDFSSWLGGY

SEQ ID NO: 3

MSAPAARSASGAEAHRSRALSSPLTSWRSRVARAPQDSARLRSRCRPTSRRNAGSRAPSCPRGPGTKKRGR

ARRRPGWSLAARGAQTAARPAASALPPARCARRRARPAGAAARGCTPRLSAASPPCSASCWRRRAARAAAA

PGSPSSPASRGCARAHCAALRPLRRLRSLRWPVAAAGCSATVPGTRVSAGQRSRQGRGAQGARTWAVCRRP

SRLHPPARSRSRRAAGRCRQRNRRRRGKLWRPKGASGTAPPGNSPGHAS

PL 209 127 B1

SEQ Π) NO: 4

GTACCTTGCTTTGGGGGCGCACTAAGTACCTGCCGGGAGCAGGGGGCGCACCGGGAACTCGCAGATTTCGCC

AGTTGGGCGCACTGGGGATCTGTGGACTGCGTCCGGGGGATGGGCTAGGGGGACATGCGCACGCTTTGGGCC

TTACAGAATGTGATCGCGCCGAGGGGGAGGGCCGAAGCGTGGCGGGAGGGCGAGGCGAAGGAAGGAGGGCGT GAGAAAGGCGACGGCGGCGGCGCGGAGGAGGGTTATCTATACATTTAAAAACCAGCCGCCTGCGCCGCGCCT GCGGAGACCTGGGAGAGTCCGGCCGCACGCGCGGGACACGAGCGTCCCACGCTCCCTGGCGCGTACGGCCTG CCACCACTAGGCCTCCTATCCCCGGGCTCCAGACGACCTAGGACGCGTGCCCTGGGGAGTTGCCTGGCGGCG CCGTGCCAGAAGCCCCCTTGGGGCGCCACAGTTTTCCCCGTCGCCTCCGGTTCCTCTGCCTGCACCTTCCTG

CGGCGCGCCGGGACCTGGAGCGGGCGGGTGGATGCAGGCGCGatggacggcggcacactgcccaggtccgcg ccccctgcgccccccgtccctgtcggctgcgctgcccggcggagacccgcgtccccggaactgttgcgctgc agccggcggcggcgaccggccaccgcagagaccggaggcggcgcagcggccgtagcgcggcgcaatgagcgc gagcgcaacegcgtgaagctggtgaacttgggcttccaggcgctgcggcagcacgtgccgcacggcggcgcc agcaagaagctgagcaaggtggagacgctgcgctcagccgtggagtacatccgcgcgctgcagcgcctgctg gccgagcacgacgccgtgcgcaacgcgctggcgggagggctgaggccgcaggccgtgcggccgtctgcgccc cgcgggccgccagggaccaccccggtcgccgcctcgccctcccgcgcttcttcgtccccgggccgcgggggc agctcggagcccggctccccgcgttccgcctactcgtcggacgacagcggctgcgaaggcgcgctgagtcct gcggagcgcgagctactcgacttctccagctggttagggggctactgaGCGCCCTCGACCTAATAAGCCTCA AGCCCCGGAAACCCGAGCGAACGGGCCGGCGCGCTTCATCGCCGGGGAAGCCCGCCAAGGTGGACCGGGCCC

GCGCTCCGCCCCCAGCGAGCCGGGGACCCACCCACCACCCCCCGCACCGCCGACGCCGCCTCGTTCGTCCGG CCCAGCCTGACCAATGCCGCGGTGGAAACGGGCTTGGAGCTGGCCCCATAAGGGCTGGCGGCTTCCTCCGAC GCCGCCCCTCCCCACAGCTTCTCGACTGCAGTGGGGCGGGGGGCACCAACACTTGGAGATTTTTCCGGAGGG GAGAGGATTTTCTAAGGGCACAGAGAATCCATTTTCTACACATTAACTTGAGCTGCTGGAGGGACACTGCTG GCAAACGGAGACCTATTTTTGTACAAAGAACCCTTGACCTGGGGCGTAATAAAGATGACCTGGACCCCTGCC

CCCACTATCTGGAGTTTTCCATGCTGGCCAAGATCTGGACACGAGCAGTCCCTGAGGGGCGGGGTCCCTGGC GTGAGGCCCCCGTGACAGCCCACCCTGGGGTGGGTTTGTGGGCACTGCTGCTCTGCTAGGGAGAAGCCTGTG TGGGGCACACCTCTTCAAGGGAGCGTGAACTTTATAAATAAATCAGTTCTGTTTAAAAAAAAAAAAAAAAAA AAAACCGAGGGGGGGCCCGGAGCCAACAAA

SEQ Π) NO: 5

GGTAAACAGAACTGATTTATTTATAAAGTTCACGCTCCCTTGAAGAGGTGTGCCCCACACAGGCTTCTCCC

TAGCAGAGCAGCAGTGCCCACAAACCCACCCCAGGGTGGGCTGTCACGGGGGCCTCACGCCAGGGACCCCG

CCCCTCAGGGACTGCTCGTGTCCAGATCTTGGCCAGCATGGAAAACTCCAGATAGTGGGGGCAGGGGTCCA

GGTCATCTTTATTACGCCCCAGGTCAAGGGTTCTTTGTACAAAAATAGGTCTCCGTTTGCCAGCAGTGTCC

5 CTCCAGCAGCTCAAGTTAATGTGTAGAAAATGGATTCTCTGTGCCCTTAGAAAATCCTCTCCCCTCCGGAA AAATCTCCAAGTGTTGGTGCCCCCCGCCCCACTGCAGTCGAGAAGCTGTGGGGAGGGGCGGCGTCGGAGGA AGCCGCAGCCCATTATGGGGCCAGCTCCAAGCCCGTTTCCACCGCGGCATTGGTCAGGCTGGGCGGACGAA CGAGGCGGCGTCGGCGGTGCGGGGGGTGGTGGGTGGGTCCCCGGCTCGCTGGGGGCGGAGCAGCGGGCCGG TCCACCTGGCGGGCTCCCC

PL 209 127 B1

SEQ Π) NO: 6 _{TTTTTTTTTTTTTTTTTTTAAACAgAACTGATTTA7TTATAAAGTTCACGCTCCCTTGAAGAGGTGTGCCCC}5 ACACAGGCTTCTCCCTAGCAGAGCAGCAGTGCCCACAAACCCACCCCAGGGTGGGCTGTCACGGGGGCCTCA

CGCCAGGGACCCCGCCCCTCAGGGACTGCTCGTGTCCAGATCTTGGCCAGCATGGAAAACTCCAGATAGTGG

GGGCAGGGGTCCAGGTCATCTTTATTACGCCCCAGGTCAAGGGTTCTTTGTACAAAAATAGGTCTCCGTTTG

CCAGCAGTGTCCCTCCAGCAGCTCAAGTTAATGTGTAGAAAATGGATTCTCTGTGCCCTTAGAAAATCCTCT

CCCCTCCGGAAAAATCTCCAAGTGTTGGTGCCCCCCGCCCCACTGCAGTCGAGAAGCTGTGGGGAGGGGCGG

CGTCGGAGGAAGCCGCCAGCCCTTATGGGGCCAGCTCCAAGCCCGTTTCCACCGCGGCATTGGTCAGGCTGG GCCGGACGAACGAGGCGGCGTCGGCGGTGCGGGGGGTGGTGGGTGGGTCCCCGGCTCGCTGGGGGCGGAGCG CGGGCCGGTCCACCTGGCGGGCTCCCCGGCGATGAGCGCGCCGGCCGCTCGCTCGGCTTCCGGGGCTGAGGC TCATAGGTCGAGGGCGCTCAGTAGCCCCCTAACCAGCTGGAGAAGTCGAGTAGCTCGCGCTCCGCAGGACTC AGCGCGCCTTCGCAGCCGCTGTCGTCCGACGAGTAGGCGGAACGCGGGGAGCCGGGCTCCGAGCTGCCCCCG

CGGCCCGGGGACGAAGAAGCGCGGGAGGGCGAGGCGGCGACCGGGGTGGTCCCTGGCGGCCCGCGGGGCGCA GACGGCCGCACGGCCTGCGGCCTCAGCCCTCCCGCCAGCGCGTTGCGCACGGCGTCGTGCTCGGCCAGCAGG CGCTGCAGCGCGCGGATGTACTCCACGGCTGAGCGCAGCGTCTCCACCTTGCTCAGCTTCTTGCTGGCGCCG CCGTGCGGCACGTGCTGCCGCAGCGCCTGGAAGCCCAAGTTCACCAGCTTCACGCGGTTGCGCTCGCGCTCA TTGCGCCGCGCTACGGCCGCTGCGCCGCCTCCGGTCTCTGCGGTGGCCGGTCGCCGCCGCCGGCTGCAGCGC

AACAGTTCCGGGGACGCGGGTCTCCGCCGGGCAGCGCAGCCGACAGGGACGGGGGGCGCAGGGGGCGCGGAC CTGGGCAGTGTGCCGCCGTCCATCGCGCCTGCATCCACCCGCCCGCTCCAGGTCCCGGCGCGCCGCAGGAAG GTGCAGGCAGAGGAACCGGAGGCGACGGGGAAAACTGTGGCGCCCCAAGGGGGCTTCTGGCACGGCGCCGCC AGGCAACTCCCCAGGGCACGCGTCCTAGGTCGTCTGGAGCCCGGGGATAGGAGGCCTAGTGGTGGCAGGCCG TACGCGCCAGGGAGCGTGGGACGCTCGTGTCCCGCGCGTGCGGCCGGACTCTCCCAGGTCTCCGCAGGCGCG

GCGCAGGĆGGCTGGTTTTTAAATGTATAGATAACCCTCCTCCGCGCCGCCGCCGTCGCCTTTCTCACGCCCT CCTTCCTTCGCCTCGCCCTCCCGCCACGCTTCGCCCTCCCCCTCGCGCGATCACATTCTGTAAGGCCCAAAG CGTGCGCATGTCCCCCTAGCCCATCCCCCGGACGCAGTCCACAGATCCCCAGTGCGCCCAACTGGCGAAATC TGCGAGTTCCCGGTGCGCCCCCTGCTCCCGGCAGGTACTTAGTGCGCCCCCAAAGCAAGGTAC

SEQ ID NO: 7

MCRKWILCALRKSSPLRKNLQVLVPPAPLQSRSCGEGRRRRKPPALMGPAPSPFPPRHWSGWAGRTRRRRR CGGWWVGPRLAGGGARARSTLAGFPGDEARRPVRSGFRGLRLIRSRALSSPLTSWRSRVARAPQDSARLRS RCRPTSRRNAGSRAPSCPRGPGTKKRGRARRRPGWSLAARGAQTAARFAASALPPARCARRRARPAGAAAR GCTPRLSAASPPCSAS CWRRRAARAAAAPGS PSS PASRGCARAHCAALRPLRRLRS LRWPVAAAGCSATVP

GTRVSAGQRSRQGRGAQGARTWAVCRRPSRLHPPARSRSRRAAGRCRQRNRRRRGKLWRPKGASGTAPPGN SPGHAS

PL 209 127 B1

SEQ Π) NO: 8

ATGGATCCAAACACTGTGTCAAGCTTTCAGGTAGATTGCTTTCTTTGGCATGTCCGCAAACGAGTTGCAGAC

CAAGAACTAGGTGATGCCCCATTCCTTGATCGGCTTCGCCGAGATCAGAAATCCCTAAGAGGAAGGGGCAGC

ACcCTcGGTCTGGACATCGAGACAGCCACACGTGCTGGAAAGCAGATAGtGGAGCGGAttctGAAAGAAGAA

TCCGATGAGGCACTTAAAATGACCATGGACGGCGGCACACTGCCCAGGTCCGCGCCCCCTGCGCCCCCCGTC

CCTGTCGGCTGCGCTGCCCGGCGGAGACCCGCGTCCCCGGAACTGTTGCGCTGCAGCCGGCGGCGGCGACCG GCCACCGCAGAGACCGGAGGCGGCGCAGCGGCCGTAGCGCGGCGCAATGAGCGCGAGCGCAACCGCGTGAAG CTGGTGAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAGCAAGAAGCTGAGCAAG GTGGAGACGCTGCGCTCAGCCGTGGAGTACATCCGCGCGCTGCAGCGCCTGCTGGCCGAGCACGACGCCGTG CGCAACGCGCTGGCGGGAGGGCTGAGGCCGCAGGCCGTGCGGCCGTCTGCGCCCCGCGGGCCGCCAGGGACC

ACCCCGGTCGCCGCCTCGCCCTCCCGCGCTTCTTCGTCCCCGGGCCGCGGGGGCAGCTCGGAGCCCGGCTCC CCGCGTTCCGCCTACTCGTCGGACGACAGCGGCTGCGAAGGCGCGCTGAGTCAGCGCTGAGTCAGCGCTGAGTCAGCGCTGAGTCTACGCGcat

SEQ Π) NO: 9

ATGGATGCAAACACTGTGTCAAGCTTTCAGGTAGATTGCTTTCTTTGGCATGTCCGCAAACGAGTTGCAGA CCAAGAACTAGGTGATGCCCCATTCCTTGATCGGCTTCGCCGAGATCAGAAATCCCTAAGAGGAAGGGGCA GCACCCTCGGTCTGGACATCGAGACAGCCACACGTGCTGGAAAGCAGATAGTGGAGCGGATTCTGAAAGAA GAATCCGATGAGGCA.CTTAAAATGACCATGGACGGCGGCACCCTGCCGCGTTCCGCGCCGCCGGCGCCGCC AGTTCCGGTTGGCTGCGCTGCCCGTCGCCGTCCCGCGTCCCCGGAACTGCTGCGCTGCAGCCGTCGCCGTC

GCCCGGCCACCGCAGAGACCGGAGGCGGCGCAGCGGCCGTAGCGCGGCGCAATGAGCGCGAGCGCAACCGC GTGAAGCTGGTGAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAGCAAGAAGCT GAGCAAGGTGGAGACGCTGCGCTCAGCCGTGGAGTACATCCGCGCGCTGCAGCGCCTGCTGGCCGAGCACG ACGCCGTGCGCAACGCGCTGGCGGGAGGGCTGAGGCCGCAGGCCGTGCGGCCGTCTGCGCCCCGCGGGCCG CCAGGGACCACCCCGGTCGCCGCCTCGCCCTCCCGCGCTTCTTCGTCCCCGGGCCGCGGGGGCAGCTCGGA

GCCCGGCTCCCCGCGTTCCGCCTACICGTCGGACGACAGCGGCTGCGAAGGCGCGCTGAGTCCTGCGGAGC GCGAGCTACTCGACTrCTCCAGCTGGTTAGGGGGCTACACTAGTGGCCACCATCATTAA

SEQIDO: 10

MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIE "ATRAGKQIVERILKEE

S DEALKMTMEGGTLPRSAP PAP PVPVGCAARRRPAS PELLRCS RRRRPATAETGGGAAAVARRNERERNRVK

LVNLGFQALRQHVPHGGAS KKLS KVETLRSAVEYI RALQRLLAEHDAVRNALAGGLRPQAVRP S APRG P PGT TPVAASPSRASSSPGRGGSSEPGSPRSAYSSDDSGCEGALSPAERELLDFSSWLGHHYTSG

SEQIDNO: 11

MYSTASRSVSTLLSFLLAPPCGTCCRSAWKPKFTSFTRLRSRSLRRATAAAPPPVSAVAGRRRRLQRNSSG DAGLRRAAQPTGTGGAGGADLGSVPPSIAPASTRPLQVPARSRKVQAEEPEATGKTVAPQQLGPGVDRAPSARQAGPGVDRAPSARQAGPGVDRAPSARQAGPGVDRAGARGPGVDRAGARG

PL 209 127 B1

SEQ ID NO: 12

MEAHLDWYGVPGLQEASDACPRESCSSALPEAREGANVHFPPHPVPREHFSCAAPELVAGAQGLNASLMDG

GALPRLMPTSSGVAGACAARRRQASPELLRCSRRRRSGATEASSSSAAVARRNERERNRVKLVNLGFQALR

QHVPHGGANKKLSKVETLRSAVEYIRALQRI, LAEHDAVRAALAGGLLTPATPPSDECAQPSASPASASLSC

ASTSPSPDRLGCSEPTSPRSAYSSEESSCEGELSPMEQELLDFSSWLGGY

SEQ ID NO: 13

GCCCGGAGCATGGAAGCACGTCAGCTAGGCCATGAACTGCACCCGGGAGGGGTGGGGGTGGAAGCGCACGG

TGTCAGCTTTGCAGAATGTGTACACCAAGGGGAGGGCGAGGCGAAGGAAGGAGGGCGTAAGAAAGGAGGCG

GGGAGCGCCTGACAGCACGCGCGGGACACGAGAGTACCACGCTTCCCTACTCTTTTCAGACCTTGACTGGT

ACGGGGTCCCAGGACTGCAGGAGGCCAGCGACGCGTGCCCTAGGGAGTCCTGCAGCAGTGCCCTGCCTGAG

GCCCGTGAAGGTGCAAACGTCCACTTCCCACCGCACCCGGTTCCTCGCGAGCACTTTTCCTGTGCCGCACC

AGAACTCGTAGCAGGGGCCCAGGGGCTGAATGCAAGCTTGATGGACGGCGGCGCGCTGCCCAGACTCATGC

CCACCTCGTCTGGAGTCGCTGGAGCCTGCGCTGCTCGGCGGAGACAAGCGTCTCCGGAATTGCTGCGCTGC AGCCGGCGGCGGCGATCTGGAGCAACCGAGGCCAGCAGCAGCTCGGCGTCCGTGGCACGCCGCAATGAGCG CGAGCGCAACCGCGTAAAGCTGGTAAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCG CCAACAAGAAGCTGAGTAAGGTGGAGACGCTGCGCTCCGCGGTAGAGTACATTCGTGCGCTGCAGCGGCTG CTCGCAGAGCACGACACGGTGCGGCCGGNGCTCGCTGGGGGGCTGTTAACACCCGCTACTCCGCCGTCCGA

TGAGTGCACGCAGCCCTCTGCCTCCCCTGCCAGCGGGTCTCTGTCCTGCGCCTCTACGTCTCCGTCCCGGA CCCTGGGCTGCTCTGAGCCTACCTCCCCGCGCTCCGCCTACTCGTCGGGAGGAAAGCAGCAGCAGTGCGTCGGAGTGCGTCAGCAGTAGGCGTA

SEQ ID NO: 14

MESHFNWYGVPRLQKASDACPRESCSSALPEAREGANVKFPPHPVPREHFSCGA? KPVAGAPALNASLMDG GALPRLVPTSSGVAGACTARRRPPSPELLRCSRRRRSGATEASSSSAAVARRNERERNRVKLVNLGFQALR QHVPHGGANKKLSKVETLRSAVEYIRALQRLLAEHDAVRAALSGGLLTPATRPSDVCTQPSASPASASLSC TSTSPDRLGCSEPAS PRSAYSSEDS S CEGETYPMGQMFDFSNWLGGY

SEQ Π) NO-.15

TTCACCCGGCTGCAAGCGCTAGGTGTACGGAGACCTGGCAGCTCTTGGGGCTTAAGGACTGAGCRCCAGAG

CCGGTGGAGGTTCCTGTGGAGTACATTCGGACCCTCTCACAGCCCCCGAGAGTGCGGGACGTGCGGAGCGC

AGTTCGGGATCTGCACTCGAGGACTTGTCGAGGACGCATTAAGCTAAGCATCTGCTCGGAGCATGGAATCG

CACTTTAACTGGTACGGGGTCCCAAGGCTCCAGAAGGCTAGCGACGCGTGCCCTAGGGAATCCTGCAGCAG

TGCCCTGCCTGAGGCCCGTGAAGGTGCGAACGTCCACTTCCCACCGCACCCGGTTCCTCGCGAGCACTTTT CCTGTGGCGCACCGAAACCCGTAGCGGGGGCCCCGGCGCTGAATGCAAGCTTGATGGACGGCGGCGCGCTG CCCAGACTCGTGCCCACCTCGTCTGGAGTCGCTGGAGCCTGCACTGCTCGGCGGAGACCCCCGTCCCCGGA

PL 209 127 B1

ACTGCTTCGCTGCAGCCGACGGCGGCGATCGGGAGCAACCGAGGCCAGCAGCAGCTCGGCGGCCGTGGCAC

GCCGCAATGAGCGTGAGCGCAACCGCGTAAAGCTGGTAAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTG

CCGCACGGCGGCGCCAACAAGAAGCTGAGTAAGGTGGAGACGCTGCGCTCCGCGGTAGAGTACATCCGTGC

GCTGCAGCGGCTGCTAGCAGAGCACGACGCGGTGCGTGCTGCGCTCTCTGGGGGTCTATTAACACCCGCTA

CTCGGCCGTCCGATGTGTGCACGCAGCCCTCCGCCTCCCCTGCCAGCGCGTCTCTGTCCTGCACCTCTACA TCCCCAGACCGCCTAGGCTGCTCCGAGCCTGCCTCTCCGCGCTCCGCCTACTCGTCGGAGGACAGCAGCTG CGAGGGAGAGACTTACCCGATGGGGCAGATGTTTGACTTTTCCAATTGGTTAGGGGGCTACTGAGCACCCC ACACCCCTAAGCTGCGTCCCTGGGTGTCCCCTGGTGGACCTACCTGCGTTTCTTGCCCAGGAAACCTGGGC CCATGCCTTACCCATGCTGTCTAGTGCAGCCTGACCAAATGCCAAGTACTGACCTCTGCTCGGCCTCCACG

CCGCGGAATGACATCTTCCATCTCCCAGTCCTTGCCGAACCAGGACTTGGAAATTTCTCAGGAGAAAGAAT TTTACAATGACAATCTGCTTTTTATCAATTAACTTGAACTGCTGGAGGACTCTGerGAAAATATGAAGAAT TATTTTTATACAAAGGATCCTTAAGCTTGGAGCACAATAAAGATGACCTCTGTCTCTCACCCCCACTGTCT AGAACTTTCCAACCTGGCCAAAGTGTGGACGGGTCGGGCCCTGAGGGCAAGATGCCTGGCTGCACCCTTCT TCCTCTTCCGAAGCCTATCCTGACGCTGATGTTTGGCCAGTGTGGGAACCCTGCTATTGCAAAGTGTACTA

TTCTATAAAAGTTGTTTTTCATTGGAAAGGAATTC

SEQ ID NO: 16 KLVNlGFQAL

SEQ Π) NO: 17 ELLDFSSWL

SEQ Π) NO: 18 RLLAEHDAV

SEQ Π) NO: 19

KLVNLGFQA

SEQIDNO: 20

EYIRALQRL

SEQ Π) NO: 21 EYIRALQRLL

SEQIDNO: 22 AVRNALAGGL

PL 209 127 B1

SEQ ID NO: 23

SEPGSPRSAY

SEQIDO: 24

VETLRSAVEY

SEQ ID NO: 25

ZRALQRLLA

SEQIDO: 26

LRPQAVRPS

SEQIDO: 27

LRQHVPHGG

SEQIDO: 28

LGFQALRQH

SEQ Π) NO: 29

VRNALAGGL

SEQ ID NO: 30

YIRALQRLL

SEQ ID NOt31 LVNLGFQAL

SEQIDNOt32

VEYIRALQR

SEQIDNOt33

LLRCSRRRR

PL 209 127 B1

<td></td><td>List</td><td>sequence</td>

<td> &lt;110&gt;</td><td colspan="2">SmithKline Beecham Biologicals sa</td>

<td> &lt;120</td><td>New relationships</td><td></td>

<td> &lt;130</td><td>BC45300</td><td></td>

<td> &lt;160</td><td> 32</td><td></td>

<td> &lt;170</td><td>FastSEQ for Windows</td><td>version 3.0</td>

<td> &lt;210</td><td> 1</td><td></td>

<td> &lt;211&gt;</td><td> 1791</td><td></td>

<td> &lt;212&gt;</td><td>GOUT</td><td></td>

<td> &lt;213&gt;</td><td>Man</td><td></td>

<td> &lt;400</td><td> 1</td><td></td>

gtaccttgct ttgggggcgc actaagtacc tgccgggagc agggggcgca ccgggaactc 60 gcagatttcg ccagttgggc gcactgggga tctgtggact gcgtccgggg gatgggctag 120 ggggacatgc gcacgctttg ggccttacag aatgtgatcg cgcgaggggg agggcgaagc 180 gtggcgggag ggcgaggcga aggaaggagg gcgtgagaaa ggcgacggcg gcggcgcgga 240 ggagggttat ctatacattt aaaaaccagc cgcctgcgcc gcgcctgcgg agacctggga 300 gagtccggcc gcacgcgcgg gacacgagcg tcccacgctc cetggcgcgt acggcctgcc 360 accactaggc ctcctatccc cgggctccag acgacctagg acgcgtgccc tggggagttg 420 cctggcggcg ccgtgccaga agcccccttg gggcgccaca gttttccccg tcgcctccgg 480 ttcctctgcc tgcaccttcc tgcggcgcgc cgggacctgg agcgggcggg tggatgcagg 540 cgcgatggac ggcggcacac tgcccaggtc cgcgccccct gcgccccccg tccctgtcgg 600 ctgcgctgcc cggcggagac ccgcgtcccc ggaactgttg cgctgcagcc ggcggcggcg 660 accggccacc gcagagaccg gaggcggcgc agcggccgta gcgcggcgca atgagcgcga 720 gcgcaaccgc gtgaagctgg tgaacttggg cttccaggcg ctgcggcagc acgtgccgca 780 cggcggcgcc agcaagaagc tgagcaaggt ggagacgctg cgctcagccg tggagtacat 840 ccgcgcgctg cagcgcctgc tggccgagca cgacgccgtg cgcaacgcgc tggcgggagg 900 gctgaggccg caggccgtgc ggccgtctgc gccccgcggg ccgccaggga ccaccccggt 960 cgccgcctcg ccctcccgcg cttcttcgtc cccgggccgc gggggcagct cggagcccgg 1020 ctccccgcgt tccgcctact cgtcggacga cagcggctgc gaaggcgcgc tgagtcctgc 1080 ggagcgcgag ctactcgact tctccagctg gttagggggc tactgagcgc cctcgaccta 1140 tgagcctcag ccccggaagc cgagcgagcg gccggcgcgc tcatcgccgg ggagcccgcc 1200 aggtggaccg gcccgcgctc cgcccccagc gagccgggga cccacccacc accccccgca 1260 ccgccgacgc cgcctcgttc gtccggccca gcctgaccaa tgccgcggtg gaaacgggct 1320 tggagctggc cccataaggg ctggcggctt cctccgacgc cgcccctccc cacagcttct 1380 cgactgcagt ggggcggggg gcaccaacac ttggagattt ttccggaggg gagaggattt 1440 tctaagggca cagagaatcc attttctaca cattaacttg agctgctgga gggacactgc 1500 tggcaaacgg agacctattt ttgtacaaag aacccttgac ctggggcgta ataaagatga 1560 cctggacccc tgcccccact atctggagtt ttccatgctg gccaagatct ggacacgagc 1620 agtccctgag gggcggggtc cctggcgtga ggcccccgtg acagcccacc ctggggtggg 1680 tttgtgggca ctgctgctct gctagggaga agcctgtgtg gggcacacct cttcaaggga 1740 gcgtgaactt tataaataaa tcagttctgt ttaaaaaaaa aaaaaaaaaa a 1791 &lt;210&gt; 2 &lt;211&gt; 193 &lt;212&gt; PRT &lt;213&gt;

<td>Underworld 1</td><td>Asp</td><td>Gly</td><td>Gly</td><td>Thr 5</td><td>Leu</td><td>Pro</td><td>Arg</td><td>Cheese</td><td>Ala 10</td><td>Pro</td><td>Pro</td><td>Ala</td><td>Pro</td><td>Pro 15</td><td>Val</td>

<td>Pro</td><td>Val</td><td>Gly</td><td>Cys twenty</td><td>Ala</td><td>Ala</td><td>Arg</td><td>Arg</td><td>Arg 25</td><td>Pro</td><td>Ala</td><td>Cheese</td><td>Pro</td><td>Glu thirty</td><td>Leu</td><td>Leu</td>

<td>Arg</td><td>Cys</td><td>Cheese 35</td><td>Arg</td><td>Arg</td><td>Arg</td><td>Arg</td><td>Pro 40</td><td>Ala</td><td>Thr</td><td>Ala</td><td>Glu</td><td>Thr 45</td><td>Gly</td><td>Gly</td><td>Gly</td>

PL 209 127 B1

<td>Ala</td><td>Ala 50</td><td>Ala</td><td>Val</td><td>Ala</td><td>Arg</td><td>Arg 55</td><td>Asn</td>

<td>Leu 65</td><td>Val</td><td>Asn</td><td>Leu</td><td>Gly</td><td>Phe 70</td><td>Gin</td><td>Ala</td>

<td>Gly</td><td>Ala</td><td>Cheese</td><td>Lys</td><td>Lys 85</td><td>Leu</td><td>Cheese</td><td>Lys</td>

<td>Glu</td><td>Tyr</td><td>How much</td><td>Arg 100</td><td>Ala</td><td>Leu</td><td>Gin</td><td>Arg</td>

<td>Arg</td><td>Asn</td><td>Ala 115</td><td>Leu</td><td>Ala</td><td>Gly</td><td>Gly</td><td>Leu 120</td>

<td>Ala</td><td>Pro 130</td><td>Arg</td><td>Gly</td><td>Pro</td><td>Pro</td><td>Gly 135</td><td>Thr</td>

<td>Arg 145</td><td>Ala</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Pro ISO</td><td>Gly</td><td>Arg</td>

<td>Pro</td><td>Arg</td><td>Cheese</td><td>Ala</td><td>Tyr 165</td><td>Cheese</td><td>Cheese</td><td>Asp</td>

<td>Cheese</td><td>Pro</td><td>Ala</td><td>Glu 180</td><td>Arg</td><td>Glu</td><td>Leu</td><td>Leu</td>

Tyr

<td>Glu</td><td>Arg</td><td>Glu</td><td>Arg 60</td><td>Asn</td><td>Arg</td><td>Val</td><td>Lys</td>

<td>Leu</td><td>Arg</td><td>Gin 75</td><td>His</td><td>Val</td><td>Pro</td><td>His</td><td>Gly 80</td>

<td>Val</td><td>Glu 90</td><td>Thr</td><td>Leu</td><td>Arg</td><td>Cheese</td><td>Ala 95</td><td>Val</td>

<td>Leu 105</td><td>Leu</td><td>Ala</td><td>Glu</td><td>His</td><td>Asp 110</td><td>Ala</td><td>Val</td>

<td>Arg</td><td>Pro</td><td>Gin</td><td>Ala</td><td>Val 125</td><td>Arg</td><td>Pro</td><td>Cheese</td>

<td>Thr</td><td>Pro</td><td>Val</td><td>Ala 140</td><td>Ala</td><td>Cheese</td><td>Pro</td><td>Cheese</td>

<td>Gly</td><td>Gly</td><td>Cheese 155</td><td>Cheese</td><td>Glu</td><td>Pro</td><td>Gly</td><td>Cheese 160</td>

<td>Asp</td><td>Cheese 170</td><td>Gly</td><td>Cys</td><td>Glu</td><td>Gly</td><td>Ala 175</td><td>Leu</td>

<td>Asp 185</td><td>Phe</td><td>Cheese</td><td>Cheese</td><td>Trp</td><td>Leu 190</td><td>Gly</td><td>Gly</td>

&lt;210&gt; 3 &lt;211&gt; 2S2 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 3

<td>Underworld 1</td><td>Cheese</td><td>Ala</td><td>Pro</td><td>Ala 5</td><td>Ala</td><td>Arg</td><td>Cheese</td><td>Ala</td><td>Cheese 10</td><td>Gly</td><td>Ala</td><td>Glu</td><td>Ala</td><td>His 15</td><td>Arg</td>

<td>Cheese</td><td>Arg</td><td>Ala</td><td>Leu twenty</td><td>Cheese</td><td>Cheese</td><td>Pro</td><td>Leu</td><td>Thr 25</td><td>Cheese</td><td>Trp</td><td>Arg</td><td>Cheese</td><td>Arg thirty</td><td>Val</td><td>Ala</td>

<td>Arg</td><td>Ala</td><td>Pro 35</td><td>Gin</td><td>Asp</td><td>Cheese</td><td>Ala</td><td>Arg 40</td><td>Leu</td><td>Arg</td><td>Cheese</td><td>Arg</td><td>Cys 45</td><td>Arg</td><td>Pro</td><td>Thr</td>

<td>Cheese</td><td>Arg 50</td><td>Arg</td><td>Asn</td><td>Ala</td><td>Gly</td><td>Cheese 55</td><td>Arg</td><td>Ala</td><td>Pro</td><td>Cheese</td><td>Cys 60</td><td>Pro</td><td>Arg</td><td>Gly</td><td>Pro</td>

<td>Gly 65</td><td>Thr</td><td>Lys</td><td>Lys</td><td>Arg</td><td>Gly 70</td><td>Arg</td><td>Ala</td><td>Arg</td><td>Arg</td><td>Arg 75</td><td>Pro</td><td>Gly</td><td>Trp</td><td>Cheese</td>

<td>Ala</td><td>Ala</td><td>Arg</td><td>Gly</td><td>Ala 85</td><td>Gin</td><td>Thr</td><td>Ala</td><td>Ala</td><td>Arg 90</td><td>Pro</td><td>Ala</td><td>Ala</td><td>Cheese</td><td>Ala 95</td>

<td>Pro</td><td>Pro</td><td>Ala</td><td>Arg 100</td><td>Cys</td><td>Ala</td><td>Arg</td><td>Arg</td><td>Arg 105</td><td>Ala</td><td>Arg</td><td>Pro</td><td>Ala</td><td>Gly 110</td><td>Ala</td>

<td>Ala</td><td>Arg</td><td>Gly 115</td><td>Cys</td><td>Thr</td><td>Pro</td><td>Arg</td><td>Leu 120</td><td>Cheese</td><td>Ala</td><td>Ala</td><td>Cheese</td><td>Pro 125</td><td>Pro</td><td>Cys</td>

<td>Ala</td><td>Cheese 130</td><td>Cys</td><td>Trp</td><td>Arg</td><td>Arg</td><td>Arg 135</td><td>Ala</td><td>Ala</td><td>Arg</td><td>Ala</td><td>Ala 140</td><td>Ala</td><td>Ala</td><td>Pro</td>

<td>Cheese 145</td><td>Pro</td><td>Cheese</td><td>Cheese</td><td>Pro</td><td>Ala 150</td><td>Cheese</td><td>Arg</td><td>Gly</td><td>Cys</td><td>Ala 155</td><td>Arg</td><td>Ala</td><td>His</td><td>Cys</td>

<td>Ala</td><td>Leu</td><td>Arg</td><td>Pro</td><td>Leu 165</td><td>Arg</td><td>Arg</td><td>Leu</td><td>Arg</td><td>Cheese 170</td><td>Leu</td><td>Arg</td><td>Trp</td><td>Pro</td><td>Val 175</td>

<td>Ala</td><td>Ala</td><td>Gly</td><td>Cy3 180</td><td>Cheese</td><td>Ala</td><td>Thr</td><td>Val</td><td>Pro 185</td><td>Gly</td><td>Thr</td><td>Arg</td><td>Val</td><td>Cheese 190</td><td>Ala</td>

<td>Gin</td><td>Arg</td><td>Cheese 195</td><td>Arg</td><td>Gin</td><td>Gly</td><td>Arg</td><td>Gly 200</td><td>Ala</td><td>Gin</td><td>Gly</td><td>Ala</td><td>Arg 205</td><td>Thr</td><td>Trp</td>

<td>Val</td><td>Cys</td><td>Arg</td><td>Arg</td><td>Pro</td><td>Cheese</td><td>Arg</td><td>Leu</td><td>His</td><td>Pro</td><td>Pro</td><td>Ala</td><td>Arg</td><td>Cheese</td><td>Arg</td>

210 215 220

Arg Arg Ala Ala Gly Arg Cys Arg Gin Arg Asn Arg Arg Arg Arg 225 230 235

Lys Leu Trp Arg Pro Lys Gly Ala Ser Gly Thr Ala Pro Pro Gly 245 250 255

Ser Pro Gly His Ala Ser 260 &lt;210&gt; 4 &lt;211&gt; 1830 &lt;212&gt; DNA ^&lt;213&gt; Man

Leu

Leu

Ala

Cheese

Gly

Ala

160

Ala

Gly

Ala

Cheese

Gly

240

Asn

GB 209 127 B1 &lt;400&gt; 4 gtaccttgct ttgggggcgc actaagtacc tgccgggagc agggggcgca ccgggaactc 60 gcagatttcg ccagttgggc gcactgggga tctgtggact gcgtccgggg gatgggctag 120 ggggacatgc gcacgctttg ggccttacag aatgtgatcg cgccgagggg gagggccgaa 180 gcgtggcggg agggcgaggc gaaggaagga gggcgtgaga aaggcgacgg cggcggcgcg 240 gaggagggtt atctatacat ttaaaaacca gccgcctgcg ccgcgcctgc ggagacctgg 300 gagagtccgg ccgcacgcgc gggacacgag cgtcccacgc tccctggcgc gtacggcctg 360 ccaccactag gcctcctatc cccgggctcc agacgaccta ggacgcgtgc cctggggagt 420 tgcctggcgg cgccgtgcca gaagccccct tggggcgcca cagttttccc cgtcgcctcc 480 ggttcctctg cctgcacctt cctgcggcgc gccgggacct ggagcgggcg ggtggatgca 540 ggcgcgatgg acggcggcac actgcccagg tccgcgcccc ctgcgccccc cgtccctgtc 600 ggctgcgctg cccggcggag acccgcgtcc ccggaactgt tgcgctgcag ccggcggcgg 660 cgaccggcca ccgcagagac cggaggcggc gcagcggccg tagcgcggcg caatgagcgc 720 gagcgcaacc gcgtgaagct ggtgaacttg ggcttccagg cgctgcggca gcacgtgccg 780 cacggcggcg ccagcaagaa gctgagcaag gtggagacgc tgcgctcagc cgtggagtac 840 atccgcgcgc tgcagcgcct gctggccgag cacgacgccg tgcgcaacgc gctggcggga 900 gggctgaggc cgcaggccgt gcggccgtct gcgccccgcg ggccgccagg gaccaccccg 960 gtcgccgcct cgccctcccg cgcttcttcg tccccgggcc gcgggggcag ctcggagccc 1020 ggctccccgc gttccgccta ctcgtcggac gacagcggct gcgaaggcgc gctgagtcct 1080 gcggagcgcg agctactcga cttctccagc tggttagggg gctactgagc gccctcgacc 1140 taataagcct caagccccgg aaacccgagc gaacgggccg gcgcgcttca tcgccgggga 1200 agcccgccaa ggtggaccgg gcccgcgctc cgcccccagc gagccgggga cccacccacc 1260 accccccgca ccgccgacgc cgcctcgttc gtccggccca gcctgaccaa tgccgcggtg 1320 gaaacgggct tggagctggc cccataaggg ctggcggctt cctccgacgc cgcccctccc 1380 cacagcttct cgactgcagt ggggcggggg gcaccaacac ttggagattt ttccggaggg 1440 gagaggattt tctaagggca cagagaatcc attttctaca cattaacttg agctgctgga 1500 gggacactgc tggcaaacgg agacctattt ttgtacaaag aacccttgac ctggggcgta 1560 ataaagatga cctggacccc tgcccccact atctggagtt ttccatgctg gccaagatct 1620 ggacacgagc agtccctgag gggcggggtc cctggcgtga ggcccccgtg acagcccacc 1680 ctggggtggg tttgtgggca ctgctgctct gctagggaga agcctgtgtg gggcacacct 1740 cttcaaggga gcgtgaactt tataaataaa tcagttctgt ttaaaaaaaa aaaaaaaaaa 1800 aaaaccgagg gggggcccgg agccaacaaa 1830 &lt;210&gt; 5 &lt;211&gt; 587 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 5 ggtaaacaga actgatttat ttataaagtt cacgctccct tgaagaggtg tgccccacac 60 aggcttctcc ctagcagagc agcagtgccc acaaacccac cccagggtgg gctgtcacgg 120 gggcctcacg ccagggaccc cgcccctcag ggactgctcg tgtccagatc ttggccagca 180 tggaaaactc cagatagtgg gggcaggggt ccaggtcatc tttattacgc cccaggtcaa 240 gggttctttg tacaaaaata ggtctccgtt tgccagcagt gtccctccag cagctcaagt 300 taatgtgtag aaaatggatt ctctgtgccc ttagaaaatc ctctcccctc cggaaaaatc 360 tccaagtgtt ggtgcccccc gccccactgc agtcgagaag ctgtggggag gggcggcgtc 420 ggaggaagcc gcagcccatt atggggccag ctccaagccc gtttccaccg cggcattggt 480 caggctgggc ggacgaacga ggcggcgtcg gcggtgcggg gggtggtggg tgggtccccg 540 gctcgctggg ggcggagcag cgggccggtc cacctggcgg gctcccc 537 &lt;210&gt; 6 &lt;211 &gt; 1791 &lt;212&gt; DNA &lt;213&gt; Human &lt;400&gt; 6 tttttttttt ttttttttta aacagaactg atttatttat aaagttcacg ctcccttgaa 60 gaggtgtgcc ccacacaggc ttctccctag cagagcagca gtgcccacaa acccacccca 120 gggtgggctg tcacgggggc ctcacgccag ggaccccgcc cctcagggac tgctcgtgtc 180 cagatcttgg ccagcatgga aaactccaga tagtgggggc aggggtccag gtcatcttta 240 ttacgcccca ggtcaagggt tctttgtaca aaaataggtc tccgtttgcc agcagtgtcc 300 ctccagcagc tcaagttaat gtgtagaaaa tggattctct gtgcccttag aaaatcctct 360 cccctccgga aaaatctcca agtgttggtg ccccccgccc cactgcagtc gagaagctgt 420 ggggaggggc ggcgtcggag gaagccgcca gcccttatgg ggccagctcc aagcccgttt 480 ccaccgcggc attggtcagg ctgggccgga cgaacgaggc ggcgtcggcg gtgcgggggg 540

GB 209 127 B1 tggtgggtgg gtccccggct cgctgggggc ggagcgcggg ccggtccacc tggcgggctc 600 cccggcgatg agcgcgccgg ccgctcgctc ggcttccggg gctgaggctc ataggtcgag 660 ggcgctcagt agccccctaa ccagctggag aagtcgagta gctcgcgctc cgcaggactc 720 agcgcgcctt cgcagccgct gtcgtccgac gagtaggcgg aacgcgggga gccgggctcc 730 gagctgcccc cgcggcccgg ggacgaagaa gcgcgggagg gcgaggcggc gaccggggtg 840 gtccctggcg gcccgcgggg cgcagacggc cgcacggcct gcggcctcag ccctcccgcc 900 agcgcgttgc gcacggcgtc gtgctcggcc agcaggcgct gcagcgcgcg gatgtactcc 960 acggctgagc gcagcgtctc caccttgctc agcttcttgc tggcgccgcc gtgcggcacg 1020 tgctgccgca gcgcctggaa gcccaagttc accagcttca cgcggttgcg ctcgcgctca 1080 ttgcgccgcg ctacggccgc tgcgccgcct ccggtctctg cggtggccgg tcgccgccgc 1140 cggctgcagc gcaacagttc cggggacgcg ggtctccgcc gggcagcgca gccgacaggg 1200 acggggggcg cagggggcgc ggacctgggc agtgtgccgc cgtccatcgc gcctgcatcc 1260 acccgcccgc tccaggtccc ggcgcgccgc aggaaggtgc aggcagagga accggaggcg 1320 acggggaaaa ctgtggcgcc ccaagggggc ttctggcacg gcgccgccag gcaactcccc 1380 agggcacgcg tcctaggtcg tctggagccc ggggatagga ggcctagtgg tggcaggccg 1440 tacgcgccag ggagcgtggg acgctcgtgt cccgcgcgtg cggccggact ctcccaggtc 1500 tccgcaggcg cggcgcaggc ggctggtttt taaatgtata gataaccctc ctccgcgccg 1560 ccgccgtcgc ctttctcacg ccctccttcc ttcgcctcgc cctcccgcca cgcttcgccc 1620 tccccctcgc gcgatcacat tctgtaaggc ccaaagcgtg cgcatgtccc cctagcccat 1680 cccccggacg cagtccacag atccccagtg cgcccaactg gcgaaatctg cgagttcccg 1740 gtgcgccccc tgctcccggc aggtacttag tgcgccccca aagcaaggta c 1791 &lt; 210&gt; 7 &lt;211&gt; 361 &lt;212&gt; PRT &lt;213&gt; human

<td></td><td colspan="2"> &lt;400&gt;</td><td> 7</td><td></td><td></td>

<td>Underworld 1</td><td>Cys</td><td>Arg</td><td>Lys</td><td>Trp 5</td><td>How much</td>

<td>Arg</td><td>Lys</td><td>Asn</td><td>Leu twenty</td><td>Gin</td><td>Val</td>

<td>Cheese</td><td>Cys</td><td>Gly 35</td><td>Glu</td><td>Gly</td><td>Arg</td>

<td>Pro</td><td>Ala 50</td><td>Pro</td><td>Cheese</td><td>Pro</td><td>Phe</td>

<td>Arg 65</td><td>Thr</td><td>Arg</td><td>Arg</td><td>Arg</td><td>Arg 70</td>

<td>Leu</td><td>Ala</td><td>Gly</td><td>Gly</td><td>Gly 85</td><td>Ala</td>

<td>Gly</td><td>Asp</td><td>Glu</td><td>Ala 100</td><td>Arg</td><td>Arg</td>

<td>Leu</td><td>How much</td><td>Arg 115</td><td>Cheese</td><td>Arg</td><td>Ala</td>

<td>Arg</td><td>Val 130</td><td>Ala</td><td>Arg</td><td>Ala</td><td>Pro</td>

<td>Arg 145</td><td>Pro</td><td>Thr</td><td>Cheese</td><td>Arg</td><td>Arg 150</td>

<td>Arg</td><td>Gly</td><td>Pro</td><td>Gly</td><td>Thr 165</td><td>Lys</td>

<td>Trp</td><td>Cheese</td><td>Leu</td><td>Ala 180</td><td>Ala</td><td>Arg</td>

<td>Cheese</td><td>Ala</td><td>Leu 195</td><td>Pro</td><td>Pro</td><td>Ala</td>

<td>Gly</td><td>Ala 210</td><td>Ala</td><td>Ala</td><td>Arg</td><td>Gly</td>

<td>Pro 225</td><td>Cys</td><td>Cheese</td><td>Ala</td><td>Cheese</td><td>Cys 230</td>

<td>Ala</td><td>Pro</td><td>Gly</td><td>Cheese</td><td>Pro 245</td><td>Cheese</td>

<td>His</td><td>Cys</td><td>Ala</td><td>Ala 260</td><td>Leu</td><td>Arg</td>

<td>Pro</td><td>Val</td><td>Ala 275</td><td>Ala</td><td>Ala</td><td>Gly</td>

<td>Cheese</td><td>Ala 290</td><td>Gly</td><td>Gin</td><td>Arg</td><td>Cheese</td>

<td>Leu</td><td>Cys</td><td>Ala</td><td>Leu 10</td><td>Arg</td><td>Lys</td>

<td>Leu</td><td>val</td><td>Pro 25</td><td>Pro</td><td>Ala</td><td>Pro</td>

<td>Arg</td><td>Arg 40</td><td>Arg</td><td>Lys</td><td>Pro</td><td>Pro</td>

<td>Pro 55</td><td>Pro</td><td>Arg</td><td>His</td><td>Trp</td><td>Cheese 60</td>

<td>Arg</td><td>Cys</td><td>Gly</td><td>Gly</td><td>Trp 75</td><td>Trp</td>

<td>Arg</td><td>Ala</td><td>Arg</td><td>Cheese 90</td><td>Thr</td><td>Leu</td>

<td>Pro</td><td>Val</td><td>Arg 105</td><td>Cheese</td><td>Gly</td><td>Phe</td>

<td>Leu</td><td>Cheese 120</td><td>Cheese</td><td>Pro</td><td>Leu</td><td>Thr</td>

<td>Gin 135</td><td>Asp</td><td>Cheese</td><td>Ala</td><td>Arg</td><td>Leu 140</td>

<td>Asn</td><td>Ala</td><td>Gly</td><td>Cheese</td><td>Arg 155</td><td>Ala</td>

<td>Lys</td><td>Arg</td><td>Gly</td><td>Arg 170</td><td>Ala</td><td>Arg</td>

<td>Gly</td><td>Ala</td><td>Gin 185</td><td>Thr</td><td>Ala</td><td>Ala</td>

<td>Arg</td><td>Cys 200</td><td>Ala</td><td>Arg</td><td>Arg</td><td>Arg</td>

<td>Cys 215</td><td>Thr</td><td>Pro</td><td>Arg</td><td>Leu</td><td>Cheese 220</td>

<td>Trp</td><td>Arg</td><td>Arg</td><td>Arg</td><td>Ala 235</td><td>Ala</td>

<td>Cheese</td><td>Pro</td><td>Ala</td><td>Cheese 250</td><td>Arg</td><td>Gly</td>

<td>Pro</td><td>Leu</td><td>Arg 265</td><td>Arg</td><td>Leu</td><td>Arg</td>

<td>Cys</td><td>Cheese 280</td><td>Ala</td><td>Thr</td><td>Val</td><td>Pro</td>

<td>Arg 295</td><td>Gin</td><td>Gly</td><td>Arg</td><td>Gly</td><td>Ala 300</td>

<td>Cheese</td><td>Cheese</td><td>Pro 15</td><td>Leu</td>

<td>Leu</td><td>Gin thirty</td><td>Cheese</td><td>Arg</td>

<td>Ala 45</td><td>Leu</td><td>Underworld</td><td>Gly</td>

<td>Gly</td><td>Trp</td><td>Ala</td><td>Gly</td>

<td>Val</td><td>Gly</td><td>Pro</td><td>Arg 80</td>

<td>Ala</td><td>Gly</td><td>Phe 95</td><td>Pro</td>

<td>Arg</td><td>Gly 110</td><td>Leu</td><td>Arg</td>

<td>Cheese 125</td><td>Trp</td><td>Arg</td><td>Cheese</td>

<td>Arg</td><td>Cheese</td><td>Arg</td><td>Cys</td>

<td>Pro</td><td>Cheese</td><td>Cys</td><td>Pro 160</td>

<td>Arg</td><td>Arg</td><td>Pro 175</td><td>Gly</td>

<td>Arg</td><td>Pro 190</td><td>Ala</td><td>Ala</td>

<td>Ala 205</td><td>Arg</td><td>Pro</td><td>Ala</td>

<td>Ala</td><td>Ala</td><td>Cheese</td><td>Pro</td>

<td>Arg</td><td>Ala</td><td>Ala</td><td>Ala 240</td>

<td>Cys</td><td>Ala</td><td>Arg 255</td><td>Ala</td>

<td>Cheese</td><td>Leu 270</td><td>Arg</td><td>Trp</td>

<td>Gly 285</td><td>Thr</td><td>Arg</td><td>Val</td>

<td>Gin</td><td>Gly</td><td>Ala</td><td>Arg</td>

PL 209 127 B1

Thr Trp Ala Val Cys Arg Arg Pro Ser Arg Leu His Pro Pro Ala Arg

305 310 . 315 320

Ser Arg Ser Arg Arg Ala Ala Gly Arg Cys Arg Gin Arg Asn Arg Arg

32S 330 335

Arg Arg Gly Lys Leu Trp Arg Pro Lys Gly Ala Ser Gly Thr Ala Pro

340 345 350

Pro Gly Asn Ser Pro Gly His Ala Ser

355 360 &lt;210&gt; 8 &lt;211&gt; 849 &lt;212&gt; DNA &lt;213&gt; Influenza viruses and human &lt;400&gt; 8 atggatccaa acactgtgtc aagctttcag gtagattgct ttctttggca tgtccgcaaa cgagttgcag accaagaact aggtgatgcc ccattccttg atcggcttcg ccgagaccag aaatccctaa gaggaagggg cagcaccctc ggtctggaca tcgagacagc cacacgtgct ggaaagcaga tagtggagcg gattctgaaa gaagaatccg atgaggcact taaaatgacc atggacggcg gcacactgcc caggtccgcg ccccctgcgc cccccgtccc tgtcggctgc gctgcccggc ggagacccgc gtccccggaa ctgttgcgct gcagccggcg gcggcgaccg gccaccgcag agaccggagg cggcgcagcg gccgtagcgc ggcgcaatga gcgcgagcgc aaccgcgtga agctggtgaa cttgggcttc caggcgctgc ggcagcacgt gccgcacggc ggcgccagca agaagctgag caaggrggag acgctgcgct cagccgtgga gtacatccgc gcgctgcagc gcctgctggc cgagcacgac gccgtgcgca acgcgctggc gggagggctg aggccgcagg ccgtgcggcc gtctgcgccc cgcgggccgc cagggaccac cccggtcgcc gcctcgccct cccgcgcttc ttcgtccccg ggccgcgggg gcagctcgga gcccggctcc ccgcgttccg cctactcgtc ggacgacagc ggctgcgaag gcgcgctgag tcctgcggag cgcgagctac tcgacttctc cagctggtta gggggctaca ctagtggcca ccatcaccat caccattaa &lt;210&gt; 9 &lt;211&gt; 849 &lt;212&gt; DNA &lt;213&gt; Influenza viruses and human &lt;400&gt; 9 atggatccaa acactgtgtc aagctttcag gtagattgct ttctttggca tgtccgcaaa cgagttgcag accaagaact aggtgatgcc ccattccttg atcggcttcg ccgagatcag aaatccctaa gaggaagggg cagcaccctc ggtctggaca tcgagacagc cacacgtgct ggaaagcaga tagtggagcg gattctgaaa gaagaatccg atgaggcact taaaatgacc atggacggcg gcaccctgcc gcgttccgcg ccgccggcgc cgccagttcc ggttggctgc gctgcccgtc gccgtcccgc gtccccggaa ctgctgcgct gcagccgtcg ccgtcgcccg gccaccgcag agaccggagg cggcgcagcg gccgtagcgc ggcgcaatga gcgcgagcgc aaccgcgtga agctggtgaa cttgggcttc caggcgctgc ggcagcacgt gccgcacggc ggcgccagca agaagctgag caaggtggag acgctgcgct cagccgtgga gtacatccgc gcgctgcagc gcctgctggc cgagcacgac gccgtgcgca acgcgctggc gggagggctg aggccgcagg ccgtgcggcc gtctgcgccc cgcgggccgc cagggaccac cccggtcgcc gcctcgccct cccgcgcttc ttcgtccccg ggccgcgggg gcagctcgga gcccggctcc ccgcgttccg cctactcgtc ggacgacagc ggctgcgaag gcgcgctgag tcctgcggag cgcgagctac tcgacttctc cagctggtta gggggctaca ctagtggcca ccatcaccat caccattaa &lt;210&gt; 10 &lt;211&gt; 282 &lt;212&gt; PRT &lt;213&gt; Influenza viruses and human

120

180

240

300

360

420

480

540

600

660

720

780

840

849

120 180 240 300 360 4 20 480 540 600 660 720 780 840 849 &lt;400&gt; 10

<td>Underworld</td><td rowspan="2">Asp</td><td>Pro</td><td>Asn</td><td>Thr</td><td>Val</td><td>Cheese</td><td>Cheese</td><td>Phe</td><td>Gin</td><td>Val</td><td>Asp</td><td>Cys</td><td>Phe</td><td>Leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>His</td><td>Val</td><td>Arg</td><td>Lys</td><td>Arg</td><td>Val</td><td>Ala</td><td>Asp</td><td>Gin</td><td>Glu</td><td>Leu</td><td>Gly</td><td>Asp</td><td>Ala</td><td>Pro</td><td>Phe</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu</td><td>Asp</td><td>Arg</td><td>Leu</td><td>Arg</td><td>Arg</td><td>Asp</td><td>Gin</td><td>Lys</td><td>Cheese</td><td>Leu</td><td>Arg</td><td>Gly</td><td>Arg</td><td>The goof</td><td>Cheese</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Thr</td><td>Leu</td><td>Gly</td><td>Leu</td><td>Asp</td><td>How much</td><td>Glu</td><td>Thr</td><td>Ala</td><td>Thr</td><td>Arg</td><td>Ala</td><td>Gly</td><td>Lys</td><td>Gin</td><td>How much</td>

PL 209 127 B1

<td></td><td> 50</td><td> 55</td><td> 60</td>

<td>Val 65</td><td>Glu Arg Ile Leu</td><td>Lys Glu 70</td><td>Glu Ser Asp Glu Ala Leu Lys Met Thr 75 80</td>

<td>Underworld</td><td>Asp Gly Gly Thr 85</td><td>Leu Pro</td><td>Arg Ser Ala Pro Pro Ala Pro Pro Val 90 95</td>

<td>Pro</td><td>Val Gly Cys Ala 100</td><td>Ala Arg</td><td>Arg Arg Pro Ala Ser Pro Glu Leu Leu 105 110</td>

<td>Arg</td><td>Cys Ser Arg Arg 115</td><td>Arg Arg</td><td>Pro Ala Thr Ala Glu Thr Gly Gly Gly 120 125</td>

<td>Ala</td><td>Ala Ala Val Ala 130</td><td>Arg Arg 135</td><td>Asn Glu Arg Glu Arg Asn Arg Val Lys 140</td>

<td>Leu 145</td><td>Val Asn Leu Gly</td><td>Phe Gin 150</td><td>Ala Leu Arg Gin His Val Pro His Gly 155 160</td>

<td>Gly</td><td>Ala Ser Lys Lys 165</td><td>Leu Ser</td><td>Lys Val Glu Thr Leu Arg Ser Ala Val 170 175</td>

<td>Glu</td><td>Tyr Ile Arg Ala 180</td><td>Leu Gin</td><td>Arg Leu Leu Ala Glu His Asp Ala Val 185 190</td>

<td>Arg</td><td>Asn Ala Leu Ala 195</td><td>Gly Gly</td><td>Leu Arg Pro Gin Ala Val Arg Pro Ser 200 205</td>

<td>Ala</td><td>Pro Arg Gly Pro 210</td><td>Pro Gly 215</td><td>Thr Thr Pro Val Ala Ala Ser Pro Ser 220</td>

<td>Arg 225</td><td>Ala Cheese Cheese Cheese</td><td>Pro Gly 230</td><td>Arg Gly Gly Cheese Ser Glu Pro Gly Ser 235 240</td>

<td>Pro</td><td>Arg Ser Ala Tyr 245</td><td>Cheese Cheese</td><td>Asp Asp Ser Gly Cys Glu Gly Ala Leu 250 255</td>

<td>Cheese Tyr</td><td colspan="2">Pro Ala Glu Arg Glu Leu 260 Thr Ser Gly His His His 275 &lt;210&gt; 11 &lt;211&gt; 193 &lt;212&gt; PRT &lt;213&gt; human &lt;400&gt; 11</td><td>Leu Asp Phe Cheese Cheese Trp Leu Gly Gly 265 270 His His His 280</td>

<td>Underworld 1</td><td>Tyr Ser Thr Ala 5</td><td>Glu Arg</td><td>Val Cheese Thr Leu Leu Cheese Phe Leu Cheese 10 15</td>

<td>Leu</td><td>Ala Pro Pro Cys 20</td><td>Gly Thr</td><td>Cys Cys Arg Ser Ala Trp Lys Pro Lys 25 30</td>

<td>Phe</td><td>Thr Ser Phe Thr 35</td><td>Arg Leu</td><td>Arg Ser Arg Ser Leu Arg Arg Ala Thr 40 45</td>

<td>Ala</td><td>Ala Ala Pro Pro 50</td><td>Pro Val 55</td><td>Cheese Ala Val Ala Gly Arg Arg Arg Arg 60</td>

<td>Leu 65</td><td>Gin Arg Asn Ser</td><td>Gly Cheese 70</td><td>Asp Ala Gly Leu Arg Arg Ala Ala Gin 75 30</td>

<td>Pro</td><td>Thr Gly Thr Gly 85</td><td>Gly Ala</td><td>Gly Gly Ala Asp Leu Gly Ser Val Pro 90 95</td>

<td>Pro</td><td>Ile Ala Pro 100 cheese</td><td>Ala Ser</td><td>Thr Arg Pro Leu Gin Val Pro Ala Arg 105 110</td>

<td>Arg</td><td>Arg Lys Val Gin 115</td><td>Ala Glu</td><td>Glu Pro Glu Ala Thr Gly Lys Thr Val 120 125</td>

<td>Ala</td><td>Pro Gin Gly Gly 130</td><td>Phe Trp 135</td><td>His Gly Ala Ala Arg Gin Leu Pro Arg 140</td>

<td>Ala 145</td><td>Arg Val Leu Gly</td><td>Arg Leu 150</td><td>Glu Pro Gly Asp Arg Arg Pro Ser Gly 155 160</td>

<td>Gly</td><td>Arg Pro Tyr Ala 165</td><td>Pro Gly</td><td>Val Gly Arg Cheese Cys Pro Ala Arg Cheese 170 175</td>

<td>Ala</td><td>Ala Gly Leu Ser</td><td>Gin Val</td><td>Cheese Ala Gly Ala Ala Gin Ala Ala Gly</td>

180 185 190

Phe &lt;210&gt; 12 &lt;211&gt; 263 &lt;212&gt; PRT

PL 209 127 B1

<td></td><td colspan="2"> &lt;213&gt;</td><td colspan="13">Mouse</td>

<td></td><td colspan="2"> &lt;400&gt;</td><td> 12</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Underworld</td><td>Glu</td><td>Ala</td><td>His</td><td>Leu</td><td>Asp</td><td>Trp</td><td>Tyr</td><td>Gly</td><td>Val</td><td>Pro</td><td>Gly</td><td>Leu</td><td>Gin</td><td>Glu</td><td>Ala</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Cheese</td><td>Asp</td><td>Ala</td><td>Cys</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Cheese</td><td>Cys</td><td>Cheese</td><td>Cheese</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Glu</td><td>Ala</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Arg</td><td>Glu</td><td>Gly</td><td>Ala</td><td>Asn</td><td>Val</td><td>His</td><td>Phe</td><td>Pro</td><td>Pro</td><td>His</td><td>Pro</td><td>Val</td><td>Pro</td><td>Arg</td><td>Glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>His</td><td>Phe</td><td>Cheese</td><td>Cys</td><td>Ala</td><td>Ala</td><td>Pro</td><td>Glu</td><td>Leu</td><td>Val</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Gin</td><td>Gly</td><td>Leu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Asn</td><td>Ala</td><td>Cheese</td><td>Leu</td><td>Underworld</td><td>Asp</td><td>Gly</td><td>Gly</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Arg</td><td>Leu</td><td>Underworld</td><td>Pro</td><td>Thr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Cheese</td><td>Cheese</td><td>Gly</td><td>Val</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Cys</td><td>Ala</td><td>Ala</td><td>Arg</td><td>Arg</td><td>Arg</td><td>Gin</td><td>Ala</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Pro</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Cys</td><td>Cheese</td><td>Arg</td><td>Arg</td><td>Arg</td><td>Arg</td><td>Cheese</td><td>Gly</td><td>Ala</td><td>Thr</td><td>Glu</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Ala</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Ala</td><td>Ala</td><td>Val</td><td>Ala</td><td>Arg</td><td>Arg</td><td>Asn</td><td>Glu</td><td>Arg</td><td>Glu</td><td>Arg</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Asn</td><td>Arg</td><td>Val</td><td>Lys</td><td>Leu</td><td>Val</td><td>Asn</td><td>Leu</td><td>Gly</td><td>Phe</td><td>Gin</td><td>Ala</td><td>Leu</td><td>Arg</td><td>Gin</td><td>His</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Val</td><td>Pro</td><td>His</td><td>Gly</td><td>Gly</td><td>Ala</td><td>Asn</td><td>Lys</td><td>Lys</td><td>Leu</td><td>Cheese</td><td>Lys</td><td>Val</td><td>Glu</td><td>Thr</td><td>Leu</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Arg</td><td>Cheese</td><td>Ala</td><td>Val</td><td>Glu</td><td>Tyr</td><td>How much</td><td>Arg</td><td>Ala</td><td>Leu</td><td>Gin</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Ala</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>His</td><td>Asp</td><td>Ala</td><td>Val</td><td>Arg</td><td>Ala</td><td>Ala</td><td>Leu</td><td>Ala</td><td>Gly</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Thr</td><td>Pro</td><td>Ala</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Thr</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Asp</td><td>Glu</td><td>Cys</td><td>Ala</td><td>Gin</td><td>Pro</td><td>Cheese</td><td>Ala</td><td>Cheese</td><td>Pro</td><td>Ala</td><td>Cheese</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Ala</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Ala</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>Pro</td><td>Cheese</td><td>Pro</td><td>Asp</td><td>Arg</td><td>Leu</td><td>Gly</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Cys</td><td>Cheese</td><td>Glu</td><td>Pro</td><td>Thr</td><td>Cheese</td><td>Pro</td><td>Arg</td><td>Cheese</td><td>Ala</td><td>Tyr</td><td>Cheese</td><td>Cheese</td><td>Glu</td><td>Glu</td><td>Cheese</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Cheese</td><td>Cys</td><td>Glu</td><td>Gly</td><td>Glu</td><td>Leu</td><td>Cheese</td><td>Pro</td><td>Underworld</td><td>Glu</td><td>Gin</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Asp</td><td>Phe</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

Ser Ser Trp Leu Gly Gly Tyr 260 &lt;210&gt; 13 &lt;211&gt; 1051 &lt;212&gt; DNA &lt;213&gt; Mouse &lt;400&gt; 13 gcccggagca tggaagcacg tcagctaggc catgaactgc acccgggagg ggtgggggtg 60 gaagcgcacg gtgtcagctt tgcagaatgt gtacaccaag gggagggcga ggcgaaggaa 120 ggagggcgta agaaaggagg cggtggcggg gcggaggaga ttatctatac tttttaaaaa 190 aaaggagcct cttagccgcg taaaggagac ttggggagcg cctgacagca cgcgcgggac 240 acgagagtac cacgcttccc tactcttttc agaccttgac tggtacgggg tcccaggact 300 gcaggaggcc agcgctccagcgcggt gccgggcc agcgctccagcgcgc gcc 360 tgaaggtgca aacgtccact tcccaccgca cccggttcct cgcgagcact tttcctgtgc 420 cgcaccagaa ctcgtagcag gggcccaggg gctgaatgca agcttgatgg acggcggcgc 480 gctgcccaga ctcatgccca cctcgtctgg agtcgctgga gcctgcgctg ctcggcggag 540 acaagcgtct ccggaattgc tgcgctgcag ccggcggcgg cgatctggag caaccgaggc 600 cagcagcagc tcggcgtccg tggcacgccg caatgagcgc gagcgcaacc gcgtaaagct 660 ggtaaacttg ggcttccagg cgctgcggca gcacgtgccg cacggcggcg ccaacaagaa 720 gctgagtaag gtggagacgc tgcgctccgc ggtagagtac attcgtgcgc tgcagcggct 780 gctcgcagag cacgacacgg tgcggccggn gctcgctggg gggctgttaa cacccgctac 840 tccgccgtcc gatgagtgca cgcagccctc tgcctcccct gccagcgggt ctctgtcctg 900 cgcctctacg tctccgtccc ggaccctggg ctgctctgag cctacctccc cgcgctccgc 960 ctactcgtcg gaggaaagca gctgcgaggg agagetaagc ccgatggagc aggagctgct tgacttttcc agttggttag ggggctactg 1020 and 1051 &lt;210&gt; 14 &lt;211&gt; 260 &lt;212&gt; PRT

PL 209 127 B1 ^&lt;213&gt; Rat

<td></td><td colspan="2"> &lt;400&gt;</td><td colspan="13"> 14</td>

<td>Underworld</td><td>Glu</td><td>Cheese</td><td>His</td><td>Phe</td><td>Asn</td><td>Trp</td><td>Tyr</td><td>Gly</td><td>Val</td><td>Pro</td><td>Arg</td><td>Leu</td><td>Gin</td><td>Lys</td><td>Ala</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Cheese</td><td>Asp</td><td>Ala</td><td>Cys</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Cheese</td><td>Cys</td><td>Cheese</td><td>Cheese</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Glu</td><td>Ala</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Arg</td><td>Glu</td><td>Gly</td><td>Ala</td><td>Asn</td><td>Val</td><td>His</td><td>Phe</td><td>Pro</td><td>Pro</td><td>His</td><td>Pro</td><td>Val</td><td>Pro</td><td rowspan="2">Arg</td><td>Glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td>

<td>His</td><td>Phe</td><td>Cheese</td><td>Cys</td><td>Gly</td><td>Ala</td><td>Pro</td><td>Lys</td><td>Pro</td><td>Val</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Pro</td><td>Ala</td><td>Leu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Asn</td><td>Ala</td><td>Cheese</td><td>Leu</td><td>Underworld</td><td>Asp</td><td>Gly</td><td>Gly</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Arg</td><td>Leu</td><td>Val</td><td>Pro</td><td>Thr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Cheese</td><td>Cheese</td><td>Gly</td><td>Val</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Cys</td><td>Thr</td><td>Ala</td><td>Arg</td><td>Arg</td><td rowspan="2">Arg</td><td>Pro</td><td>Pro</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Pro</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Arg</td><td>Cys</td><td>Cheese</td><td>Arg</td><td>Arg</td><td>Arg</td><td>Arg</td><td>Cheese</td><td rowspan="2">Gly</td><td>Ala</td><td>Thr</td><td>Glu</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Ala</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Ala</td><td>Ala</td><td>Val</td><td>Ala</td><td>Arg</td><td>Arg</td><td>Asn</td><td>Glu</td><td rowspan="2">Arg</td><td>Glu</td><td>Arg</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td>

<td>Asn</td><td>Arg</td><td>Val</td><td>Lys</td><td>Leu</td><td>Val</td><td>Asn</td><td>Leu</td><td>Gly</td><td>Phe</td><td>Gin</td><td>Ala</td><td>Leu</td><td>Arg</td><td>Gin</td><td>His</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Val</td><td>Pro</td><td>His</td><td>Gly</td><td>Gly</td><td>Ala</td><td>Asn</td><td>Lys</td><td>Lys</td><td>Leu</td><td>Cheese</td><td>Lys</td><td>Val</td><td>Glu</td><td>Thr</td><td>Leu</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Arg</td><td>Cheese</td><td>Ala</td><td>Val</td><td>Glu</td><td>Tyr</td><td>How much</td><td>Arg</td><td>Ala</td><td>Leu</td><td>Gin</td><td>Arg</td><td>Leu</td><td>Leu</td><td>Ala</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>His</td><td>Asp</td><td>Ala</td><td>Val</td><td>Arg</td><td>Ala</td><td>Ala</td><td>Leu</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Thr</td><td>Pro</td><td>Ala</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Thr</td><td>Arg</td><td>Pro</td><td>Cheese</td><td>Asp</td><td>Val</td><td>Cys</td><td>Thr</td><td>Gin</td><td>Pro</td><td>Cheese</td><td>Ala</td><td>Cheese</td><td>Pro</td><td>Ala</td><td>Cheese</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Ala</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Thr</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>Pro</td><td>Asp</td><td>Arg</td><td>Leu</td><td rowspan="2">Gly</td><td rowspan="2">Cys</td><td>Cheese</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td>

<td>Glu</td><td>Pro</td><td>Ala</td><td>Cheese</td><td>Pro</td><td>Arg</td><td>Cheese</td><td>Ala</td><td>Tyr</td><td>Cheese</td><td>Cheese</td><td>Glu</td><td rowspan="2">Asp</td><td>Cheese</td><td>Cheese</td><td>Cys</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td> 240</td>

<td>Glu</td><td>Gly</td><td>Glu</td><td>Thr</td><td>Tyr</td><td>Pro</td><td>Underworld</td><td>Gly</td><td>Gin</td><td>Underworld</td><td>Phe</td><td>Asp</td><td>Phe</td><td>Cheese</td><td>Asn</td><td rowspan="2">Trp</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td>

<td>Leu</td><td colspan="2">Gly Gly</td><td>Tyr</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 15 &lt;211&gt; 1526 &lt;212&gt; DNA &lt;213&gt; Rat &lt;400&gt; 15 ttcacccggc tgcaagcgct aggtgtacgg agacctggca gctcttgggg cttaaggact 60 gagcrccaga gccggtggag gttcctgtgg agtacattcg gaccctctca cagcccccga 120 gagtgcggga cgtgcggagc gcagttcggg atctgcactc gaggacttgt cgaggacgca 180 ttaagctaag catctgctcg gagcatggaa tcgcacttta actggtacgg ggtcccaagg 240 ctccagaagg ctagcgacgc gtgccctagg gaatcctgca gcagtgccct gcctgaggcc 300 cgtgaaggtg cgaacgtcca cttcccaccg cacccggttc ctcgcgagca cttttcctgt 360 ggcgcaccga aacccgtagc gggggccccg gcgctgaatg caagcttgat ggacggcggc 420 gcgctgccca gactcgtgcc cacctcgtct ggagtcgctg gagcctgcac tgctcggcgg 480 agacccccgt ccccggaact gcttcgctgc agccgacggc ggcgatcggg agcaaccgag 540 gccagcagca gctcggcggc cgtggcacgc cgcaatgagc gtgagcgcaa ccgcgtaaag 600 ctggtaaact tgggcttcca ggcgctgcgg cagcacgtgc cgcacggcgg cgccaacaag 660 aagctgagta aggtggagac gctgcgctcc gcggtagagt acatccgtgc gctgcagcgg 720 ctgctagcag agcacgacgc ggtgcgtgct gcgctctctg ggggtctatt aacacccgct 780 actcggccgt ccgatgtgtg cacgcagccc tccgcctccc ctgccagcgc gtctctgtcc 840 tgcacctcta catccccaga ccgcctaggc tgctccgagc ctgcctctcc gcgctccgcc 900 tactcgtcgg aggacagcag ctgcgaggga gagacttacc cgatggggca gatgtttgac 960 ttttccaatt ggttaggggg ctactgagca ccccacaccc ctaagctgcg tccctgggtg 1020 tcccctggtg gacctacctg cgtttcttgc ccaggaaacc tgggcccatg ccttacccat 1080 gctgtctagt gcagcctgac caaatgccaa gtactgacct ctgctcggcc tccacgccgc 1140 ggaatgacat cttccatctc ccagtccttg ccgaaccagg acttggaaat ttctcaggag 1200 aaagaatttt acaatgacaa tctgcttttt atcaattaac ttgaactgct ggaggactct 1260 gctgaaaata tgaagaatta tttttataca aaggatcctt aagcttggag cacaataaag 1320

GB 209 127 B1 atgacctctg tctctcaccc ccactgtcta gaactttcca acctggccaa agtgtggacg 1380 ggtcgggccc tgagggcaag atgcctggct gcacccttct tcctcttccg aagcctatcc 1440 tgacgctgat gtttggccag tgtgggaacc ctgctattgc aaagtgtact attctataaa 1500 agttgttttt cattggaaag GAATTC 1526 &lt;210&gt; 1S &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 16

Lys Leu Val Asn Leu Gly Phe Gin Ala Leu 1 S 10 &lt;210&gt; 17 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 17

Glu Leu Leu Asp Phe Ser Ser Trp Leu 1 5 &lt;210&gt; 18 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 18

Arg Leu Leu Ala Glu His Asp Ala Val 1 5 &lt;210&gt; 19 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 19

Lys Leu Val Asn Leu Gly Phe Gin Ala 1 5 &lt;210&gt; 20 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 20

Glu Tyr Ile Arg Ala Leu Gin Arg Leu 1 5 &lt;210&gt; 21 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 21

Glu Tyr Ile Arg Ala Leu Gin Arg Leu Leu 1 S 10 &lt;210&gt; 22 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 22

Ala Val Arg Asn Ala Leu Ala Gly Gly Leu

PL 209 127 B1

<td colspan="2"> 1</td><td colspan="3"> 5</td><td colspan="2"> 10</td>

<td></td><td> &lt;210&gt;</td><td> 23</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;211&gt;</td><td> 10</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;212&gt;</td><td>PRT</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;213&gt;</td><td>Man</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;400&gt;</td><td> 23</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>Glu Pro</td><td>Gly Ser Pro</td><td>Arg</td><td>Cheese</td><td>Ala</td><td>Tyr</td>

<td> 1</td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td>

<td></td><td> &lt;210&gt;</td><td> 24</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;211&gt;</td><td> 10</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;212&gt;</td><td>PRT</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;213&gt;</td><td>Man</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;400&gt;</td><td> 24</td><td></td><td></td><td></td><td></td>

<td>Val</td><td>Glu Thr</td><td>Leu Arg Ser</td><td>Ala</td><td>Val</td><td>Glu</td><td>Tyr</td>

<td> 1</td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td>

<td></td><td> &lt;210&gt;</td><td> 25</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;211&gt;</td><td> 9</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;212&gt;</td><td>PRT</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;213&gt;</td><td>Man</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;400&gt;</td><td> 25</td><td></td><td></td><td></td><td></td>

<td>How much</td><td>Arg Al</td><td>Leu Gin Arg</td><td>Leu</td><td>Leu</td><td>Ala</td><td></td>

<td> 1</td><td></td><td> 5</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;210&gt;</td><td> 26</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;211&gt;</td><td> 9</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;212&gt;</td><td>PRT</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;213&gt;</td><td>Man</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;400&gt;</td><td> 26</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Arg Pro</td><td>Gin Ala Val</td><td>Arg</td><td>Pro</td><td>Cheese</td><td></td>

<td> 1</td><td></td><td> 5</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;210&gt;</td><td> 27</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;211&gt;</td><td> 9</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;212&gt;</td><td>PRT</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;213&gt;</td><td>Man</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;400&gt;</td><td> 27</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Arg Gin</td><td>His Val Pro</td><td>His</td><td>Gly</td><td>Gly</td><td></td>

<td> 1</td><td></td><td> 5</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;210&gt;</td><td> 28</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;211&gt;</td><td> 9</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;212&gt;</td><td>PRT</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;213&gt;</td><td>Man</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;400&gt;</td><td> 28</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Gly Phe</td><td>Gin Ala Leu</td><td>Arg</td><td>Gin</td><td>His</td><td></td>

<td> 1</td><td></td><td> 5</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;210&gt;</td><td> 29</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;211&gt;</td><td> 9</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;212&gt;</td><td>PRT</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;213&gt;</td><td>Man</td><td></td><td></td><td></td><td></td>

<td></td><td> &lt;400&gt;</td><td> 29</td><td></td><td></td><td></td><td></td>

<td>Val</td><td>Arg Asn</td><td>Ala Leu Ala</td><td>Gly</td><td>Gly</td><td>Leu</td><td></td>

5 &lt;210&gt; 30

PL 209 127 B1 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Human &lt;400&gt; 30

Tyr Ile Arg Ala Leu Gin Arg Leu Leu 1 5 &lt;210 31 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Human &lt;400 31

Leu Val Asn Leu Gly Phe Gin Ala Leu 1 5 &lt;210&gt; 32 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Human &lt;40 0 32

Val Glu Tyr Ile Arg Ala Leu Gin Arg 1 5 &lt;210&gt; 33 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Human &lt;400 33

Leu Leu Arg Cy3 Ser Arg Arg Arg Arg