ES2549405T3 - Antibodies and related molecules that bind with PSCA proteins - Google Patents

Abstract

A monoclonal antibody or an antigen-binding fragment thereof comprising an antigen-binding site that specifically binds with a PSCA protein comprising the amino acid sequence of SEQ ID NO: 2, and wherein the monoclonal antibody comprises the sequence of amino acids of the VH region of SEQ ID NO: 47 and the VL region of SEQ ID NO: 51.

Description

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well and incubation continued for an additional 4-5 hours at 37 ° C. Fluorescence in each well was read using a 96-well fluorimeter with excitation at 530 nm and emission at 590 nm. The results show that PSCA antibodies that have an IgG1 isotype (HA1-4121) or an IgG2 isotype (HA1-5.99.1) but not an IgG4 isotype (HA1-6.46) were able to mediate complement-dependent lysis of target cells Figure 15. Generation of F (ab ’) 2 fragments of MAb Ha1-4121 by pepsin digestion. 20 mg of MAb Ha1-4121 were incubated in 20 mM sodium acetate buffer pH 4.5 with and without immobilized pepsin (Pierce. Rockford IL) during the indicated times. Intact MAb and Fc fragments digested by protein A chromatography were removed. A gel stained with Coomasie SDS-PAGE of unimplemented undigested MAb is shown, non-reduced aliquots of digested material taken at the indicated times and a reduced sample of the product of F (ab ') 2 final digested. Figure 16. Recombinant anti-PSCA human mAb binding with PSCA by flow cytometry. (16A) 293T cells were transfected with expression constructs encoding the heavy and light chains of human anti-PSCA mAbs. Supernatant was collected after 48 hours and tested for binding with PSCA. (16B) anti-PSCA human mAb was purified from hybridoma supernatant and used for PSCA binding assays. Binding with PSCA was tested as follows. Parental PC3 or PC3-PSCA cells were incubated with the anti-PSCA human mAbs described above for 30 minutes on ice. The cells were washed and incubated with anti-human Ig Ig conjugated with PE for 30 minutes on ice. The cells were washed and then tested by flow cytometry. Figure 17. Detection of PSCA protein by immunohistochemistry. PSCA protein expression was detected in tumor test samples of cancer patients using the HA1-4.117 antibody. Tissues included in paraffin were cut, fixed in formalin into 4 micrometer sections and mounted on glass slides. Sections were lowered, rehydrated and treated with antigen recovery solution (Citra Antigen Recovery Solution; BioGenex, 4600 Norris Canyon Road, San Ramon, CA, 94583) at room temperature. Sections were then incubated in fluorescein-conjugated human monoclonal anti PSCA antibody, Ha1-4,117, for 16 hours at 4 ° C. The slides were washed three times in buffer and further incubated with Rabbit anti Fluorescein for 1 hour and, after washing in buffer, they were immersed in secondary goat anti-rabbit immunoglobulin antibody conjugated with DAKO EnVision + ™ peroxidase (DAKO Corporation, Carpenteria , CA) for 30 minutes. Sections were then washed in buffer, developed using the DAB kit (SIGMA Chemicals), counterstained using hematoxylin, and analyzed by light field microscopy. The results show PSCA expression in prostate adenocarcinoma tumor cells (Panel A, Panel B), transitional bladder carcinoma (Panel C) and pancreatic ductal adenocarcinoma (Panel D). These results indicate that PSCA is expressed in human cancers and that antibodies directed to this antigen are useful as diagnostic reagents. Figure 18. The PSCA Ha1-4.120 MAb Inhibits the Growth of Subcutaneous Prostate Cancer Xenografts. LAPC-9AI tumor cells (2.0 x 106 cells) were injected subcutaneously into male SCID mice. Mice were randomly classified into groups (n = 10 in each group) and treatment was initiated intraperitoneally (i.p.) on Day 0 with MAb HA1-4.120 or control isotype as indicated. Animals were treated twice a week for a total of 7 doses until study day 28. Tumor growth was monitored using calibrator measurements every 3 to 4 days as indicated. The results show that the human anti-PSCA monoclonal antibody Ha1-4.120 significantly inhibited the growth of human prostate cancer xenografts implanted subcutaneously in SCID mice (p <0.05). Figure 19. PSCA Ha1-5.99 MAb Inhibits Growth of Prostate Cancer Xenografts Established in SCID mice. LAPC-9AI tumor cells (2.0 x 106 cells) were injected subcutaneously into male SCID mice. When the tumor volume reached 50 mm3, the mice were randomly classified into groups (n = 10 in each group) and the treatment was initiated intraperitoneally (i.p.) with HA1-5.99.1 or isotype control MAb as indicated. Animals were treated twice a week for a total of 5 doses until the day of study 14. Tumor growth was monitored using calibrator measurements every 3 to 4 days as indicated. The results show that the completely human anti-PSCA Ha1-5.99 monoclonal antibody significantly inhibited the growth of established androgen independent human prostate cancer xenografts implanted subcutaneously in SCID mice (p <0.05). Figure 20. The PSCA HA1-4.121 Mab Inhibits the Growth of Established Androgen-dependent Human Prostate Cancer Xenografts. LAPC-9AD tumor cells (2.5 x 10 6 cells) were injected subcutaneously into male SCID mice. When the tumor volume reached 40 mm3, the mice were randomly classified into groups (n = 10 in each group) and the treatment was initiated intraperitoneally (ip) with increasing concentrations of HA1-4.121 or isotype control MAb as indicated . Animals were treated twice a week with a total of 7 doses until the day of study 21. Tumor growth was monitored using measurements per calibrator every 3 to 4 days as indicated. The results of this study demonstrated that HA1-4121 inhibited the growth of prostate xenografts dependent on subcutaneous androgen established in SCID mice. The results were statistically significant for the 300 dosisg dose group on days 14, 17 and 21 (p <0.05, Kruskal-Wallis trial, double-sided with  = 0.05) and for the dose group 700 g on days 10, 14, 17 and 21 (p <0.05, Kruskal-Wallis trial, double-sided with  = 0.05). Figure 21. Androgen-dependent LAPC-9AD tumor cells, derived from patients (2.0 x 106 cells), were injected into the dorsal lobes of male SCID mouse prostates. The tumors were allowed to grow for approximately 10 days at which time the mice were randomly classified into groups. Treatment with 500 deg of HA1-4.117, HA1-4.121 or human Isotype control MAb was started 10 days after tumor implantation. The antibodies were delivered intraperitoneally twice by

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week for a total of 7 doses. Four days after the last dose, the animals were sacrificed and the primary tumors were excised and weighed. The results show that human anti-PSCA monoclonal antibodies Ha1-4.121 (p <0.01) and Ha1-4.117 (p <0.05) significantly inhibited the growth of orthopedically implanted LAPC-9AD prostate cancer xenografts in SCID mice . Figure 22. The MACA of PSCA HA1-4.121 Prolongs Survival of SCID Mice with Established Orthotopic Human Androgen Dependent Prostate Tumors. Androgen-dependent LAPC-9AD tumor cells, derived from patients (2.0 x 106 cells), were injected into the dorsal lobes of male SCID mouse prostates. The tumors were allowed to grow for approximately 9 days at which time the mice were randomly classified into groups. Animals randomly classified in the survival groups include 11 mice in the isotype control MAb and 12 mice in the group treated with HA1

4,121. The animals were treated i.p. with 1000 g of Ha1-4121 or 1000 g of isotype control MAb twice a week for a total of 9 doses. The results showed that HA1-4.121 significantly prolonged (logarithmic range test: p <0.01) the survival of SCID mice with human androgen-dependent prostate tumors. Two mice in the group treated with HA1-4121 remained without palpable tumors 110 days after the last treatment. Figure 23. Enhanced Inhibition of Prostate Tumor Growth with HA1-4.21 and Taxotere Combination Therapy. LAPC-9AI tumor cells (2 x 10 6 cells per animal) were injected subcutaneously into male SCID mice. When the tumor volume reached 65 mm3, the animals were randomly classified and assigned to four different groups (n = 10 in each group) as indicated. Starting on day 0, Ha1-4.121 or isotype control MAb of i.p. twice a week at a dose of 500 parag for a total of 6 doses. The last dose was provided on day 17. Taxotere was given intravenously at a dose of 5 mg / kg on days 0, 3 and 7. Tumor growth was monitored every 3-4 days using calibrator measurements. The results of this study demonstrate that HA1-4.121 as a single agent inhibited the growth of androgen independent prostate xenografts in SCID mice by 45% compared to the control antibody treatment only on day 28 (ANOVA / Tukey assay : p <0.05). Administration of the isotype plus taxotere control MAb inhibited tumor growth by 28% compared to the control antibody treatment alone, which was not statistically significant. Administration of HA1-4121 in combination with Taxotere potentiated the effect and resulted in a 69% inhibition of tumor growth compared to control antibody only (ANOVA / Tukey assay: p <0.01). A statistically significant difference was also demonstrated when the combination group of HA1-4.121 plus Taxotere was compared with the HA1-4.121 or MAb control groups of isotype plus Taxotere (ANOVA / Tukey test: p <0.05). Figure 24. Human PSCA MAbs Inhibit the Growth of Pancreatic Cancer Xenografts in Human SCID / HPAC Mice. Pancreatic cancer cells (2 x 106 / mouse) were injected subcutaneously into immunodeficient ICR SCID mice (Taconic Farm, Germantown, NY). Mice were randomly classified into groups (n = 10 animals / group) and treatment was initiated with the human PSCA monoclonal antibody indicated the same day. Antibodies (500 mg / mouse) were given intraperitoneally twice a week for a total of 8 doses. The results showed that human anti-PSCA monoclonal antibodies Ha1-4121, Ha1-4.117 and Ha1-1.16 significantly inhibited the growth of human pancreatic cancer xenografts implanted subcutaneously in SCID mice. Statistical analyzes were performed using a t-test (double-sided,  = 0.05). Figure 25. The MACA of PSCA HA1-4.121 Inhibits the Growth of Orthopedically Implanted Pancreatic Tumors in SCID Mice. HPAC cells (3.0 x 106 cells) were implanted orthotopically into the pancreas of SCID mice. Mice were randomly assigned to three groups (n = 9 in each group) as indicated. Treatment with HA1-4.121 (250 og or 1000 g) or isotype control MAb (1000 g) was started on the day of implantation. The antibodies were administered i.p. twice a week for a total of 10 doses. Thirteen days after the last dose, the animals were sacrificed and the primary tumors were excised and weighed. The results of this study demonstrated that HA1-4121 significantly inhibited the orthotopic growth of human pancreatic cancer xenografts in SCID mice at both dose levels examined. Treatment with 250 g and 1000 g of AGS-PSCA inhibited tumor growth in 66% and 70%, respectively (Kruskal-Wallis / Tukey trial: p <0.01 and p <0.01, respectively). Figure 26. The PSCA MAb HA1-4121 inhibits metastases. At autopsy, visible metastases to lymph nodes and distant organs were observed in the group treated with control antibody. No visible metastases were observed in any of the groups treated with HA1-4121. Lymph nodes, lungs and livers were removed from all animals and examined histologically for the presence of metastatic tumor. Sections of the lungs and lymph nodes removed from each animal with respect to human cytokeratin were stained and the number of metastases was determined microscopically. The results of the histological analysis demonstrated a significant reduction in lymph node (LN) metastases in animals treated with HA1-4121 (p = 0.0152 as detected by Fisher's exact test). The incidence of metastasis and invasion was also significantly reduced in animals treated with both concentrations of HA1-4121 (p = 0.0152 as detected by Fisher's exact test). The number of lung metastases was significantly reduced in mice treated with the dose of 1.0 mg of HA1-4121 only (p = 0.0498 as detected by Fisher's exact test). Figure 27. Human PSCA MAbs Inhibit the Growth of SW780 Bladder Tumors in SCID Mice. Human SW780 bladder cancer cells (2 x 106 / mouse) were injected subcutaneously into ICR mice

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Immunodeficient SCID (Taconic Farm, Germantown, NY). Mice were randomly classified into groups (n = 10 animals / group) and treatment with the indicated human PSCA MAb was initiated on the same day. Antibodies (250 mg / mouse) were given intraperitoneally twice a week for a total of 7 doses. The results showed that HA1-4.117 (p = 0.014), HA1-4.37 (p = 0.0056), HA1-1.78 (p = 0.001), Ha1-5.99 (p = 0.0002) and HA1-4.5 (p = 0.0008) significantly inhibited the growth of SW780 bladder tumors implanted subcutaneously in SCID mice. Statistical analyzes were performed using a t-test (double sided,  = 0.05).

Detailed description of the invention

Section Summary

I.) Definitions II.) PSCA polynucleotides

II.A.) Uses of PSCA Polynucleotides

II.A.1.) Control of Genetic Abnormalities II.A.2.) Antisense Embodiments II.A.3.) Primers and Primer Pairs II.A.4.) Isolation of Nucleic Acid Molecules Encoding PSCAII.A. 5.) Recombinant Nucleic Acid Molecules and Vector-Host Systems

III.) PSCA-related Proteins

III.A.) Embodiments of Carrier Proteins III.B.) Protein Expression Related to PSCA III.C.) Protein Modifications Related to PSCA III.D.) Uses of Proteins Related to PSCA

IV.) PSCA Antibodies V.) Cellular Immune Responses of PSCA VI.) Transgenic Animals for PSCA VII.) Methods for the Detection of PSCA VIII.) Methods for the Control of the Status of Genes Related to PSCA and Its Products IX.) Identification of Molecules That Interact with PSCA X.) Therapeutic Methods and Compositions

X.A.) Antineoplastic Vaccines X.B.) PSCA as a Target for Antibody-Based Therapy X.C.) PSCA as a Target for Cellular Immune Responses

X.C.1. Minigenes vaccines

X.C.2. Combinations of CTL Peptides with Auxiliary Peptides

X.C.3. Combinations of CTL Peptides with T Lymphocyte Sensitization Agents

X.C.4. Vaccine Compositions comprising DC Pulsed with CTL and / or HTL Peptides

X.D.) Adoptive Immunotherapy X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes

XI.) Diagnostic and Prognostic Performance of PSCA. XII.) Inhibition of PSCA Protein Function

XII.A.) Inhibition of PSCA With Intracellular Antibodies XII.B.) Inhibition of PSCA with Recombinant Proteins XII.C.) Inhibition of Transcription or Translation of PSCA XII.D.) General Considerations for Therapeutic Strategies

XIII.) Identification, Characterization and Use of PSCA Modulators XIV.) RNAi and Therapeutic Use of Small Interference RNA (siRNA) XV.) KITS / Articles of Manufacture

I.) Definitions:

Unless defined otherwise, it is intended that all technical terms, annotations and other terms

or scientific terminology used herein have the meanings usually understood by the

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experts in the field to which the present invention belongs. In some cases, terms with commonly understood meanings for clarity and / or easy reference are defined herein, and it should not necessarily be construed that the inclusion of such definitions in this document represents a substantial difference from what is generally understood in this field. Many of the techniques and procedures described or referred to herein are well understood and commonly employed using conventional methodology, by those skilled in the art, such as, for example, the widely used molecular cloning methodologies described in Sambrook et al. , Molecular Cloning: A Laboratory Manual 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with the protocols and / or parameters defined by the manufacturer unless otherwise indicated.

The terms "advanced prostate cancer", "locally advanced prostate cancer", "advanced disease" and "locally advanced disease" mean prostate cancers that have spread through the prostate capsule, and are understood to include stage disease C according to the American Urological Association (AUA) system, stage C1-C2 disease according to the Whitmore-Jewett system, and stage T3-T4 and N + disease according to the TNM system (tumor, nodule, metastasis). In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable results compared to patients who have clinically localized prostate cancer (confined to an organ). Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral edge of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is currently diagnosed pathologically after radical prostatectomy if the tumor invades or penetrates the prostate capsule, extends to the surgical margin, or invades the seminal vesicles.

It is understood that "altering the native glycosylation pattern" for purposes of the present document means deleting one or more carbohydrate residues found in native sequence PSCA (either by removing the underlying glycosylation site or by suppressing glycosylation by chemical and / or enzymatic means ) and / or add one or more glycosylation sites that are not present in the PSCA in the native sequence. In addition, the phrase includes qualitative changes in the glycosylation of native proteins, which imply a change in the nature and proportions of the various carbohydrate residues present.

The term "analog" refers to a molecule that is structurally similar or shares similar or corresponding attributes with another molecule (for example, a PSCA-related protein). For example, an analog of a PSCA protein can specifically bind to an antibody or T lymphocyte that specifically binds with PSCA.

The term "antibody" is used in the broadest sense unless otherwise stated clearly. Therefore, an "antibody" can be of natural or artificial origin such as monoclonal antibodies produced by conventional hybridoma technology. Anti-PSCA antibodies comprise monoclonal and polyclonal antibodies as well as fragments that contain the antigen-binding domain and / or one or more complementarity determining regions of these antibodies. As used herein, the term "antibody" refers to any form of antibody or fragment thereof that specifically binds with PSCA and / or shows the desired biological activity and specifically encompasses monoclonal antibodies (including full length monoclonal antibodies ), polyclonal antibodies, multispecific antibodies (eg bispecific antibodies) and antibody fragments provided they specifically bind with PSCA and / or show the desired biological activity. Any specific antibody can be used in the methods and compositions provided herein. Therefore, in one embodiment the term "antibody" encompasses a molecule comprising at least one variable region of a light chain immunoglobulin molecule and at least one variable region of a heavy chain molecule that in combination forms a binding site specific for the target antigen. In one embodiment, the antibody is an IgG antibody. For example, the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody. Antibodies useful in the present methods and compositions can be generated in cell culture, in phages, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees. Apes Therefore, in one embodiment, an antibody of the present invention is a mammalian antibody. Phage techniques can be used to isolate an initial antibody or to generate variants with altered avidity or specificity characteristics. Such techniques are routine and are well known in this field. In one embodiment, the antibody is produced by recombinant means known in this field. For example, a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect the DNA sequence that expresses at least one VL region and one VH in the host cell. Exemplary descriptions of recombinant antibody generation and production media include Delves, ANTI-BODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, et al., MONOCLONAL ANTIBODIES (Oxford University Press, 2000); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993); CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, most recent edition). An antibody of the present invention can be modified by recombinant means to increase the efficacy of the antibody by mediating the desired function. Therefore, it is within the scope of the invention that antibodies can be modified by substitutions using recombinant means. Normally, the substitutions will be conservative substitutions. For example, you can replace at least one

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amino acid in the constant region of the antibody with a different moiety. See, for example, U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol 30: 105-08 (1993). The amino acid modification includes deletions, additions, amino acid substitutions. In some cases, such changes are made to reduce unwanted activities, for example, complement-dependent cytotoxicity. Frequently, antibodies are labeled by binding, either covalently or non-covalently, a substance that provides a detectable signal. A wide variety of markers and conjugation techniques are known and are exhaustively indicated in both the scientific and patent literature. These antibodies can be screened for binding to normal or defective PSCA. See, for example, ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996). Suitable antibodies can be identified with the desired biological activities in the following in vitro assays including but not limited to: proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction and the following in-assays. live such as tumor growth inhibition. The antibodies provided herein may also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for the ability to bind with the specific antigen without inhibiting the receptor or biological binding activity of the antigen. As neutralizing antibodies, antibodies can be useful in competitive binding assays. They can also be used to quantify the PSCA or its receptor.

An "antibody fragment" is defined as at least a part of the variable region of the immunoglobulin molecule that binds to its target, that is, the antigen-binding region. In one embodiment it specifically encompasses individual anti-PSCA antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-PSCA antibody compositions with polyepitopic specificity. The antibody of the present methods and compositions may be monoclonal or polyclonal. An antibody may be in the form of an antigen-binding antibody fragment including a Fab fragment, F (ab ’) 2 fragment, a light chain variable region and the like. Fragments of intact molecules can be generated using methods well known in the art and including enzymatic digestion and recombinant media.

As used herein, any form of the "antigen" can be used to generate an antibody that is specific for PSCA. Therefore, the inducing antigen can be an individual epitope, multiple epitopes

or the complete protein alone or in combination with one or more immunogenicity enhancing agents known in this field. The inducing antigen can be an isolated full-length protein, a cell surface protein (for example, which immunizes with cells transfected with at least a part of the antigen), or a soluble protein (for example, which immunizes only with the part of extracellular domain of the protein). The antigen can be produced in a genetically modified cell. The DNA encoding the antigen can be genomic or non-genomic (for example, cDNA) and encodes at least a portion of the extracellular domain. As used herein, the term "part" refers to the minimum number of amino acids or nucleic acids, as appropriate, to constitute an immunogenic epitope of the antigen of interest. Any genetic vector suitable for transformation of the cells of interest may be employed, including but not limited to adenoviral vectors, plasmids and non-viral vectors, such as cationic lipids. In one embodiment, the antibody of the methods and compositions herein specifically binds to at least a portion of the extracellular domain of the PSCA of interest.

Antibodies or antigen binding fragments thereof provided herein may be conjugated with a "bioactive agent." As used herein, the term "bioactive agent" refers to any synthetic or naturally occurring compound that binds with the antigen and / or potency or media in the desired biological effect to enhance cell-killing toxins.

In one embodiment, the binding fragments useful in the present invention are biologically active fragments. As used herein, the term "biologically active" refers to an antibody or antibody fragment that is capable of binding with the desired antigenic epitope and directly or indirectly exerting a biological effect. Direct effects include, but are not limited to, modulation, stimulation and / or inhibition of a growth signal, modulation, stimulation and / or inhibition of an antiapoptotic signal, modulation, stimulation and / or inhibition of an apoptotic or necrotic signal. , modulation, stimulation and / or inhibition of the ADCC cascade, and modulation, stimulation and / or inhibition of the CDC cascade.

"Bispecific" antibodies are also useful in the present methods and compositions. As used herein, the term "bispecific antibody" refers to an antibody, usually a monoclonal antibody, which has binding specificities for at least two different antigenic epitopes. In one embodiment, the epitopes are of the same antigen. In another embodiment, the epitopes are from two different antigens. Methods for preparing bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the coexpression of two immunoglobulin heavy chain / light chain pairs. See, for example, Milstein et al., Nature 305: 537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical bonding. See, for example, Brennan, et al., Science 229: 81 (1985). Bispecific antibodies include bispecific antibody fragments. See, for example, Hollinger, et al., Proc. Natl. Acad. Sci. U.S.A. 90: 6444-48 (1993), Gruber, et al., J. Immunol. 152: 5368 (1994).

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Martek Biosciences, Columbia, MD; etc.).

As used herein, the term "conservative substitution" refers to amino acid substitutions that are known to those skilled in the art and can be performed in generating without altering the biological activity of the resulting molecule. Those skilled in the present art recognize that, in general, individual amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson, et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin / Cummings Pub. Co., p. 224 (4th Edition 1987). Said exemplary substitutions are preferably made in accordance with those set forth in Tables III (a-b). For example, such changes include replacing any other of these hydrophobic amino acids with any of isoleucine (I), valine (V) and leucine (L); glutamic acid (E) by aspartic acid (D) and vice versa; asparagine (N) by glutamine (Q) and vice versa; and threonine (T) by serine (S) and vice versa. Other substitutions may also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can be frequently interchangeable, such as alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can be frequently exchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations where the significant characteristic of the rest of amino acids is their charge and the different pKs of these two amino acid residues are not significant. Other changes may be considered "conservative" in particular environments (see, for example, Table III (a) in this document; pages 13-15 "Biochemistry" 2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al. , PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem May 19, 1995; 270 (20): 11882-6). Other substitutions are also permissible and can be determined empirically or in accordance with known conservative substitutions.

The term "cytotoxic agent" refers to a substance that inhibits or prevents cell expression activity, cell function and / or causes cell destruction. The expression is intended to include radioactive isotopes, chemotherapeutic agents and toxins such as toxic small molecules or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof. Examples of cytotoxic agents include, but are not limited to, auristatins, auristatin e, auromycins, maitansinoids, yttrium, bismuth, ricin, ricin A chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, bromidide , mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, A-chain, A-chain, modeccin A chain, alpha-sarcin, gelonin, Mitogelin, retstrictocin, phenomycin, enomycin, curicin, crotine, calicheamycin, Saponaria officinalis inhibitor, and glucocorticoids and other chemotherapeutic agents, as well as radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, Bi212 and radioactive isotopes of Lu including Lu177. Antibodies can also be conjugated with an antineoplastic prodrug activating enzyme capable of converting the prodrug into its active form.

As used herein, the term "diabody" refers to small antibody fragments with two antigen binding sites, said fragments comprising a heavy chain variable domain (VH) connected with a variable light chain (VL domain) ) in the same polypeptide chain (VH-VL). Using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-48 (1993).

The "gene product" is used herein to indicate a peptide / protein or mRNA. For example, a "gene product of the invention" is sometimes referred to herein as a "cancer amino acid sequence", "cancer protein", "a cancer protein listed in Table I", a "mRNA of cancer ”,“ mRNA of a cancer listed in Table I ”, etc. In one embodiment, the cancer protein is encoded by a nucleic acid of Figure 1. The cancer protein may be a fragment or, alternatively, be the full length protein encoded by the nucleic acids of Figure 1. In a embodiment, a cancer amino acid sequence is used to determine the identity or similarity of sequences. In another embodiment, the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of Figure

1. In another embodiment, the sequences are sequence variants as further described herein.

"Heteroconjugate" antibodies are useful in the present methods and compositions. As used herein, the term "heteroconjugated antibody" refers to two covalently bound antibodies. Such antibodies can be prepared using methods known in synthetic protein chemistry, including crosslinking agents. See, for example, U.S. Patent No. 4,676,980.

"High performance" assays with respect to the presence, absence, quantification or other properties of particular nucleic acids or protein products are well known to those skilled in the art. Similarly, binding assays and indicator gene assays are equally well known. Therefore, for example, US Patent No. 5,559,410 discloses high performance scanning methods for proteins; U.S. Patent No. 5,585,639 discloses high performance scanning methods for

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defined in this document.

Cancer modulators can also be nucleic acids. Nucleic acid modulating agents may be naturally occurring nucleic acids, random nucleic acids or "deviated" random nucleic acids. For example, prokaryotic or eukaryotic genome digestion products can be used in an approach analogous to that indicated above for proteins.

The term "monoclonal antibody", as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in smaller quantities. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. In one embodiment, the polyclonal antibody contains a plurality of monoclonal antibodies with different specificities, affinities or avidities within a single antigen containing multiple antigenic epitopes. The "monoclonal" modifier indicates as the character of the antibody that is obtained from a substantially homogeneous population of antibodies, and should not be construed to require production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be made by the hybridoma method described first in Kohler et al., Nature 256: 495 (1975), or they can be made by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991), for example. These monoclonal antibodies will usually bind with at least one Kd of about 1 µM, more usually at least about 300 nM, usually at least about 30 nM, preferably at least about 10 nM, more preferably at least about 3 nM or better, usually determined by ELISA.

A "motif", as in the biological motif of a PSCA-related protein, refers to any pattern of amino acids that is part of the primary sequence of a protein, which is associated with a particular function (eg protein-protein interaction, interaction protein-DNA, etc.) or modification (for example, that is phosphorylated, glucosyl or amide), or location (e.g., secretory sequence, nuclear localization sequence, etc.) or a sequence that correlates with being immunogenic, either in a humoral or cellular way. A motive may be contiguous or capable of aligning with certain positions that are generally correlated with a certain function or property. In the context of HLA motifs, "motif" refers to the pattern of residues in a peptide of different length, usually a peptide of about 8 to about 13 amino acids for a HLA motif of class I and about 6 to about 25 amino acids. for a class II HLA motif, which is recognized by a particular HLA molecule. The peptide motifs for HLA binding are normally different for each protein encoded by each human HLA allele and differ in the pattern of primary and secondary anchor moieties. Reasons for frequent occurrence are presented in Table V.

A "pharmaceutical excipient" comprises a material such as an adjuvant, a vehicle, buffering and pH adjusting agents, tonicity adjusting agents, wetting agents, preservatives and the like.

"Pharmaceutically acceptable" refers to a non-toxic, inert composition that is physiologically compatible with humans or other mammals.

The term "polynucleotide" means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of one of the types of nucleotides and is understood to include mono and double stranded forms of DNA and / or RNA. In the art, this term is often used interchangeably with "oligonucleotide." A polynucleotide may comprise a nucleotide sequence disclosed herein in which thymidine (T), as shown for example in Figure 1, may also be uracil (U); This definition belongs to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four main bases in RNA is uracil (U) instead of thymidine (T).

The term "polypeptide" means a polymer of at least about 4, 5, 6, 7 or 8 amino acids. Throughout the specification, conventional three-letter or one-letter designations for amino acids are used. In the art, this term is often used interchangeably with "peptide" or "protein."

An "HLA primary anchor moiety" is an amino acid at a specific position along a peptide sequence that is understood to provide a point of contact between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor moieties within a peptide of defined length generally define a "motive" for an immunogenic peptide. It is understood that these residues fit in close contact with the peptide binding groove of an HLA molecule, with its side chains embedded in specific pockets of the binding groove. In one embodiment, for example, the primary anchoring moieties for a class I HLA molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a peptide epitope of 8, 9, 10, 11 or 12 residues according to the invention. As an alternative, in another

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embodiment, the primary anchoring moieties of a peptide are linked with a class II HLA molecule and spaced apart, rather than with the terminal end of a peptide, in which the peptide is generally at least 9 amino acids of length. The primary anchor positions for each motive and supermotive are shown in Table IV (a). For example, analogous peptides can be created by altering the presence or absence of particular moieties in the primary and / or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and / or population coverage of a peptide comprising a particular motif or supermotivative of HLA.

The "radioisotopes" include, but are not limited to the following (exemplary non-limiting uses are also shown in Table IV (I)).

By "random" or grammatical equivalents as applied herein to nucleic acids and proteins it is understood that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. These random peptides (or nucleic acids, discussed herein) can incorporate any amino acid or nucleotide in any position. The synthetic process can be designed to generate random proteins or nucleic acids, to allow the formation of all or most of the possible combinations along the length of the sequence, thereby forming a library of random candidate bioactive protein agents.

In one embodiment, a library is "completely random", without sequence preferences or constants in any position. In another embodiment, the library is a "random deviated" library. That is, some positions within the sequence remain constant, or are selected from a limited number of possibilities. For example, nucleotide or amino acid residues are randomly selected within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, residues with steric deviation (either small or large), towards the creation of nucleic acid binding domains , the creation of cysteines, for crosslinking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or purines, etc.

A "recombinant" DNA or RNA molecule is a DNA or RNA molecule that has undergone molecular manipulation in vitro.

As used herein, the term "single chain Fv" or "scFv" or "single chain" antibody refers to antibody fragments comprising the VH and VL domains of the antibody, in which these domains are present in a single chain. polypeptide In general, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

Non-limiting examples of "small molecules" include compounds that bind with or interact with PSCA, ligands including hormones, neuropeptides, chemokines, odorizers, phospholipids and functional equivalents thereof that bind with and preferably inhibit the function of PSCA protein. Said non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably below about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules are physically associated with, or bind with, PSCA protein; they are not found in metabolic pathways of natural origin; and / or are more soluble in aqueous solutions than non-aqueous solutions.

As used herein, the term "specific" refers to the selective binding of the antibody with the target antigen epitope. Antibodies can be tested for binding specificity by comparing binding with appropriate antigen with binding with irrelevant antigen or mixture of antigens under a given set of conditions. If the antibody binds with the appropriate antigen at least 2, 5, 7 and preferably 10 times more than the irrelevant antigen or mixture of antigens then it is considered to be specific. In one embodiment, a specific antibody is one that only binds with the PSCA antigen, but does not bind with the irrelevant antigen. In another embodiment, a specific antibody is one that binds with the human PSCA antigen but does not bind with a non-human PSCA antigen with 70%, 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid homology with the PSCA antigen. In another embodiment, a specific antibody is one that binds with the human PSCA antigen and binds with the murine PSCA antigen, but with a greater degree of binding with the human antigen. In another embodiment, a specific antibody is one that binds with the human PSCA antigen and binds with the primate PSCA antigen, but with a greater degree of binding with the human antigen. In another embodiment, the specific antibody binds with the human PSCA antigen and any non-human PSCA antigen, but with a greater degree of binding than the human antigen or any combination thereof.

The "stringency" of the hybridization reactions can easily be determined by a person skilled in the art, and in general it is an empirical calculation that depends on the length of the probe, the washing temperature and the salt concentration. In general, longer probes require higher temperatures for proper hybridization,

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The HLA class I peptides of the invention can be mixed with, or bound to, class II HLA peptides, to facilitate the activation of both cytotoxic T lymphocytes and auxiliary T lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting cells, for example, dendritic cells.

The term "variant" refers to a molecule that shows a variation with respect to a type or standard described, such as a protein that has one or more different amino acid residues at the corresponding position or positions of a specifically described protein ( for example, the PSCA protein shown in Figure 1. An analogue is an example of a variant protein, splicing isoforms and single nucleotide polymorphisms (SNPs) are additional examples of variants.

"PSCA-related proteins" as disclosed herein include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated / generated and characterized without undue experimentation following the highlighted methods. in this document or readily available in the art. Also included are fusion proteins that combine parts of different PSCA proteins or fragments thereof, as well as fusion proteins of a PSCA protein and a heterologous polypeptide. Said PSCA proteins are collectively referred to as PSCA-related proteins, proteins of the invention or PSCA. The term "PSCA-related protein" refers to a polypeptide fragment or a PSCA protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 , 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 330, 335, 339 or more amino acids.

II.) PSCA polynucleotides

Polynucleotides corresponding to or complementary to all or a part of a PSCA gene, mRNA and / or coding sequence are disclosed, preferably in isolation, including polynucleotides encoding a PSCA-related protein and fragments thereof, DNA, RNA, DNA / RNA hybrid and related molecules, polynucleotides or oligonucleotides complementary to a PSCA gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize with a PSCA gene, mRNA or with a polynucleotide encoding PSCA (collectively, "PSCA polynucleotides"). In all cases when reference is made to this section, T can also be U in Figure 1.

PSCA polynucleotides include: a PSCA polynucleotide having the sequence shown in Figure 1, the nucleotide sequence of PSCA as shown in Figure 1 in which T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 1; or at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 1 in which T is U.

Polynucleotides encoding relatively long portions of a PSCA protein are also within the scope of the invention. For example, polynucleotides encoding from about amino acid 1 (or 20, 30 or 40, etc.) to about amino acid 20, (or 30, 40 or 50, etc.) of the PSCA protein "or variant" shown in Figure 1 or Figure 3 can be generated by various techniques well known in this field. These polynucleotide fragments can include any part of the PSCA sequence as shown in Figure 1.

II.A.) Uses of PSCA Polynucleotides

II.A.1. Control of genetic abnormalities

The polynucleotides of the preceding paragraphs have several different specific uses. The human PSCA gene is mapped to the chromosomal location set forth in the Example entitled "PSCA Chromosomal Mapping." For example, because the PSCA gene is mapped on this chromosome, polynucleotides encoding different regions of PSCA proteins are used to characterize cytogenetic abnormalities of this chromosomal location, such as abnormalities that have been identified to be associated with various cancers. In certain genes, various chromosomal abnormalities have been identified including rearrangements, such as frequent cytogenetic abnormalities in several different cancers (see for example Krajinovic et al., Mutat. Res. 382 (3-4): 8183 (1998); Johansson et al. , Blood 86 (10): 3905-3914 (1995) and Finger et al., PNAS 85 (23): 9158-9162 (1988)). Therefore, polynucleotides encoding specific regions of PSCA proteins provide new tools that can be used to delimit, more precisely than previously possible, cytogenetic abnormalities in the chromosomal region encoding PSCA that can contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need for the technique of expanding the sensitivity of chromosomal scanning to identify more subtle and less common chromosomal abnormalities (see for example Evans et al., Am. J. Obstet. Gynecol 171 (4): 1055 -1057 (1994)).

In addition, as PSCA has been shown to be highly expressed in prostate and other cancers, PSCA polynucleotides are used in methods that assess the status of PSCA gene products in normal tissues against cancer. Typically, polynucleotides encoding specific regions of PSCA proteins are used to assess the presence of disturbances (such as deletions, insertions, point mutations or

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alterations that result in the loss of an antigen etc.) in specific regions of the PSCA gene, such as regions that contain one or more motifs. Exemplary assays include RT-PCR assays as well as single stranded conformation polymorphism (SSCP) analysis (see, for example, Marrogi et al., J. Cutan. Pathol. 26 (8): 369-378 (1999)), both of which use polynucleotides that encode specific regions of a protein to examine these regions within the protein.

II.A.2. Antisense Performances

Also disclosed herein are genomic DNA, cDNA, ribozymes and antisense molecules, as well as nucleic acid molecules based on an alternative main chain, or that includes alternative bases, either derived from natural or synthesized sources, and include molecules capable of inhibit the expression of PSCA RNA or protein. For example, the antisense molecules can be RNA or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind to DNA or RNA in a base pair dependent manner. One skilled in the art can readily obtain these kinds of nucleic acid molecules using PSCA polynucleotides and polynucleotide sequences disclosed herein.

Antisense technology involves the administration of exogenous oligonucleotides that bind with a target polynucleotide located within the cells. The term "antisense" refers to the fact that said oligonucleotides are complementary to their intracellular targets, for example, PSCA. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1: 1-5 (1988). The PSCA antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, mentioned above), which show inhibitory action of enhanced cancer cell growth. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a non-binding oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos can be prepared by treating the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, for example, Iyer, R.

P. et al., J. Org. Chem. 55: 4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112: 1253-1254 (1990). Additional PSCA antisense oligonucleotides include morpholino antisense oligonucleotides known in the art (see, for example, Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).

The PSCA antisense oligonucleotides disclosed herein can normally be RNA or DNA that is stably complementary to and hybridizes with the first 100 'codons 5' or the last 100 'codons 3' of a PSCA genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. The use of a complementary oligonucleotide from this region allows selective hybridization with PSCA mRNA and not with mRNA that specifies other protein kinase regulatory subunits. The PSCA antisense oligonucleotides disclosed herein may be fragments of 15 to 30 units of the antisense DNA molecule having a sequence that hybridizes with PSCA mRNA. Optionally, the PSCA antisense oligonucleotide is a 30-unit oligonucleotide that is complementary to a region in the first 10 ′ codons or the last 10 ″ PSCA codons. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of PSCA expression, see, for example, L. A. Couture and D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3. Primers and Primer Pairs

Other nucleotides disclosed herein include primers and primer pairs that allow specific amplification of polynucleotides of the invention or any specific part thereof, and probes that specifically or selectively hybridize with nucleic acid molecules of the invention or any part. from the same. The probes can be labeled with a detectable label, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a PSCA polynucleotide in a sample and as a means to detect a cell expressing a PSCA protein.

Examples of such probes include polypeptides comprising all or part of the human PSCA cDNA sequence shown in Figure 1. Examples of primer pairs capable of specifically amplifying PSCA mRNA are also described in the examples. As will be understood by one skilled in the art, a large number of different primers and probes can be prepared based on the sequences provided herein and effectively used to amplify and / or detect a PSCA mRNA.

The PSCA polynucleotides disclosed herein are useful for various purposes, including but not limited to their use as probes and primers for amplification and / or detection of the gene or genes of PSCA, mRNA or fragments thereof; as reagents for the diagnosis and / or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of PSCA polypeptides; as tools to modulate or inhibit the expression of the PSCA gene or genes and / or translation of the PSCA transcript or transcripts; and as therapeutic agents.

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The use of any probe is disclosed herein as described herein to identify and isolate a PSCA or PSCA-related nucleic acid sequence from a source of natural origin, such as humans or other mammals, as well as the isolated nucleic acid sequence itself, which would comprise all or most of the sequences found in the probe used.

II.A.4. Isolation of Nucleic Acid Molecules Encoding PSCA

The PSCA cDNA sequences described herein allow the isolation of other polynucleotides encoding a PSCA gene product or products, as well as the isolation of polynucleotides encoding homologs of PSCA gene products, isoforms cut and spliced alternately, allelic variants and mutant forms of a PSCA gene product as well as polynucleotides encoding PSCA-related protein analogs. Various molecular cloning methods that can be used to isolate full-length cDNAs encoding a PSCA gene are well known (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems (eg, Lambda ZAP Express, Stratagene). Phage clones containing PSCA gene cDNAs can be identified by scanning with a labeled PSCA cDNA or a fragment thereof. For example, in one embodiment, a PSCA cDNA (for example, Figure 1) or a part thereof can be synthesized and used as a probe to recover overlapping and full-length cDNAs corresponding to a PSCA gene. A PSCA gene itself can be isolated by scanning genomic DNA libraries, bacterial artificial chromosome libraries (BAC), yeast artificial chromosome libraries (YAC) and the like, with PSCA DNA primers or probes.

II.A.5. Recombinant Nucleic Acid Molecules and Host-Vector Systems

Recombinant DNA or RNA molecules containing a PSCA polynucleotide, a fragment, analog or homologue thereof, including but not limited to phage, plasmids, phagemids, cosmids, YAC, BAC, as well as various viral vectors and are disclosed herein. non-viral well known in the art, and cells transformed or transfected with said recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al., 1989, mentioned above).

A host-vector system comprising a recombinant DNA molecule containing a PSCA polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell is also disclosed herein. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (for example, a cell that can be infected by baculovirus such as an Sf9 cell or HighFive). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPr1, other transferable or transducible prostate cancer cell lines, primary cells (PrEC), as well as several mammalian cells commonly used for the expression of recombinant proteins (for example, COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the PSCA coding sequence or a fragment, analog or homolog thereof can be used to generate PSCA proteins or fragments thereof using any variety of host-vector systems commonly used and widely known in the art.

A wide range of host-vector systems suitable for the expression of PSCA proteins or fragments thereof are available, see for example, Sambrook et al., 1989, mentioned above; Current Protocols in Molecular Biology, 1995, mentioned above. Preferred vectors for mammalian expression include but not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRhtkneo (Muller et al., 1991, MCB 11: 1785). Using these expression vectors, PSCA can be expressed in several prostate and non-prostate cancer cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPr1. Host-vector systems are useful for the production of a PSCA protein or fragment thereof. Such host-vector systems can be used to study the functional properties of PSCA and mutations or analogs of PSCA.

A recombinant human PSCA protein or an analogue or homologue or fragment thereof can be produced by mammalian cells transfected with a construct encoding a PSCA-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding PSCA or a fragment, analog or homolog thereof, a PSCA-related protein is expressed in 293T cells, and the recombinant PSCA protein is isolated using conventional purification methods ( for example, affinity purification using anti PSCA antibodies). Alternatively, a PSCA coding sequence is subcloned into the retroviral vector pSRMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 and rat-1 to establish cell lines that express PSCA. Various other expression systems well known in the art can also be employed. Expression constructs encoding a phase bound leader peptide with a PSCA coding sequence can be used for the generation of a secreted form of recombinant PSCA protein.

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As discussed herein, the redundancy of the genetic code allows variation in PSCA gene sequences. In particular, it is known in the art that specific host species frequently have specific codon preferences, and therefore the disclosed sequence can be adapted as preferred for a desired host. For example, preferred analog codon sequences usually have uncommon codons (ie, codons having a frequency of use of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. The coding preferences for a specific species are calculated, for example, using codon usage tables available on the INTERNET such as the web address URL dna.affrc.go.jp/∼nakamura/codon.html.

It is known that additional sequence modifications enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, intron / exon splicing and splicing site signals, transposon-like repeats and / or other such well-characterized sequences that are deleterious for gene expression. The GC content of the sequence is adjusted to average levels for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a consensus sequence of translation initiation at the beginning of the open reading phase, as described in Kozak, Mol. Cell Biol., 9: 5073-5080 (1989). Those skilled in the art understand that the general rule that eukaryotic ribosomes begin translation exclusively in the next 5 'AUG codon is canceled only under very unusual conditions (see, for example, Kozak PNAS 92 (7): 2662-2666 , (1995) and Kozak NAR 15 (20): 8125-8148 (1987)).

III.) PSCA-related proteins

PSCA-related proteins are also disclosed herein. Specific examples of PSCA proteins comprise a polypeptide having all or a part of the amino acid sequence of human PSCA as shown in Figure 1, preferably Figure 1A. Alternatively, examples of PSCA proteins comprise variant, homologous or analogous polypeptides that have alterations in the PSCA amino acid sequence shown in Figure 1.

A PSCA polypeptide may include: a PSCA polypeptide having a sequence shown in Figure 1, a peptide sequence of a PSCA as shown in Figure 1 in which T is U; at least 10 contiguous nucleotides of a polypeptide having the sequence as shown in Figure 1; or at least 10 contiguous peptides of a polypeptide having the sequence shown in Figure 1 in which T is U.

Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can often be made on a protein without altering the conformation or function of the protein. The proteins may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions.

The disclosures herein include a wide variety of variants or analogs accepted in the art of PSCA proteins such as polypeptides having amino acid insertions, deletions and substitutions. Variants of PSCA can be made using methods known in the art such as directed mutagenesis, alanine scanning and PCR mutagenesis. Targeted mutagenesis (Carter et al., Nucl. Acids Res., 13: 4331 (1986); Zoller et al., Nucl. Acids Res., 10: 6487 (1987)), cassette mutagenesis (Wells et al. , Gene, 34: 315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)) or other known techniques in cloned DNA to produce the variant DNA of PSCA.

Scanning amino acid analysis can also be used to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is normally a preferred screening amino acid among this group because it eliminates the side chain beyond the beta carbon and is less likely to alter the main chain conformation of the variant. Alanine is also normally preferred because it is the most common amino acid. In addition, it is frequently found in both interned and exposed positions (Creighton, The Proteins, (W. H. Freeman & Co., N. Y.); Chothia, J. Mol. Biol., 150: 1 (1976)). If the alanine substitution does not produce sufficient amounts of variant, an isostatic amino acid can be used.

As defined herein, variants, analogs or homologs of PSCA have the distinctive attribute of having at least one epitope that is "cross-reactive" with a PSCA protein having an amino acid sequence of Figure 1 As used in this phrase, "cross-reactive" means that an antibody or T lymphocyte that specifically binds to a PSCA variant also specifically binds to a PSCA protein that has an amino acid sequence set forth in Figure 1. A polypeptide is no longer a variant of a protein shown in Figure 1, when it no longer contains any epitope that can be recognized by an antibody or T lymphocyte that specifically binds with the starting PSCA protein. Those skilled in the art understand that antibodies that recognize proteins bind with epitopes of various sizes, and a grouping of the order of approximately four or five amino acids, contiguous or not, is considered a number.

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typical of amino acids in a minimal epitope. See, for example, Nair et al., J. Immunol 2000 165 (12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26 (9): 865-73; Schwartz et al., J Immunol (1985) 135 (4): 2598-608.

Other classes of PSCA-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity to an amino acid sequence of Figure 1, or a fragment thereof. Another specific class of PSCA protein variants or analogs comprises one or more of the biological motifs of PSCA described herein or currently known in the art. Therefore, the disclosures herein encompass analogs of PSCA fragments (nucleic acid or amino acid) that have altered functional properties (eg, immunogenic) in relation to the starting fragment. It should be appreciated that the current motifs or that become part of the technique should be applied to the amino acid or nucleic acid sequences of Figure 1.

As discussed herein, the polypeptides may have less than the complete amino acid sequence of a PSCA protein shown in Figure 1. For example, the peptides / proteins may have any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a PSCA protein shown in Figure 1.

PSCA-related proteins are generated using conventional peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a PSCA-related protein. In one example, nucleic acid molecules provide a means to generate defined fragments of a PSCA protein (or variants, homologs or analogs thereof).

III.A.) Reasons for Carrier Protein

Additional illustrative disclosures herein include PSCA polypeptides comprising amino acid residues of one or more of the biological motifs contained within a PSCA polypeptide sequence set forth in Figure 1. Various motifs are known in the art, and a motif can be evaluated in the art. protein with respect to the presence of such motives by several publicly available Internet sites (see, for example, URLs: pfam.wustl.edu/; searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html; psort.ims.utokyo.ac.jp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html; expasy.ch/tools/scnpsitl.html; Epimatrix ™ and Epimer ™, Brown University, brown .edu / Research / TB-HIV_Lab / epimatrix / epimatrix.html; and BIMAS, bimas.dcrt.nih.gov/.).

Subsequent carrier sequences of all PSCA variant proteins are shown and identified in Tables V-XVIII and XXII-LI.

Table IV (h) sets out several reasons for frequent occurrence based on pfam searches (see URL pfam.wustl.edu/). The columns in Table IV (h) list (1) the abbreviation of the name of the motive, (2) the percentage of identity found among the different members of the motive family, (3) name or description of the motive and (4) most common function; Location information is included if the reason is relevant to the location.

Polypeptides comprising one or more of the PSCA motifs discussed above are useful for elucidating the specific characteristics of a malignant phenotype in view of the observation that the PSCA motifs discussed above are associated with growth deregulation and why PSCA is overexpressed in certain cancers (see, for example, Table I). Casein kinase II, cAMP and cAMP-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see for example Chen et al., Lab Invest., 78 (2 ): 165-174 (1998); Gaiddon et al., Endocrinology 136 (10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24 (6): 1119-1126 (1996); Peterziel et al ., Oncogene 18 (46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5 (2): 305-309 (1998)). In addition, both glycosylation and myristoylation are modifications of proteins also associated with cancer and cancer progression (see for example Dennis et al., Biochem. Biophys. Acta 1473 (1): 21-34 (1999); Raju et al. , Exp. Cell Res. 235 (1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see for example Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).

In another disclosure, the proteins may comprise one or more of the immunoreactive epitopes identified according to methods accepted in the art, such as the peptides set forth in Tables V-XVIII and XXII-LI. CTL epitopes can be determined using specific algorithms to identify peptides within a PSCA protein that are capable of optimally binding with specific HLA alleles (eg, Table IV; Epimatrix ™ and Epimer ™, Brown University, URL brown.edu/Research /TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URL bimas.dcrt.nih.gov/). In addition, processes to identify peptides that have sufficient binding affinity for HLA molecules and that correlate with being immunogenic epitopes are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.

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Principles for creating analogues of said epitopes to modulate immunogenicity are also known in the art. For example, we start with an epitope that carries a CTL or HTL motif (see, for example, the HLA Class I and HLA Class II motifs / supermotives in Table IV). The epitope is made analogous by replacing an amino acid in one of the specific positions, and replacing it with another specific amino acid for that position. For example, based on the moieties defined in Table IV, a deleterious moiety can be substituted in favor of any other moiety, such as a preferred moiety; replace a less preferred moiety with a preferred moiety; or substitute a preferred residue that originally appeared with another preferred residue. Substitutions may occur at primary anchor positions or at other positions in a peptide; see, for example, Table IV.

Various references reflect the technique regarding the identification and generation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 97/33602 from Chesnut et al .; Sette, Immunogenetics 1999 50 (3-4): 201-212; Sette et al., J. Immunol. 2001 166 (2): 1389-1397; Sidney et al., Hum. Immunol 1997 58 (1): 12-20; Kondo et al., Immunogenetics 1997 45 (4): 249-258; Sidney et al., J. Immunol. 1996 157 (8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255: 1261-3 (1992); Parker et al., J. Immunol. 149: 3580-7 (1992); Parker et al., J. Immunol. 152: 163-75 (1994)); Kast et al., 1994 152 (8): 3904-12; Borras-Cuesta et al., Hum. Immunol 2000 61 (3): 266-278; Alexander et al., J. Immunol. 2000 164 (3); 164 (3): 16251633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J. Immunol. 1991 147 (8): 2663-2669; Alexander et al., Immunity 1994 1 (9): 751-761 and Alexander et al., Immunol. Res. 1998 18 (2): 79-92.

Related disclosures include polypeptides comprising combinations of the different motifs set forth in Tables IV (a), IV (b), IV (c), IV (d), and IV (h), and / or one or more of the predicted CTL epitopes of Tables V-XVIII and XXII-LI, and / or one or more of the predicted HTL epitopes of Tables XLVIII-LI, and / or one or more of the T lymphocyte binding motifs known in The technique. Some polypeptides do not contain insertions, deletions or substitutions within the motifs or within the intermediate sequences of the polypeptides. In addition, polypeptides that include several N-terminal and / or C-terminal amino acid residues on one side of these motifs (for example, to include a major part of the polypeptide architecture in which the motif is located) may be desirable. Normally, the number of N-terminal and / or C-terminal amino acid residues on one side of a motif is between about 1 and about 100 amino acid residues, preferably between 5 and about 50 amino acid residues.

PSCA-related proteins are made in many ways, preferably in isolation. A purified PSCA protein molecule will be substantially without other proteins or molecules that alter the binding of PSCA with the antibody, T lymphocyte or other ligand. The nature and degree of isolation and purification will depend on the intended use. PSCA-related proteins may include purified, functional PSCA-related proteins and soluble, functional PSCA-related proteins. In one disclosure, a soluble, functional PSCA protein or fragment thereof retains the ability to bind with the antibody, T lymphocyte or other ligand.

The disclosures herein also provide PSCA proteins that comprise biologically active fragments of a PSCA amino acid sequence shown in Figure 1. Such proteins show properties of the starting PSCA protein, such as the ability to induce the generation of antibodies that are specifically bind with an epitope associated with the starting PSCA protein; to bind with said antibodies; to induce the activation of HTL or CTL; and / or to be recognized by HTL or CTL that also specifically bind with the starting protein.

PSCA-related polypeptides containing particularly interesting structures can be predicted and / or identified using various analytical techniques well known in this field, including, for example, the methods of analysis of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus -Schultz or Jameson-Wolf,

or based on immunogenicity. Fragments containing such structures are particularly useful in the generation of subunit specific anti-PSCA antibodies or T lymphocytes or in the identification of cellular factors that bind with PSCA. For example, hydrophilicity profiles can be generated and immunogenic peptide fragments can be identified, using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 3824-3828. Hydropaticity profiles can be generated, and immunogenic peptide fragments can be identified using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157: 105-132. Percentage profiles (%) of Accessible Remains can be generated and immunogenic peptide fragments identified using the method of Janin J., 1979, Nature 277: 491-492. Average Flexibility profiles can be generated and immunogenic peptide fragments can be identified, using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res.

32: 242-255. Beta spin profiles can be generated, and immunogenic peptide fragments can be identified, using the method of Deleage, G., Roux B., 1987, Protein Engineering 1: 289-294.

CTL epitopes can be determined using specific algorithms to identify peptides within a PSCA protein that are capable of optimally binding with specific HLA alleles (for example, using the SYFPEITHI site in the web URL syfpeithi.bmi-heidelberg.com/; Listings in Table IV (A) - (E); Epimatrix ™ and Epimer ™, Brown University, URL (brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt. nih.gov/). Illustrating this, PSCA peptide epitopes that occur in the context of human MHC Class I molecules have been predicted, for example, HLA-A1, A2, A3, A11, A24, B7 and B35 (see, for example,

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PSCA-related proteins can also be modified to form a chimeric molecule comprising PSCA fused to another amino acid sequence or heterologous polypeptide. Said chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule may have a protein as disclosed herein fused with another tumor-associated antigen or fragment thereof. Alternatively, a protein according to the disclosures herein may comprise a fusion of fragments of a PSCA sequence (amino acids or nucleic acid) so that a molecule is created which, in its entire length, is not homologous directly from the amino acid or nucleic acid sequences shown in Figure 1. Said chimeric molecule may comprise multiple of the same PSCA sub-sequence. A chimeric molecule may comprise a fusion of a PSCA-related protein with an epitope marker of polyhistidine, which provides an epitope with which the immobilized nickel can be selectively bound, with cytokines or with growth factors. The epitope marker is generally placed at the amino or carboxyl terminus of a PSCA protein. In an alternative example, the chimeric molecule may comprise a fusion of a PSCA-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also called "immunoadhesin"), said fusion could be with the Fc region of an IgG molecule. Ig fusions preferably include the substitution of a soluble form (suppressed or inactivated transmembrane domain) of a PSCA polypeptide instead of at least one variable region within an Ig molecule. The immunoglobulin fusion may include the hinge, CH2 and CH3 regions or the hinge, CH1, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, for example, United States Patent No.

5,428,130 issued June 27, 1995.

III.D.) Uses of Proteins related to PSCA

The proteins disclosed herein have several different specific uses. As PSCA is highly expressed in prostate and other cancers, PSCA-related proteins are used in methods that assess the status of PSCA gene products in cancerous versus normal tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a PSCA protein are used to assess the presence of disturbances (such as deletions, insertions, point mutations, etc.) in those regions (such as regions containing one or more motifs). Exemplary assays use antibodies or T lymphocytes that target PSCA-related proteins that comprise the amino acid residues of one or more of the biological motifs contained within a PSCA polypeptide sequence to assess the characteristics of this region in normal tissues versus cancerous or to induce an immune response to the epitope. Alternatively, PSCA-related proteins containing amino acid residues of one or more of the biological motifs in a PSCA protein are used to explore for factors that interact with that region of PSCA.

PSCA protein fragments / subsequences are particularly useful for generating and characterizing domain-specific antibodies (eg, antibodies that recognize an extracellular or intracellular epitope of a PSCA protein), to identify agents or cellular factors that bind with PSCA or a domain particular structural structure, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic tests, cancer vaccines and methods to prepare such vaccines.

Proteins encoded by the PSCA genes, or by analogs, homologs or fragments thereof, have various uses, including but not limited to generating antibodies and in methods to identify ligands and other agents and cellular constituents that bind with a gene product of PSCA Antibodies induced against a PSCA protein or fragment thereof are useful in diagnostic and prognostic assays, and image capture methodologies in the treatment of human cancers characterized by PSCA protein expression, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods to treat patients with said cancers. Proteins or nucleic acids related to PSCA are also used in the generation of HTL or CTL responses.

Various immunological assays useful for the detection of PSCA proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods and the like. Antibodies can be labeled and used as immunological image capture reagents capable of detecting cells expressing PSCA (for example, in radio-scintigraphic image capture methods). PSCA proteins are also particularly useful in the generation of cancer vaccines, as further described herein.

IV.) PSCA antibodies

One aspect of the invention provides antibodies that bind with PSCA-related proteins. Preferred antibodies specifically bind with a PSCA-related protein and do not bind (or weakly bind) with peptides or proteins that are not PSCA-related proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Tris buffered saline solution containing 25 mM Tris and 150 mM NaCl; or normal saline (0.9% NaCl);

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4) animal serum such as human serum; or 5) a combination of any of 1) to 4); these reactions preferably take place at pH 7.5, alternatively in a range of pH 7.0 to 8.0, or alternatively in a pH range of 6.5 to 8.5; these reactions also taking place at a temperature between 4 ° C and 37 ° C. For example, antibodies that bind with PSCA can bind with PSCA-related proteins such as homologs.

or analogues thereof.

PSCA antibodies of the invention are particularly useful in cancer diagnostic and prognostic assays (see, for example, Table I) and image capture methodologies. Similarly, such antibodies are useful in the treatment, diagnosis and / or prognosis of prostate and other cancers, to the extent that PSCA is also expressed or overexpressed in these other cancers. In addition, intracellularly expressed antibodies (eg, single chain antibodies) are therapeutically useful in the treatment of cancers in which PSCA expression is involved, such as advanced or metastatic prostate cancers or other advanced or metastatic cancers.

The antibodies of the invention can also be used in various immunological assays useful for the detection and quantification of PSCA and mutant PSCA-related proteins. Such assays may comprise one or more PSCA antibodies capable of recognizing and binding with a PSCA-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA) and the like.

Non-antibody immunological assays also comprise T lymphocyte immunogenicity assays (inhibitors or stimulators) as well as major histocompatibility complex (MHC) binding assays.

In addition, immunological image capture methods capable of detecting prostate cancer and other cancers expressing PSCA are also disclosed herein, including but not limited to methods of capturing radio-scintigraphic images using labeled PSCA antibodies. Such trials are clinically useful in the detection, control and prognosis of cancers that express PSCA such as prostate cancer.

PSCA antibodies are also used in methods to purify a PSCA-related protein and to isolate PSCA homologs and related molecules. For example, a method for purifying a PSCA-related protein comprises incubating a PSCA antibody, which has been coupled with a solid matrix, with a lysate or other solution containing a PSCA-related protein under conditions that allow the PSCA antibody binds with PSCA-related protein; wash the solid matrix to remove impurities; and eluting the PSCA-related protein from the coupled antibody. Other uses of PSCA antibodies include generating anti-idiotypic antibodies that mimic a PSCA protein.

Various methods for the preparation of antibodies are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a PSCA-related protein, peptide or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, PSCA fusion proteins, such as a PSCA GST fusion protein, can also be used. In a particular embodiment, a GST fusion protein is produced comprising all or most of the amino acid sequence of Figure 1, and then used as an immunogen to generate appropriate antibodies. In another embodiment, a PSCA-related protein is synthesized and used as an immunogen.

In addition, nude DNA immunization techniques known in this field (with or without purified PSCA-related protein or cells expressing PSCA) are used to generate an immune response to the encoded immunogen (for a review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of a PSCA protein as shown in Figure 1 can be analyzed to select specific regions of the PSCA protein to generate antibodies. For example, hydrophobicity and hydrophilicity analyzes of a PSCA amino acid sequence are used to identify hydrophilic regions in the PSCA structure. Regions of a PSCA protein that show immunogenic structure, as well as other regions and domains, can be readily identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz analysis or Jameson-Wolf. Hydrophilic profiles can be generated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci.

USES. 78: 3824-3828. Hydropaticity profiles can be generated using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157: 105-132. Percentage profiles (%) of Accessible Remains can be generated using the method of Janin J., 1979, Nature 277: 491-492. Average flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32: 242-255. Beta turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1: 289-294. Therefore, each region identified by any of these programs or methods is within the scope of the present invention. Preferred methods for generating PSCA antibodies are further illustrated by means of the examples provided herein. Methods for preparing are well known in the art

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a protein or polypeptide for use as an immunogen. Methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein, are also well known in the art. In some circumstances, direct conjugation is used using, for example, carbodiimide reagents; in other cases, binding reagents such as those provided by Pierce Chemical Co., Rockford, IL, are effective. Administration of a PSCA immunogen is frequently performed by injection for a suitable period of time and with use of a suitable adjuvant, as understood in the art. During the immunization program, antibody titres can be taken to determine the adequacy of antibody formation.

Monoclonal antibodies to PSCA can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using conventional hybridoma technology from Kohler and Milstein or modifications that immortalize antibody-producing B lymphocytes, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a PSCA-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and the antibodies produced either from in vitro cultures or ascites fluid.

The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that specifically bind to the desired regions of a PSCA protein can also be produced in the context of antibodies with chimeric region grafts or complementarity determinant (CDR) of multiple species origins. Humanized or human PSCA antibodies can also be produced and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies are well known, substituting corresponding human antibody sequences with one or more of the non-human antibody CDRs (see, for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies include phage and transgenic presentation methods (for a review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human PSCA monoclonal antibodies can be generated using cloning technologies that employ large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. : Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Same reference., Pp 65 -82). Fully human PSCA monoclonal antibodies can also be produced using technically modified transgenic mice to contain human immunoglobulin gene loci as described in PCT Patent Application WO98 / 24893, Kucherlapati and Jakobovits et al., Published December 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7 (4): 607-614; United States patents

6,162,963 issued on December 19, 2000; 6,150,584 issued on November 12, 2000; and 6,114,598 issued on September 5, 2000). This method avoids the required in vitro manipulation with phage display technology and effectively produces authentic high affinity human antibodies.

The reactivity of PSCA antibodies with a PSCA-related protein can be established by several well-known means, including Western blotting, immunoprecipitation, ELISA and FACS analysis using, as appropriate, PSCA-related proteins, cells expressing PSCA or extracts of the same. A PSCA antibody or fragment thereof can be labeled with a detectable label or conjugated with a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. In addition, specific bispecific antibodies are generated for two or more PSCA epitopes using methods generally known in the art. Homodimeric antibodies can also be generated by crosslinking techniques known in this field (eg, Wolff et al., Cancer Res. 53: 2560-2565).

In one embodiment, the invention provides monoclonal antibodies identified as Hal-4,121, sent (by Federal Express) to the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108 on May 4, 2005 and given assigned the reference number PTA-6701. Monoclonal antibodies identified as Ha1-1.16, Ha1-5.99, Ha1-4.117, Ha1-4.20, Ha1-4.37 that were sent (by Federal Express) to the American Type Culture Collection (ATCC), P.O. are also disclosed herein. Box 1549, Manassas, VA 20108 on May 4, 2005 and were assigned Reference numbers PTA-6698, PTA-6703, PTA-6699, PTA-6700 and PTA-6702 respectively.

V.) Cellular Immune Responses to PSCA

The mechanism by which T lymphocytes recognize antigens has been delimited. The effective peptide epitope vaccine compositions disclosed herein induce therapeutic or prophylactic immune responses in very broad segments of the world population. For an understanding of the value and efficacy of compositions that induce cellular immune responses, a brief review of the technology is provided.

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Immunology related.

A complex of an HLA molecule and a peptide antigen acts as the ligand recognized by HLA-restricted T lymphocytes (Buus, S. et al., Cell 47: 1071, 1986; Babbitt, BP et al., Nature 317: 359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7: 601, 1989; Germain, RN, Annu. Rev. Immunol. 11: 403, 1993). Throughout the study of antigen analogs with substituted individual amino acids and the sequencing of naturally processed peptides, endogenously bound, critical moieties have been identified that correspond to motifs required for specific binding with HLA antigen molecules and are exposed in Table IV (see also, for example, Southwood, et al., J. Immunol. 160: 3363, 1998; Rammensee, et al., Immunogenetics 41: 178, 1995; Rammensee et al., SYFPEITHI, access through from the Web at the URL (134.2.96.221/scripts.hlaserver.dll/home.htm); Sette, A. and Sidney, J. Curr. Opin. Immunol. 10: 478, 1998; Engelhard, VH, Curr. Opin Immunol 6: 13, 1994; Sette, A. and Gray, HM, Curr. Opin. Immunol. 4: 79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6: 52, 1994; Ruppert et al., Cell 74: 929-937, 1993; Kondo et al., J. Immunol. 155: 4307-4312, 1995; Sidney et al., J. Immunol. 157: 3480-3490, 1996; Sidney et al. ., Human Immunol. 45: 79-93, 1996; Sette, A. and Sidney, J. Immunogenetics Nov 1999; 50 (3-4): 201-12, Review).

In addition, X-ray crystallographic analyzes of HLA-peptide complexes have revealed pockets within the peptide junction / groove of HLA molecules that accommodate, in a specific allele manner, residues carried by peptide ligands; These residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, for example, Madden, DR Annu. Rev. Immunol. 13: 587, 1995; Smith, et al., Immunity 4: 203, 1996; Fremont et al., Immunity 8: 305, 1998; Stern et al. , Structure 2: 245, 1994; Jones, EY Curr. Opin. Immunol. 9: 75, 1997; Brown, JH et al., Nature 364: 33, 1993; Guo, HC et al., Proc. Natl. Acad. Sci. USA

90: 8053, 1993; Guo, H. C. et al., Nature 360: 364, 1992; Silver, M. L. et al., Nature 360: 367, 1992; Matsumura, M. et al., Science 257: 927, 1992; Madden et al., Cell 70: 1035, 1992; Fremont, D. H. et al., Science 257: 919,1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219: 277, 1991).

Consequently, the definition of HLA binding motifs specific to class I and class II alleles or supermotives of class I or class II allows the identification of regions within a protein that are correlated with binding to an HLA antigen or antigens private individuals

Therefore, by an HLA reason identification process, candidates for epitope-based vaccines have been identified; said candidates can be further evaluated by HLA-peptide binding assays to determine the binding affinity and / or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmation work can be performed to select, among these vaccine candidates, epitopes with preferred characteristics with respect to population coverage and / or immunogenicity.

Various strategies can be used to assess cellular immunogenicity, including:

1) Evaluation of cultures of primary T lymphocytes from normal individuals (see, for example, Wentworth, PA et al., Mol. Immunol. 32: 603, 1995; Celis, E. et al., Proc. Natl. Acad Sci. USA 91: 2105, 1994; Tsai, V. et al., J. Immunol. 158: 1796, 1997; Kawashima, I. et al., Human Immunol. 59: 1, 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBL) of normal subjects with a test peptide in the presence of in vitro antigen presenting cells over a period of several weeks. Peptide-specific T lymphocytes are activated during this time and are detected using, for example, a 51Cr or lymphokine release assay that involves peptide sensitized target cells. 2) Immunization of transgenic mice for HLA (see, for example, Wentworth, PA et al., J. Immunol. 26: 97, 1996; Wentworth, PA et al., Int. Immunol. 8: 651, 1996; Alexander, J. et al., J. Immunol. 159: 4753, 1997). For example, in such methods Freund's incomplete adjuvant peptides are administered subcutaneously to transgenic mice for HLA. Several weeks after immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for about a week. Peptide specific T lymphocytes are detected using, for example, a 51 Cr release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen. 3) Demonstration of recall T-cell responses from immune individuals who have been successfully vaccinated and / or chronically ill patients (see, for example, Rehermann, B. et al., J. Exp. Med. 181: 1047, 1995; Doolan, DL et al., Immunity 7: 97, 1997; Bertoni, R. et al., J. Clin. Invest. 100: 503, 1997; Threlkeld, SC et al., J. Immunol. 159: 1648 , 1997; Diepolder, HM et al., J. Virol. 71: 6011, 1997). Consequently, recall responses are detected by culturing PBL from subjects who have been exposed to the antigen due to a disease and therefore have generated a "naturally" immune response or from patients who have been vaccinated against the antigen. PBL of subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of "memory" T lymphocytes, as compared to "virgin" T lymphocytes. At the end of the culture period, T lymphocyte activity is detected using assays including 51 Cr release involving peptide sensitized targets, T lymphocyte proliferation or lymphokine release.

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VI.) Transgenic Animals for PSCA

Nucleic acids encoding a PSCA-related protein can also be used to generate transgenic animals or knock out animals which, in turn, are useful in the development and exploration of therapeutically useful reagents. According to established techniques, cDNA encoding PSCA can be used to clone genomic DNA encoding PSCA. The cloned genomic sequences can then be used to generate transgenic animals that contain cells that express DNA encoding PSCA. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 issued April 12, 1988 and 4,870,009 issued on September 26, 1989. Normally, incorporation of PSCA transgenes would be directed to particular cells with tissue specific enhancers.

Transgenic animals that include a copy of a transgene encoding PSCA can be used to examine the effect of increased expression of DNA encoding PSCA. Such animals can be used as test animals for reagents that are believed to confer protection from, for example, pathological conditions associated with their overexpression. According to the present disclosure, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human PSCA homologs can be used to construct a "knock out" animal for PSCA that has a defective or altered gene encoding PSCA as a result of homologous recombination between the endogenous gene encoding PSCA and altered genomic DNA encoding introduced PSCA in an animal embryonic cell. For example, cDNA encoding PSCA can be used to clone genomic DNA encoding PSCA according to established techniques. A part of the genomic DNA encoding PSCA can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to control integration. Typically, several kilobases of unchanged flanking DNA (at both 5 'and 3' ends) are included in the vector (see, for example, Thomas and Capecchi, Cell, 51: 503 (1987) for a description of homologous recombination vectors ). The vector is introduced into an embryonic stem cell line (for example, by electroporation) and cells in which the introduced DNA has been homologously recombined with the endogenous DNA are selected (see, for example, Li et al., Cell , 69: 915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or a rat) to form aggregation chimeras (see, for example, Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.

J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted in a suitable pseudo-pregnant female substitute animal, and the embryo is completed to create a knock out animal. The offspring that houses homologously recombined DNA in their germ cells can be identified by conventional techniques and used to raise animals in which all cells of the animal contain homologously recombined DNA. Knock out animals can be characterized, for example, by their ability to defend against certain pathological conditions or by their development of pathological conditions due to the absence of a PSCA polypeptide.

VII.) Methods for the detection of PSCA

Another disclosure herein refers to methods for detecting PSCA polynucleotides and PSCA-related proteins, as well as methods for identifying a cell expressing PSCA. The PSCA expression profile makes it a diagnostic marker for metastatic disease. Consequently, the status of PSCA gene products provides useful information to predict various factors including susceptibility to advanced stage disease, rate of progression and / or aggressiveness of the tumor. As discussed in detail herein, the status of PSCA gene products in patient samples can be analyzed by various protocols that are well known in the art including immunohistochemical analysis, the diversity of Northern blotting techniques including in situ hybridization. , RT-PCR analysis (for example in microdissected laser capture samples), Western blot analysis and tissue matrix analysis.

More particularly, the disclosure provides assays for the detection of PSCA polynucleotides in a biological sample, such as serum, bone, prostate and other tissues, urine, semen, cell preparations and the like. Detectable PSCA polynucleotides include, for example, a PSCA gene or fragment thereof, PSCA mRNA, alternative splicing variant PSCA mRNA and recombinant DNA or RNA molecules containing a PSCA polynucleotide. Several methods for amplifying and / or detecting the presence of PSCA polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.

A method of detecting a PSCA mRNA in a biological sample may comprise producing cDNA of the sample by reverse transcription using at least one primer; amplify the cDNA produced in this way using PSCA polynucleotides as sense and antisense primers to amplify PSCA cDNAs therein; and detect the presence of the amplified PSCA cDNA. Optionally, the sequence of the amplified PSCA cDNA can be determined.

A method of detecting a PSCA gene in a biological sample may comprise first isolating DNA.

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genomic of the sample; amplify the isolated genomic DNA using PSCA polynucleotides as sense and antisense primers; and detect the presence of the amplified PSCA gene. Any variety of appropriate sense and antisense probe combinations can be designed from a PSCA nucleotide sequence (see, for example, Figure 1) and used for this purpose.

Assays for detecting the presence of a PSCA protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations and the like are also disclosed herein. Methods for detecting a PSCA-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a PSCA-related protein in a biological sample comprises first contacting the sample with a PSCA antibody, a PSCA reactive fragment thereof, or a recombinant protein containing a region. antigen binding of a PSCA antibody; and then detect PSCA-related protein binding in the sample.

Methods for identifying a cell expressing PSCA are also within the scope of the disclosures. An assay to identify a cell that expresses a PSCA gene may comprise detecting the presence of PSCA mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled PSCA ribosonades, Northern blotting and related techniques) and various amplification assays. of nucleic acids (such as RT-PCR using complementary primers specific for PSCA, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay to identify a cell that expresses a PSCA gene comprises detecting the presence of PSCA-related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are used for the detection of PSCA-related proteins and cells expressing PSCA-related proteins.

PSCA expression analysis is also useful as a tool to identify and evaluate agents that modulate PSCA gene expression. For example, PSCA expression is positively regulated in prostate cancer and is expressed in tissue cancers listed in Table I. The identification of a molecule or biological agent that inhibits the expression or overexpression of PSCA in cells. Cancer has therapeutic value. For example, said agent can be identified using a scan that quantifies PSCA expression by RT-PCR, nucleic acid hybridization or antibody binding.

VIII.) Methods to Control the Status of Genes Related to PSCA and Its Products

It is known that oncogenesis is a multistage process in which cell growth is progressively deregulated and cells progress from a normal to precancerous physiological state and then cancerous states (see, for example, Alers et al., Lab Invest. 77 (5 ): 437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)). In this context, the examination of a biological sample with respect to tests of deregulated cell growth (such as expression of aberrant PSCA in cancers) allows the early detection of said aberrant physiological, before a pathological state such as cancer has progressed to stage in which the therapeutic options are more limited and / or the prognosis is worse. In such tests, the PSCA status in a biological sample of interest can be compared, for example, with the PSCA status in a corresponding normal sample (for example a sample of that individual or alternatively another individual who is not affected by a pathology). An alteration in the state of PSCA in the biological sample (compared to the normal sample) provides evidence of deregulated cell growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, a predetermined normative value such as a predetermined normal level of mRNA expression can also be used (see, for example, Grever et al., J. Comp. Neurol. 9 Dec 1996; 376 (2): 306-14 and US Patent No. 5,837,501) to compare the status of PSCA in a sample.

The term "state" in this context is used in accordance with its accepted meaning in the art and refers to the condition or state of a gene and its products. Normally, those skilled in the art use various parameters to assess the condition or condition of a gene and its products. These include, but are not limited to, the location of expressed gene products (including the location of cells expressing PSCA) as well as the level, and the biological activity of expressed gene products (such as PSCA mRNA, polynucleotides and polypeptides). Normally, an alteration of the PSCA status comprises a change in the location of PSCA and / or cells expressing PSCA and / or an increase in the expression of mRNA and / or PSCA protein.

The status of PSCA in a sample can be analyzed by several means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis in microdissected samples with laser capture, Western blot analysis and analysis of tissue matrices. Typical protocols are found to assess the status of a gene and gene products of PSCA, for example, in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Transfer), 4 (Southern Transfer), 15 (Immunoblot) and 18 (PCR analysis). Therefore, the status of PSCA in a biological sample is evaluated by various methods used by experts in the field including, but not limited to Southern analysis.

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genomic (to examine, for example perturbations in a PSCA gene), Northern analysis and / or PSCA mRNA PCR analysis (to examine, for example, alterations in polynucleotide sequences or levels of PSCA mRNA expression), and Western and / or immuohistochemical analysis (to examine, for example, alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in levels of PSCA protein expression and / or associations of PSCA proteins with polypeptide binding partners). Detectable PSCA polynucleotides include, for example, a PSCA gene or fragment thereof, PSCA mRNA, alternative splicing variants, PSCA mRNA and recombinant DNA or RNA molecules containing a PSCA polynucleotide.

The PSCA expression profile makes it a diagnostic marker for local and / or metastatic disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, PSCA status provides useful information to predict susceptibility to particular disease stages, progression and / or tumor aggressiveness. Methods and assays for determining the status of PSCA and diagnosing cancers expressing PSCA are disclosed herein, such as tissue cancers listed in Table I. For example, because PSCA mRNA is expressed to such a high extent. In prostate and other cancers in relation to normal prostate tissue, assays evaluating levels of transcripts of mRNA or PSCA proteins can be used in a biological sample to diagnose a disease associated with PSCA deregulation, and can provide prognostic information useful for defining appropriate therapeutic options.

The expression status of PSCA provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, which predict susceptibility to various stages of disease and / or to gauge tumor aggressiveness. In addition, the expression profile makes it useful as an image capture reagent for metastatic disease. Accordingly, various molecular prognostic and diagnostic methods are disclosed herein to examine the status of PSCA in biological samples such as those of individuals who suffer, or are suspected of suffering from a pathology characterized by deregulated cell growth, such as cancer.

As described above, the status of PSCA in a biological sample can be examined by several procedures well known in the art. For example, the status of PSCA in a biological sample taken from a specific location in the body can be examined by evaluating the sample with respect to the presence or absence of cells expressing PSCA (for example those expressing mRNA or PSCA proteins). This test may provide evidence of deregulated cell growth, for example, when cells expressing PSCA are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the state of PSCA in a biological sample they are frequently associated with deregulated cell growth. Specifically, an indicator of deregulated cell growth is the metastasis of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, deregulated cell growth tests are important for example because hidden lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, for example , Murphy et al., Prostate 42 (4): 315-317 (2000); Su et al., Semin. Surg. Oncol. 18 (1): 17-28 (2000) and Freeman et al., J Urol Aug 1995 154 (2 Pt 1): 474-8).

Methods for controlling PSCA gene products are disclosed herein by determining the status of PSCA gene products expressed by cells of an individual suspected of having a disease associated with deregulated cell growth (such as hyperplasia or cancer) and comparing then the state determined in this way with the status of PSCA gene products in a corresponding normal sample. The presence of aberrant PSCA gene products in the test sample in relation to the normal sample provides an indication of the presence of deregulated cell growth within the individual's cells.

Useful assays for determining the presence of cancer in an individual are disclosed herein, comprising detecting a significant increase in PSCA protein or mRNA expression in a cell sample.

or tissue test in relation to the levels of expression in the cell or the corresponding normal tissue. The presence of PSCA mRNA can be evaluated, for example, in tissues including but not limited to those listed in Table I. The presence of significant PSCA expression in any of these tissues is useful to indicate the appearance, presence and / or severity. in a cancer, since the corresponding normal tissues do not express mRNA of PSCA or express it at lower levels.

In a related disclosure, the PSCA status is determined at the level of proteins rather than at the level of nucleic acid. For example, said method comprises determining the level of PSCA protein expressed by cells in a test tissue sample and comparing the level determined in this way with the level of PSCA expressed in a corresponding normal sample. The presence of PSCA protein can be evaluated, for example, using immunohistochemical methods. PSCA antibodies or binding partners capable of detecting PSCA protein expression are used in various assay formats well known in the art for this purpose.

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The status of nucleotide and amino acid sequences of PSCA in a biological sample can be evaluated to identify disturbances in the structure of these molecules. These disturbances may include insertions, deletions, substitutions and the like. Such evaluations are useful because disturbances in nucleotide and amino acid sequences are observed in a large number of proteins associated with a deregulated growth phenotype (see, for example, Marrogi et al., 1999, J. Cutan. Pathol. 26 (8 ): 369-378). For example, a mutation in the PSCA sequence may be indicative of the presence or promotion of a tumor. Such trials therefore have diagnostic or predictive value in which a mutation in PSCA indicates a potential loss of function or increased tumor growth.

A wide variety of assays for observing disturbances in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequence of PSCA gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing disturbances in nucleotide and amino acid sequences such as single stranded polymorphism analysis are well known in the art (see, for example, U.S. Patent No. 5,382,510 issued September 7, 1999 and 5,952,170 issued January 17, 1995).

Additionally, the methylation status of a PSCA gene in a biological sample can be examined. Aberrant demethylation and / or hyperventilation of CpG islands occurs in 5 ’gene regulatory regions that frequently appear in immortalized and transformed cells, and may result in altered expression of various genes. For example, hypermethylation of the glutathione S-transferase promoter of class pi (a protein expressed in normal prostate but not expressed in> 90% of prostate carcinomas) seems to permanently silence the transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al., Am. J. Pathol. 155 (6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev., 1998, 7: 531-536). In another example, expression of the LAGE-I tumor-specific gene is induced (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) by deoxy-azacitidine in lymphoblast cells, suggesting that Tumor expression is due to demethylation (Lethe et al., Int. J. Cancer 76 (6): 903-908 (1998)). Various assays to examine the methylation status of a gene are well known in the art. For example, methylation-sensitive restriction enzymes that cannot cleave sequences containing methylated CpG sites can be used in Southern hybridization approaches to assess the methylation status of CpG islands. In addition, MSP (methylation-specific PCR) can rapidly profile the methylation status of all CpG sites present on a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using specific primers for methylated vs. nonmethylated DNA. Protocols that involve methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.

Gene amplification is an additional method to assess the status of PSCA. Gene amplification is measured in a sample directly, for example, by Southern blotting or conventional Northern blotting to quantify mRNA transcription (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77: 5201 5205), transfer point (DNA analysis) or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies that recognize specific double strands are used, including double strands of DNA, double strands of RNA, and double strands of hybrid RNA or double strands of protein and DNA. The antibodies in turn are labeled and the assay is carried out in which the double chain binds to a surface, so that after the formation of the double chain to the surface, the presence of double bound antibody can be detected chain.

Biopsy tissue or peripheral blood may be conveniently tested for the presence of cancer cells using, for example, Northern analysis, point transfer or RT-PCR to detect PSCA expression. The presence of PSCA mRNA amplifiable by RT-PCR provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and treatment of several human solid tumors. In the field of prostate cancer, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25: 373-384; Ghossein et al., 1995,

J. Clin. Oncol. 13: 1195-2000; Heston et al., 1995, Clin. Chem. 41: 1687-1688).

An assessment of the susceptibility that an individual has to develop cancer is disclosed herein. A method for predicting cancer susceptibility may comprise detecting PSCA mRNA or PSCA protein in a tissue sample, indicating its presence susceptible to cancer, in which the degree of expression of PSCA mRNA correlates with the degree of susceptibility. In one method, the presence of PSCA in prostate or other tissue is examined, providing the presence of PSCA in the sample an indication of susceptibility to prostate cancer (or the appearance or existence of a prostate tumor). Similarly, the integrity of amino acid and nucleotide sequences of PSCA in a biological sample can be evaluated to identify disturbances of the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in PSCA gene products in the sample is an indication of the

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susceptibility to cancer (or the appearance or existence of a tumor).

Methods for calibrating tumor aggressiveness are also disclosed herein. A method for calibrating the aggressiveness of a tumor may comprise determining the level of PSCA mRNA or PSCA protein expressed by tumor cells, comparing the level determined in this way with the level of PSCA mRNA or PSCA protein expressed in a corresponding normal tissue taken. of the same individual or a normal tissue reference sample, in which the degree of expression of PSCA mRNA or PSCA protein in the tumor sample in relation to the normal sample indicates the degree of aggressiveness. In one method, the aggressiveness of a tumor is evaluated by determining the degree to which PSCA is expressed in tumor cells, indicating higher levels of expression more aggressive tumors. Another method is the evaluation of the integrity of amino acid and nucleotide sequences of PSCA in a biological sample, to identify disturbances in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more disturbances indicates more aggressive tumors.

Methods for observing the progression of a malignant tumor in an individual over time are disclosed herein. Methods to observe the progression of an individual's malignant tumor over time may comprise determining the level of PSCA mRNA or PSCA protein expressed by cells in a tumor sample, comparing the level determined in this way with the level of PSCA mRNA or PSCA protein expressed in an equivalent tissue sample taken from the same individual at a different time, in which the degree of expression of PSCA mRNA or PSCA protein in the tumor sample over time provides information on the progression of the Cancer. In one method, the progression of a cancer is evaluated by determining the expression of PSCA in the tumor cells over time, in which an expression increased over time indicates a progression of the cancer. The integrity of the nucleotide and amino acid sequences of PSCA in a biological sample can also be evaluated to identify disturbances in the structure of these molecules such as insertions, deletions, substitutions and the like, in which the presence of one or more perturbations indicates a cancer progression

The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. Methods for observing a coincidence between the expression of the PSCA gene and PSCA gene products (or disturbances in the PSCA gene and PSCA gene products) and a factor that is associated with malignancy, such as a malignancy, are also disclosed herein. means to diagnose and predict the state of a tissue sample. A wide variety of factors associated with malignancy can be used, such as the expression of genes associated with malignancy (eg, expression of PSA, PSCA and PSM for prostate cancer, etc.) as well as general cytological observations (see, for example, Bocking et al., 1984, Anal. Quant. Cytol. 6 (2): 74-88; Epstein, 1995, Hum. Pathol. 26 (2): 223-9; Thorson et al., 1998, Mod. Pathol. 11 (6): 543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23 (8): 91824). Methods to observe a coincidence between the expression of the PSCA gene and PSCA gene products (or disturbances in the PSCA gene and PSCA gene products) and another factor that is associated with malignancy are useful, for example, because the presence A set of specific factors that coincide with the disease provides crucial information to diagnose or predict the state of a tissue sample.

Methods to observe a coincidence between the expression of the PSCA gene and PSCA gene products (or disturbances in the PSCA gene and PSCA gene products) and another factor associated with malignancy may involve detecting overexpression of mRNA or PSCA protein in a tissue sample, detect overexpression of mRNA or PSA protein in a tissue sample (or expression of PSCA or PSM) and observe a coincidence of overexpression of mRNA or PSCA protein and mRNA and PSA protein (or expression of PSCA or PSM). The expression of PSCA and PSA mRNA in prostate tissue can be examined, in which the coincidence of overexpression of PSCA and PSA mRNA in the sample indicates the existence of prostate cancer, susceptibility to prostate cancer or on the appearance or State of a prostate tumor.

Methods for detecting and quantifying expression of PSCA mRNA or protein are described herein, and conventional nucleic acid and protein detection and quantification technologies are well known in the art. Conventional methods for detection and quantification of PSCA mRNA include in situ hybridization using labeled PSCA ribosonates, Northern blotting and related techniques using PSCA polynucleotide probes, RT-PCR analysis using PSCA specific primers and other amplification type detection methods. , such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify PSCA mRNA expression. Any variety of primers capable of amplifying PSCA can be used for this purpose, including but not limited to the various sets of primers specifically described herein. Polyclonal or monoclonal antibodies specifically reactive with the wild-type PSCA protein can be used in an immunohistochemical biopsy tissue assay.

IX.) Identification of molecules that interact with PSCA

The PSCA protein and nucleic acid sequences disclosed herein allow a person skilled in the art to identify proteins, small molecules and other agents that interact with PSCA, as well.

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He is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63: 231-237; Fong et al., 1997, J. Immunol. 159: 3113-3117).

Such methods can be easily practiced using a PSCA-related protein or a nucleic acid molecule encoding PSCA and recombinant vectors capable of expressing and presenting the PSCA immunogen (which typically comprises several T-cell or antibody epitopes). Those skilled in the art understand that a wide variety of vaccine systems for immunoreactive epitope delivery are known in the art (see, for example, Heryln et al., Ann Med Feb 1999 31 (1): 66-78; Maruyama et al., Cancer Immunol Immunother Jun 2000 49 (3): 123-32). Briefly, said methods for generating an immune response (for example cell-mediated and / or humoral) in a mammal, comprise the steps of: exposing the mammalian immune system to an immunoreactive epitope (for example an epitope present in a PSCA protein shown in Figure 1 or analogue or homologue thereof) so that the mammal generates an immune response that is specific for that epitope (for example it generates antibodies that specifically recognize that epitope).

The complete PSCA protein, immunogenic regions or epitopes thereof can be combined and supplied by various means. Such vaccine compositions may include, for example, lipopeptides (eg, Vitiello, A. et al., J. Clin. Invest. 95: 341, 1995), peptide compositions encapsulated in poly (DLlactide-co-glycolide) microspheres. ("PLG") (see, for example, Eldridge, et al., Molec. Immunol. 28: 287-294, 1991: Alonso et al., Vaccine 12: 299-306, 1994; Jones et al., Vaccine 13 : 675-681, 1995), peptide compositions contained in immunostimulatory complexes (ISCOM) (see, for example, Takahashi et al., Nature 344: 873-875, 1990; Hu et al., Clin Exp Immunol. 113: 235- 243, 1998), multiple antigenic peptide (MAP) systems (see for example Tam,

J. P., Proc. Natl. Acad. Sci. U.S.A. 85: 5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196: 17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, normally crystallized peptides, viral delivery vectors (Perkus, ME et al., In: Concepts in vaccine development, Kaufmann, SHE, ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320: 535, 1986; Hu, SL et al., Nature 320: 537, 1986; Kieny, M.-P. et al., AIDS Bio / Technology 4: 790, 1986; Top, FH et al., J. Infect. Dis. 124: 148, 1971; Chanda, PK et al., Virology 175: 535, 1990), particles of viral or synthetic origin (eg, Kofler, N. et al., J. Immunol. Methods. 192: 25, 1996; Eldridge, JH et al., Sem. Hematol. 30: 16, 1993; Falo, LD, Jr. et al., Nature Med. 7: 649, 1995), adjuvants (Warren , HS, Vogel, FR, and Chedid, LA Annu. Rev. Immunol. 4: 369, 1986; Gupta, RK et al., Vaccine 11: 293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148: 1585, 1992; Rock, KL, Immunol. Today 17: 131, 1996), or naked or absorbed cDNA in a particle (Ulmer, JB et al., Science 259: 1745, 1993; Robinson, H. L., Hunt, L.A., and Webster, R. G., Vaccine 11: 957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., P. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12: 923, 1994 and Eldridge, J. H. et al., Sem. Hematol 30: 16, 1993). Toxin-directed delivery technologies may also be used, also known as receptor-mediated address, such as those of Avant Immunotherapeutics, Inc. (Needham, Massachusetts).

In patients with cancer associated with PSCA, the vaccine compositions may also be used in conjunction with other treatments used for cancer, for example, surgery, chemotherapy, drug therapies, radiation therapies, etc. including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identify peptides within the PSCA protein that bind to corresponding HLA alleles (see for example, Table IV; Epimer ™ and Epimatrix ™, Brown University (URL brown.edu/Research /TB-HIV_Lab/epimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI in URL syfpeithi.bmi-heidelberg.com/) A PSCA immunogen may contain one or more sequences of amino acids identified using techniques well known in this field, such as the sequences shown in Tables V-XVIII and XXII-LI or a peptide of 8, 9, 10 or 11 amino acids specified by a motive / supermotivation of HLA Class I ( for example, Table IV (A), Table IV (D), or Table IV (E)) and / or a peptide of at least 9 amino acids comprising a motif / supermotive of Class II HLA (for example, Table IV ( B) or Table IV (C)) As can be seen in the art, the HLA Class I binding groove is essentially closed so that only peptides of a particular size range can fit in the groove and bind, generally Class I HLA epitopes are 8, 9, 10 or 11 amino acids in length. In contrast, the HLA Class II binding groove is essentially open; therefore a peptide of about 9 can be attached

or more amino acids with a HLA Class II molecule. Due to differences in the binding groove between HLA Class I and II, the HLA Class I motifs are length specific, that is, position two of a Class I motif is the second amino acid in an amino to carboxyl direction. of the peptide. The amino acid positions in a Class II motif are only relative to each other, not to the general peptide, that is, additional amino acids can be attached to the terminal amino and / or carboxyl ends of a motif carrier sequence. Class II HLA epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length, or of More than 25 amino acids.

A wide variety of methods for generating an immune response in a mammal are known in the art (for example as the first stage in hybridoma generation). The methods to generate a response

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Ex vivo vaccines

Various ex vivo strategies can also be used to generate an immune response. One approach involves the use of antigen presenting cells (APCs) such as dendritic cells (DC) to present the PSCA antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, costimulator of B7 and IL-12, and are therefore highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with prostate-specific membrane antigen (PSMA) peptides are being used in a Phase I clinical trial to stimulate the immune systems of prostate cancer patients (Tjoa et al., 1996 , Prostate 28: 65-69; Murphy et al., 1996, Prostate 29: 371-380). Therefore, dendritic cells can be used to present PSCA peptides to T lymphocytes in the context of MHC class I or II molecules. In one method, autologous dendritic cells are pulsed with PSCA peptides capable of binding with MHC class I and / or class II molecules. In another method, dendritic cells are pulsed with the complete PSCA protein. Another strategy involves the technical modification of the overexpression of a PSCA gene in dendritic cells using various implementation vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4: 17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56: 3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res. 57: 28652869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186: 1177-1182). Cells expressing PSCA can also be technically modified to express immune modulators, such as GM-CSF, and used as immunization agents.

X.B.) PSCA as a Diana for Antibody-based Therapy

PSCA is an attractive target for therapeutic strategies based on antibodies. Several antibody strategies for targeting both extracellular and intracellular molecules are known in the art (see, for example, complement-mediated destruction and ADCC as well as the use of intrabodies). Because PSCA is expressed by cancer cells of various lineages in relation to corresponding normal cells, systemic administration of immunoreactive compositions to PSCA is prepared which show excellent sensitivity without toxic, non-specific and / or non-target effects by binding the immunoreactive composition with non-target organs and tissues. Antibodies specifically reactive with PCA domains are useful for treating cancers that express PSCA systemically, either as conjugates with a toxin or therapeutic agent or as naked antibodies capable of inhibiting cell proliferation or function.

PSCA antibodies can be introduced into a patient so that the antibody binds with PSCA and modulates a function, such as an interaction with a binding partner, and consequently mediates the destruction of tumor cells and / or inhibits the growth of tumor cells The mechanisms by which said antibodies exert a therapeutic effect may include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of PSCA, inhibition of ligand binding or signal transduction pathways, modulation of differentiation of tumor cells, alteration of profiles of tumor angiogenesis factors and / or apoptosis. Examples include Rituxan® for Non-Hodgkin's Lymphoma, Herceptin® for metastatic breast cancer and Erbitux® for colorectal cancer.

Those skilled in the art understand that antibodies can be used to specifically target and bind to immunogenic molecules such as an immunogenic region of a PSCA sequence shown in Figure 1. In addition, those skilled in the art understand that it is routine to conjugate antibodies with agents. cytotoxic agents (see, for example, Slevers et al. Blood 93: 11 3678-3684 (June 1, 1999)). When cytotoxic and / or therapeutic agents are delivered directly to cells, such as by conjugating them with antibodies specific for a molecule expressed by that cell (eg, PSCA), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.

A wide variety of compositions and methods for using cytotoxic antibody antibody conjugates to kill cells are known in the art. In the context of cancers, typical methods involve administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and / or therapeutic agent bound to a targeting agent (for example an anti PSCA antibody) that binds with a marker (eg PSCA) expressed, accessible to the junction or located on cell surfaces. A typical embodiment is a method of delivering a cytotoxic and / or therapeutic agent to a cell expressing PSCA, which comprises conjugating the cytotoxic agent with an antibody that immunospecifically binds with a PSCA epitope and exposing the cell to the antibody-agent conjugate. . Another illustrative embodiment is a method of treating an individual suspected of suffering from metastatic cancer, which comprises a step of parenterally administering to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic agent and / or therapeutic

Cancer immunotherapy can be performed using anti PSCA antibodies according to various approaches that have been successfully used in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18: 133-138), multiple myeloma (Ozaki et al., 1997, Blood 90: 3179-3186, Tsunenari et al., 1997, Blood 90: 2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52: 2771-2776), B lymphocyte lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19: 93-101), leukemia

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carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, alpha interferon, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargasa, pentostatin, pipobroman, plicamycin, thiophenozone, stiphetozoan, thiophenozole, streptozoa, thiophenozole, streptozoa, thiophenozole, streptozoa, thiophenozole, streptozoa, thiophenozole, streptozoa, thiophenozone of uracil, vinorelbine, chlorambucil, taxol and combinations thereof.

The radiation source, used in combination with a PSCA antibody, can be external or internal to the patient being treated. When the source is external to the patient, the therapy is known as external beam radiation therapy (EBRT). When the radiation source is internal to the patient, the treatment is called brachytherapy (BT).

The radiation is administered according to well known conventional techniques using conventional equipment manufactured for this purpose, such as AECL Theratron and Varian Clinac. The radiation dose depends on numerous factors as is well known in the art. These factors include the organ that is treated, the healthy organs in the radiation path that could be inadvertently adversely affected, the patient's tolerance for radiation therapy and the area of the body that needs treatment. The dose will normally be between 1 and 100 Gy and more particularly between 2 and 80 Gy. Some doses that have been indicated include 35 Gy for the spinal cord, 15 Gy for the kidneys, 20 Gy for the liver and 65-80 Gy for the prostate. It should be emphasized, however, that the invention is not limited to any particular dose. The dose will be determined by the attending physician according to the particular factors in a given situation, including the factors mentioned above.

The distance between the source of the external radiation and the point of entry into the patient can be any distance that represents an acceptable balance between destruction of target cells and minimization of side effects. Normally, the source of external radiation is between 70 and 100 cm from the point of entry into the patient.

Brachytherapy is generally carried out by placing the radiation source in the patient. Normally, the radiation source is placed approximately 0-3 cm from the tissue in question. Known techniques include interstitial, intercavitary and surface brachytherapy. Radioactive seeds can be implanted permanently or temporarily. Some typical radioactive atoms that have been used in permanent implants include iodine 125 and radon. Some typical radioactive atoms that have been used in temporary implants include radium, cesium 137 and iridium 192. Some additional radioactive atoms that have been used in brachytherapy include americium 241 and gold 198. The radiation dose for brachytherapy may be the same as has been mentioned above for external beam radiation therapy. In addition to the factors mentioned above to determine the dose of external beam radiotherapy, the nature of the radioactive atom used is also taken into account to determine the dose of brachytherapy.

X.C.) PSCA as a Diana for Cellular Immune Responses

Vaccines and methods for preparing vaccines containing an immunogenically effective amount of one or more HLA-binding peptides as described herein are further disclosed herein. In addition, vaccines according to the disclosures herein encompass compositions of one or more of the disclosed peptides. A peptide can be present in a vaccine individually. Alternatively, the peptide may exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. The polymers have the advantage of an increased immunological ratio and, when different peptide epitopes are used to compose the polymer, the additional ability to induce antibodies and / or CTL that react with different antigenic determinants of the tumor-related organism or peptide to which it is directed. an immune response The composition can be a region of natural origin of an antigen or can be prepared, for example, recombinantly or by chemical synthesis.

Vehicles that can be used with vaccines disclosed herein are well known in the art, and include, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-acid glutamic, influenza, hepatitis B virus core protein and the like. Vaccines may contain a physiologically tolerable diluent (i.e. acceptable) such as water,

or saline, preferably phosphate buffered saline. Vaccines also normally include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be initiated by conjugating peptides of the invention with lipids such as tripalmitoyl-S-glycerylcysteinylseryl serine (P3CSS). In addition, it has been found that an adjuvant such as oligonucleotides containing synthetic cytosine-guanine phosphorothiolated (CpG) increases CTL responses 10 to 100 times (see, for example, Davila and Celis, J. Immunol. 165: 539-547 (2000)).

After immunization with a peptide composition, by injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal or other suitable routes, the host's immune system responds to the vaccine producing large amounts of CTL and / or HTL specific for the desired antigen. Consequently, the host becomes at least partially immune to the subsequent development of cells that express or overexpress PSCA antigen, or derive at least some therapeutic benefit when the antigen was associated with tumor.

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X.C.1. Minigenes vaccines

Several different approaches are available that allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides disclosed herein are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means for administering nucleic acids encoding the disclosed peptides uses minigenes constructs encoding a peptide comprising one or multiple epitopes of the invention.

The use of multi-epitope minigenes is described later and in Ishioka et al., J. Immunol. 162: 3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71: 2292, 1997; Thomson, S. A. et al., J. Immunol. 157: 822, 1996; Whitton, J.

L. et al., J. Virol. 67: 348, 1993; Hanke, R. et al., Vaccine 16: 426, 1998. For example, a multi-epitopic DNA plasmid encoding PSCA derived from motif-bearing and / or super-motive carrier epitopes, the PADRE universal helper T-cell epitope ™ or multiple HTL epitopes of PSCA (see for example, Tables V-XVIII and XXII to LI) and a signal sequence translocation to the endoplasmic reticulum. A vaccine can also comprise epitopes that are derived from other TAA.

The immunogenicity of a multi-epitope minigene can be confirmed in transgenic mice to assess the magnitude of CTL induction responses against the epitopes tested. In addition, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Therefore, these experiments can show that the minigene serves both to: 1.) generate a CTL response and to 2.) that induced CTLs recognize cells that express the encoded epitopes.

For example, to create a DNA sequence that encodes the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes can be translated in reverse. A human codon usage chart can be used to guide the choice of codons for each amino acid. These DNA sequences encoding epitopes can be directly linked, so that when translated, a continuous polypeptide sequence is created. To optimize expression and / or immunogenicity, additional elements may be incorporated into the minigene design. Examples of amino acid sequences that can be translated in reverse and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence, and / or a signal of endoplasmic reticulum direction. In addition, the HLA presentation of CTL and HTL epitopes can be improved by including synthetic flanking sequences (for example of poly alanine) or of natural origin adjacent to the CTL or HTL epitopes; These major peptides comprising the epitope or epitopes are within the scope of the invention.

The minigene sequence can be converted to DNA by assembling oligonucleotides that encode the plus and minus chains of the minigene. Overlapping oligonucleotides (30-100 bases in length) can be synthesized, phosphorylated, purified and hybridized under appropriate conditions using well known techniques. The ends of the oligonucleotides can be linked, for example, using T4 DNA ligase. This synthetic minigene, which encodes the epitope polypeptide, can then be cloned into a desired expression vector.

Conventional regulatory sequences well known to those skilled in the art are preferably included in the vector to ensure expression in target cells. Several vector elements are desirable: a promoter with a downstream cloning site for insertion of the minigene; a polyadenylation signal for termination of effective transcription; an origin of E. coli replication; and a selectable marker of E. coli (eg, resistance to ampicillin or kanamycin). Numerous promoters can be used for this purpose, for example, the human cytomegalovirus (hCMV) promoter. See, for example, U.S. Patent Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize the expression of minigenes and immunogenicity. In some cases, introns are required for effective gene expression, and one could be incorporated

or more synthetic or naturally occurring introns in the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells can also be taken into account for increased minigenes expression.

Once an expression vector has been selected, the minigene is cloned in the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using conventional techniques. The orientation and DNA sequence of the minigene, as well as the other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells that house the correct plasmid can be stored as a master cell bank and a work cell bank. In addition, immunostimulatory sequences (ISS or CpG) appear to play a role in the immunogenicity of DNA vaccines. These sequences can be included in the vector, outside the minigene coding sequence, if immunogenicity is to be enhanced.

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In some embodiments, a bicistronic expression vector can be used that allows the production of both epitopes encoded by minigenes and a second protein (included to enhance or reduce immunogenicity). Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine inducing molecules (e.g., LeIF), costimulatory molecules or for HTL responses, DR pan binding proteins (PADRE ™, Epimmune, San Diego, CA). Auxiliary epitopes (HTL) can be linked with intracellular direction signals and expressed separately from expressed CTL epitopes; this allows the direction of the HTL epitopes to a different cell compartment from that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HTL class II pathway, thereby improving the induction of HTL. Unlike the induction of HTL or CTL, the specific reduction of the immune response by coexpression of immunosuppressive molecules (for example TGF-) may be beneficial in certain diseases.

Therapeutic amounts of plasmid DNA can be produced, for example, by fermentation in E. coli, followed by purification. The aliquots of the work cell bank are used to inoculate culture medium, and cultivate until saturation in shake flasks or in a bioreactor according to well known techniques. Plasmid DNA can be purified using conventional bioseparation technologies such as solid phase anion exchange resins provided by QIAGEN, S.A. (Valencia, California). If necessary, supercoiled DNA can be isolated from the linear and open circular forms using gel electrophoresis or other methods.

Purified plasmid DNA for injection can be prepared using various formulations. The simplest of these is the reconstitution of lyophilized DNA in a sterile phosphate buffered saline (PBS). This approach, known as "naked DNA" is currently used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigenes DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. Various methods have been described, and new techniques may be made available. Cationic lipids, glycolipids and fusogenic liposomes can also be used in the formulation (see, for example, as described in WO 93/24640 Mannino and Gould-Fogerite, BioTechniques 6 (7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84: 7413 (1987)). In addition, peptides and compounds collectively referred to as protective, interactive, non-condensing compounds (PINC) could also complex with purified plasmid DNA to influence variables such as stability, intramuscular dispersion or traffic to specific organs or cell types.

Sensitization of target cells can be used as a functional assay for the expression and presentation of HLA class I of CTL epitopes encoded by minigenes. For example, plasmid DNA is introduced into a mammalian cell line that is suitable as a target for conventional CTL chromium release assays. The transfection method used will depend on the final formulation. Electroporation can be used for "naked" DNA, while cationic lipids allow direct transfection in vitro. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell separation (FACS). These cells are then labeled with chromium 51 (51 Cr) and are used as target cells for epitope-specific CTL lines; The cytolysis, detected by 51 Cr release indicates both production of and presentation of HLA of CTL epitopes encoded by minigenes. The expression of HTL epitopes can be evaluated in an analogous manner using assays to evaluate HTL activity.

In vivo immunogenicity is a second approach to functional assays of minigen DNA formulations. Transgenic mice that express appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are dependent on the formulation (eg, IM for DNA in PBS, intraperitoneal (i.p.) for DNA in complex with lipids). Twenty-one days after immunization, splenocytes are collected and stimulated for a week in the presence of peptides encoding each epitope that is tested. Next for CTL effector cells, assays are performed for cytolysis of 51 Cr labeled cells, loaded with peptides using conventional techniques. The lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to epitopes encoded by minigenes, demonstrates the role of DNA vaccine for in vivo induction of CTL. The immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, nucleic acids can be administered using ballistic delivery as described, for example, in US Patent No. 5,204,253. Using this technique, particles comprised only of DNA are administered. In a further alternative embodiment, the DNA can adhere to particles, such as gold particles.

Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, for example, an expression construct encoding epitopes as disclosed herein can be incorporated into a viral vector such as vaccinia.

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X.C.2. Combinations of CTL Peptides with Auxiliary Peptides

Vaccine compositions comprising CTL peptides may be modified as disclosed herein, for example, made analogous, to provide desired attributes, such as improved serum half-life, extended population coverage or enhanced immunogenicity.

For example, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide with a sequence containing at least one epitope that is capable of inducing a helper T lymphocyte response. Although a CTL peptide may be directly linked with an auxiliary T peptide, CTL epitope / HTL epitope conjugates are often linked by a spacer molecule. The spacer is normally comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, that are substantially uncharged under physiological conditions. The spacers are normally selected from, for example, Ala, Gly or other neutral spacers of non-polar amino acids or neutral polar amino acids. It will be understood that it is not necessary that the optionally present spacer be comprised of the same moieties and therefore may be a hetero or homo oligomer. When present, the spacer will usually be at least one or two debris, more usually three to six debris and sometimes 10 or more debris. The CTL peptide epitope can be linked to the auxiliary peptide T epitope either directly or by a terminal or carboxyl terminal spacer of the CTL peptide. The amino terminal end of the immunogenic peptide or the auxiliary peptide T can be acylated.

HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include amino acids D to increase their protease resistance and thereby extend their serum half-life, or they can be conjugated with other molecules such as lipids, proteins, carbohydrates and the like to increase their biological activity. For example, an auxiliary T peptide can be conjugated to one or more palmitic acid chains at the amino or carboxyl terminus.

X.C.3. Combinations of CTL Peptides with T Lymphocyte Sensitization Agents

It may be desirable to include in the pharmaceutical compositions as disclosed herein at least one component that sensitizes B lymphocytes or T lymphocytes. Lipids have been identified as agents capable of sensitizing CTL in vivo. For example, palmitic acid residues can be linked to the  and  amino groups of a lysine residue and then linked, for example, by one or more linkage moieties such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, with an immunogenic peptide. The lipidated peptide can then be administered directly into a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, for example, incomplete Freund's adjuvant. A particularly effective immunogenic composition may comprise palmitic acid linked to  and  amino groups of Lys, which binds, for example, Ser-Ser, to the amino terminal end of the immunogenic peptide.

As another example of lipid sensitization of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glyceryl-cysteinylseryl serine (P3CSS) can be used to sensitize virus-specific CTLs when covalently bound with an appropriate peptide (see , for example, Deres, et al., Nature 342: 561, 1989). The peptides can be coupled with P3CSS, for example and the lipopeptide administered to an individual to specifically sensitize an immune response to the target antigen. In addition, because the induction of neutralizing antibodies can also be sensitized with epitopes conjugated with P3CSS, two such compositions can be combined to more effectively induce both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions comprising Pulsed DC with CTL and / or HTL Peptides

An example of a vaccine composition according to the disclosures herein comprises the ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or DC isolated therefrom, from the patient's blood. A pharmaceutical product can be used to facilitate the collection of DC, such as Progenipoietin ™ (Pharmacia-Monsanto, San Louis, Missouri) or GM-CSF IL-4. After pressing the DC with peptides and before reinfusion in patients, the DCs are washed to remove unbound peptides. A vaccine may comprise peptide-pulsed DCs that exhibit pulsed peptide epitopes in complex with HLA molecules on their surfaces.

DCs can be pulsed ex vivo with a peptide cocktail, some of which stimulate CTL responses to PSCA. Optionally, an auxiliary T-lymphocyte (HTL) peptide, such as a low-natural or artificial HLA Class II peptide, may be included to facilitate the CTL response. Therefore, a vaccine can be used to treat a cancer that expresses or overexpresses PSCA.

X.D.) Adoptive Immunotherapy

Antigenic PSCA-related peptides are used to induce an ex vivo CTL and / or HTL response as well. The resulting CTL or HTL cells can be used to treat tumors in patients who do not respond to other conventional forms of therapy, or will not respond to therapeutic vaccine peptide or nucleic acid according

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specific pattern of tissue expression as its overexpression in certain cancers as described for example in the Example entitled "Analysis of PSCA expression in normal tissues, and patient test samples").

PSCA can be made analogous to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and control the presence of prostate cancer (see, for example, Merrill et al., J. Urol 163 (2): 503-5120 (2000); Polascik et al., J. Urol. Aug; 162 (2): 293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91 ( 19): 1635-1640 (1999)). Various other diagnostic markers are also used in similar contexts including p53 and K-ras (see, for example, Tulchinsky et al., Int J Mol Med Jul 1999 4 (1): 99102 and Minimoto et al., Cancer Detect Prev 2000 ; 24 (1): 1-12). Therefore, the present disclosure of PSCA polynucleotides and polypeptides (as well as PSCA polynucleotide probes and anti PSCA antibodies used to identify the presence of these molecules) and their properties allow those skilled in the art to use these molecules in methods that are analogous to those used, for example, in various diagnostic tests aimed at examining the conditions associated with cancer.

Typical examples of diagnostic methods using PSCA polynucleotides, polypeptides, reactive T lymphocytes and antibodies are analogous to well established diagnostic test methods that employ, for example, PSA polynucleotides, polypeptides, reactive T lymphocytes and antibodies. For example, like PSA polynucleotides, they are used as probes (for example in Northern analysis, see, for example, Sharief et al., Biochem. Mol. Biol. Int. 33 (3): 567-74 (1994 )) and primers (for example in PCR analysis, see, for example, Okegawa et al., J. Urol. 163 (4): 1189-1190 (2000)) to observe the presence and / or mRNA level of PSA in control methods of PSA overexpression or prostate cancer metastasis, the PSCA polynucleotides described herein can be used in the same way to detect PSCA overexpression or prostate metastasis and other cancers expressing this gen. Alternatively, like PSA polypeptides they are used to generate antibodies specific for PSA that can then be used to observe the presence and / or level of PSA proteins in methods to control PSA protein overexpression (see, for example, Stephan et al., Urology 55 (4): 560-3 (2000)) or prostate cell metastasis (see, for example, Alanen et al., Pathol. Res. Pract. 192 (3): 233-7 (1996 )), the PSCA polypeptides described herein can be used to generate antibodies for use in the detection of PSCA overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.

Specifically, because metastases involve the movement of cancer cells from an organ of origin (such as the lung or prostate gland, etc.) to a different area of the body (such as a lymph node), tests that examine a biological sample with respect to the presence of cells expressing PSCA polynucleotides and / or polypeptides to provide evidence of metastasis. For example, when it is discovered that a biological sample of tissue that does not normally contain cells that express PSCA (lymph nodes) contains cells that express PSCA such as the expression of PSCA seen in LAPC4 and LAPC9, isolated xenografts of lymph node and bone metastases , respectively, this finding is indicative of metastasis.

Alternatively, PSCA polynucleotides and / or polypeptides can be used to provide cancer evidence, for example, when it is discovered that cells in a biological sample that do not normally express PSCA or express PSCA at a different level express PSCA or have an increased expression of PSCA (see, for example, the expression of PSCA in cancers listed in Table I and in patient samples etc. shown in the attached Figures). In such tests, those skilled in the art may also wish to generate complementary metastatic tests by testing the biological sample with respect to the presence of a second tissue-restricted marker (in addition to PSCA) such as PSA, PSCA etc. (See, for example, Alanen et al., Pathol. Res. Pract. 192 (3): 233-237 (1996)).

The use of immunohistochemistry to identify the presence of a PSCA polypeptide within a tissue section may indicate an altered state of certain cells within that tissue. It is well understood in the art that the ability of an antibody to localize in a polypeptide that is expressed in cancer cells is a way of diagnosing the presence of disease, disease stage, progression and / or aggressiveness of the tumor. Said antibody can also detect an altered distribution of the polypeptide within the cancer cells, as compared to corresponding non-malignant tissue.

The PSCA polypeptide and immunogenic compositions are also useful in view of the phenomena of the localization of altered subcellular protein in pathologies. The alteration of cells from the normal state to the patient causes changes in cell morphology and is frequently associated with changes in the location / distribution of subcellular proteins. For example, cell membrane proteins that are expressed in a polarized manner in normal cells can be altered in disease, resulting in the distribution of the protein in a non-polar manner over the entire cell surface.

The phenomenon of the subcellular protein localization altered in a pathology has been demonstrated with the expression of MUC1 and Her2 proteins through the use of immunohistochemical means. Normal epithelial cells have a typical apical distribution of MUC1, in addition to some supranuclear localization of glycoprotein, while malignant lesions frequently demonstrate a pattern of apolar staining (Diaz et al, The Breast Journal, 7;

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40-45 (2001); Zhang et al, Clinical Cancer Research, 4; 2669-2676 (1998): Cao, et al, The Journal of Histochemistry and Cytochemistry, 45: 1547-1557 (1997)). In addition, normal breast epithelium is negative for the Her2 protein or shows only a basolateral distribution while malignant cells can express the protein over the entire cell surface (De Potter, et al, International Journal of Cancer, 44; 969-974 (1989): McCormick, et al, 117; 935-943 (2002)). Alternatively, the protein distribution can be altered from a surface-only location to include diffuse cytoplasmic expression in the pathology. This example can be seen with MUC1 (Diaz, et al, The Breast Journal, 7: 40-45 (2001)).

The alteration in the location / distribution of a protein in the cell, as detected by immunohistochemical methods, can also provide valuable information regarding the favorableness of certain treatment modalities. This last point is illustrated by a situation in which a protein can be intracellular in normal tissue, but with a cell surface in malignant cells; cell surface localization makes cells favorably susceptible to antibody-based diagnostic and treatment regimens. When such alteration of the protein location appears for PSCA, the PSCA protein and related immune responses are very useful. Consequently, the ability to determine whether the alteration of the subcellular protein localization has occurred for 24P4C12 makes the PSCA protein and related immune responses very useful. The use of PSCA compositions allows experts in the field to make important diagnostic and therapeutic decisions.

Immunohistochemical reagents specific for PSCA are also useful for detecting metastases from tumors expressing PSCA when the polypeptide appears in tissues in which PSCA is not normally produced.

Therefore, PSCA polypeptides and antibodies resulting from immune responses thereto are useful in various important contexts such as diagnostic, prognostic, preventive and / or therapeutic purposes known to those skilled in the art.

Just as the polynucleotide fragments and polynucleotide variants of PSA are employed by those skilled in the art for use in methods of controlling PSA, polynucleotide fragments and polynucleotide variants of PSCA are used in an analogous manner. In particular, typical PSA polynucleotides used in methods to control PSA are probes or primers consisting of fragments of the PSA cDNA sequence. Illustrating this, the primers used to amplify a PSA polynucleotide by PCR must include less of the complete PSA sequence to act in the polymerase chain reaction. In the context of such PCR reactions, those skilled in the art generally create various different polynucleotide fragments that can be used as primers to amplify different parts of a polynucleotide of interest or to optimize amplification reactions (see, for example, Caethane-Anolles , G. Biotechniques 25 (3): 472-476, 478480 (1998); Robertson et al., Methods Mol. Biol. 98: 121-154 (1998)). A further illustration of the use of such fragments is provided in the Example entitled "Analysis of PSCA expression in normal tissues and patient test samples", in which a PSCA polynucleotide fragment is used as a probe to show the expression of PSCA RNA in cancer cells. In addition, variant polynucleotide sequences are normally used as primers and probes for the corresponding mRNAs in PCR and Northern analysis (see, for example, Sawai et al., Fetal Diagn. Ther. Nov-Dec 1996 11 (6): 407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)). The polynucleotide fragments and variants are useful in this context when they are capable of binding with a target polynucleotide sequence (for example, a PSCA polynucleotide shown in Figure 1 or variant thereof) under conditions of high stringency.

In addition, PSA polypeptides that contain an epitope that can be recognized by an antibody or T lymphocyte that specifically binds to that epitope are used in methods to control PSA. Fragments of polypeptides and analogs or variants of PSCA polypeptides can also be used in an analogous manner. This practice of using polypeptide fragments or variants of polypeptides to generate antibodies (such as antibodies or anti-PSA T lymphocytes) is typical in the art with a wide variety of systems such as fusion proteins that are used by practitioners (see , for example, Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this context, each epitope or epitopes act to provide the architecture with which an antibody or T lymphocyte is reactive. Typically, those skilled in the art create various different polypeptide fragments that can be used to generate specific immune responses for different parts of a polypeptide. of interest (see, for example, U.S. Patent No. 5,840,501 and U.S. Patent No. 5,939,533). For example, it may be preferable to use a polypeptide comprising one of the biological motifs of PSCA discussed herein or a motif carrier sub-sequence that is readily identified by one skilled in the art based on motifs available in the art. Fragments, variants or analogs of polypeptides are normally useful in this context as long as they comprise an epitope capable of generating an antibody or T-cell specific for a target polypeptide sequence (for example a PSCA polypeptide shown in Figure 1).

As shown herein, PSCA polynucleotides and polypeptides (as well as PSCA polynucleotide probes and anti-PSCA antibodies or T lymphocytes used to identify the presence of these molecules) show specific properties that make them useful in the diagnosis of cancers. such as those listed in Table I. Diagnostic assays that measure the presence of PSCA gene products are used,

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WO98 / 16628 and US Patent 6,107,540 describe various models of human prostate cancer xenografts capable of recapitulating the development of primary tumors, micrometastases and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure the inhibition of tumor formation, tumor regression or metastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful in the evaluation of therapeutic compositions. In one example, xenografts of mice bearing tumors treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared with mice bearing untreated control xenografts. The degree to which apoptotic foci are found in the tumors of the treated mice provides an indication of therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the above methods may be formulated in pharmaceutical compositions comprising a suitable vehicle for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the antitumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of several conventional pharmaceutical vehicles such as sterile phosphate buffered saline solutions, bacteriostatic water and the like (see, in general, Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Therapeutic formulations can be solubilized and administered by any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorganic, orthotopic and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of conserved bacteriostatic water, sterile unconserved water and / or diluted in polyvinylchloride or polyethylene bags containing 0.9% Sterile Chloride for sterile injection, USP. Therapeutic protein preparations may be lyophilized and stored as sterile powders, preferably in vacuo, and then reconstituted in bacteriostatic water (containing, for example, benzyl alcohol preservative) or in sterile water before injection.

Dosages and administration protocols for the treatment of cancers using the above methods will vary according to the method and the target cancer, and will in general depend on several other factors appreciated in the art.

XIII.) Identification, Characterization and Use of PSCA Modulators

Methods to identify and use modulators

Scanning can be performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, preferably generating the phenotype associated therewith. In another example, having identified differentially important genes expressed in a particular state; scans are performed to identify modulators that alter the expression of individual genes, either by increasing or by reducing. Scanning can be performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, scans are performed to identify agents that bind with and / or modulate the biological activity of the gene product.

In addition, scans are performed for genes that are induced in response to a candidate agent. After identifying a modulator (one that suppresses a cancer expression pattern that leads to normal expression pattern, or a cancer gene modulator that leads to gene expression as in normal tissue) a scan is performed to identify genes that are modulated specifically in response to the agent. Comparison of expression profiles between normal tissue and agent treated cancer tissue reveals genes that are not expressed in normal tissue or cancer tissue, but are expressed in agent treated tissue and vice versa. These agent specific sequences are identified and used by methods described herein for cancer genes or proteins. In particular, these sequences and the proteins they encode are used in the labeling or identification of agent treated cells. In addition, antibodies against agent-induced proteins are induced and used to direct new therapeutic products to the treated cancer tissue sample.

Identification and exploration tests related to modulator:

Assays related to gene expression

Proteins, nucleic acids and antibodies of the invention may be used in screening assays and as disclosed herein herein. Proteins, antibodies, nucleic acids, modified proteins and cells containing these cancer-associated sequences are used in screening assays, such as evaluating the effect of drug candidates on a "gene expression profile," expression profile. of polypeptides or alteration of biological function. In one example, expression profiles are used, preferably

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together with high-performance scanning techniques to allow control for expression profile genes after treatment with a candidate agent (eg, Davis, GF, et al, J Biol Screen 7: 69 (2002); Zlokarnik, et al ., Science 279: 84-8 (1998); Heid, Genome Res 6: 986-94, 1996).

Proteins, antibodies, nucleic acids, modified cancer proteins and cells containing native or modified cancer proteins or cancer genes are used in screening assays. That is, methods to explore with respect to compositions that modulate the cancerous phenotype or a physiological function of a cancer protein of the invention are disclosed herein. This is done in a gene itself or by evaluating the effect of pharmacological candidates on a "gene expression profile" or biological function. Expression profiles may be used, preferably in conjunction with high performance scanning techniques to allow control after treatment with a candidate agent, see Zlokamik, mentioned above.

Various assays are directed to the genes and proteins of the invention. Assays are processed at a single nucleic acid or protein level. That is, having identified a particular gene as positively regulated in cancer, test compounds are explored with respect to the ability to modulate gene expression or with respect to binding with the cancer protein as disclosed herein. "Modulation" in this context includes an increase or decrease in gene expression. The preferred amount of modulation will depend on the original change of gene expression in normal tissue versus tissue undergoing cancer, with changes of at least 10%, preferably 50%, more preferably 100-300% and in some embodiments 300-1000% or plus. Therefore, if a gene shows a fourfold increase in cancer tissue compared to normal tissue, an approximately fourfold reduction is often desired; similarly, a tenfold reduction in cancer tissue compared to normal tissue, a target value of a tenfold increase in expression by the test compound is often desired. Modulators that exacerbate the type of gene expression seen in cancer are also useful, for example, as a positively regulated target in further analyzes.

The amount of gene expression is controlled using nucleic acid probes and the quantification of levels of gene expression is controlled, or, alternatively, a gene product itself, for example, by the use of antibodies to the cancer protein and immunoassays. conventional. Proteomics and separation techniques also allow quantification of expression.

Expression control to identify compounds that modify gene expression

The control of gene expression, that is, an expression profile, can be controlled simultaneously with respect to several entities. Such profiles will normally involve one or more of the genes of Figure 1. In this example, for example, cancer nucleic acid probes are linked with biomicroplates to detect and quantify cancer sequences in a particular cell. Alternatively, PCR can be used. Therefore, a series can be used, for example, wells of a microtiter plate with primers distributed in desired wells. A PCR reaction can then be performed and analyzed for each well.

Expression control is performed to identify compounds that modify the expression of one or more cancer-associated sequences, for example, a polynucleotide sequence set forth in Figure 1. In general, an assay modulator is added to the cells prior to analysis. . In addition, scans are also provided to identify agents that modulate cancer, modulate cancer proteins of the invention, bind with a cancer protein as disclosed herein, or interfere with the binding of a cancer protein and an antibody. or another union partner.

High performance screening methods may involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Said "combinatorial chemical libraries" are then explored in one or more trials to identify members of the library (particular chemical species or subclasses) that exhibit a desired characteristic activity. Compounds identified in this way can act as conventional "candidate compounds", as screening compounds or as therapeutic products.

Combinatorial libraries of potential modulators can be explored for an ability to bind with a cancer polypeptide or to modulate activity. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a "candidate compound") with some desirable activity or property, for example, inhibitory activity, creating variants of the candidate compound, and evaluating the property and activity of those compounds variants. Frequently, high performance scanning methods (HTS) are used for such analysis.

As noted above, gene expression control is conveniently used to test candidate modulators (eg, protein, nucleic acid or small molecule). After the candidate agent has been added and the cells are allowed to incubate for a period, the sample containing a target sequence for analysis is added, for example, to a biomicroplate.

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Orthotopic cells that express sequences associated with cancer. Mice are then separated into groups, including control groups and treated experimental groups (for example treated with a modulator). After a suitable period of time, preferably 4-8 weeks, tumor growth is measured (for example, by volume or by its two major dimensions, or weight) and compared with the control. Tumors that have a statistically significant reduction (using, for example, Student's T test) are said to have inhibited growth.

In vitro tests to identify and characterize modulators

Assays can be performed to identify compounds with in vitro modulating activity. For example, a cancer polypeptide is first contacted with a potential modulator and incubated for a suitable period of time, for example, 0.5 to 48 hours. In one example, cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA. The protein level is measured using immunoassays such as Western blotting, ELISA and the like with an antibody that selectively binds to the cancer polypeptide or a fragment thereof. For mRNA measurement, amplification is preferred, for example, using PCR, CSF or hybridization assays, for example, Northern hybridization, RNAse protection, point transfer. The level of protein or mRNA is detected using direct or indirectly labeled detection agents, for example, fluorescent or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies and the like, as described herein.

Alternatively, an indicator gene system can be devised using a cancer protein promoter operably linked to an indicator gene such as luciferase, green fluorescent protein, CAT, or P-gal. The indicator construct is normally transfected into a cell. After treatment with a potential modulator, the amount of transcription, translation, or activity of the reporter gene is measured according to techniques known to those skilled in the art (Davis GF, mentioned above; Gonzalez, J. and Negulescu, P. Curr Opin. Biotechnol. 1998: 9: 624).

As outlined above, in vitro scans are performed on individual genes and gene products. That is, having identified that a differentially expressed particular gene is important in a particular state, modulation scan of the expression of the gene or the gene product itself is performed.

Exploration can be performed with respect to modulators of the expression of a specific gene or genes. Normally, the expression of only one or some genes is evaluated. Explorations can be designed to first find compounds that bind with differentially expressed proteins. These compounds are then evaluated with respect to the ability to modulate differentially expressed activity. In addition, once initial candidate compounds have been identified, variants can be further explored to better assess structural activity relationships.

Binding assays to identify and characterize modulators

In binding assays in accordance with the disclosures herein, a purified or isolated gene product of the invention is generally used. For example, antibodies are generated for a protein of the invention, and immunoassays are processed to determine the amount and / or location of the protein. Alternatively, cells comprising cancer proteins are used in the assays.

Therefore, the methods comprise combining a cancer protein as disclosed herein and a candidate compound such as a ligand, and determining the binding of the compound with the cancer protein of the invention. Some examples use human cancer protein; Animal models of human disease can also be developed and used. In addition, other analogous mammalian proteins can also be used as appreciated by those skilled in the art. In addition, in some embodiments, variant or derived cancer proteins are used.

In general, the cancer protein as disclosed herein, or the ligand, binds non-diffusely to an insoluble support. The support can, for example, be one that has areas that receive isolated samples (a microtiter plate, a matrix, etc.). Insoluble supports can be made of any composition with which the compositions can be attached, easily separated from the soluble material, and is otherwise compatible with the general scanning method. The surface of said supports can be solid or porous and in any convenient way.

Examples of suitable insoluble supports include microtiter plates, matrices, membranes and beads. These are usually made of glass, plastic (for example, polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon ™, etc. Microtiter plates and matrices are especially convenient because a large number of tests can be carried out simultaneously, using small amounts of reagents and samples. The particular way of joining the composition with the support is not crucial as long as it is compatible with the reagents and general methods disclosed herein, maintains the activity of the composition and is not diffusible. Preferred methods of binding include the use of antibodies that do not sterically block the

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that site, agents that generally do not bind with modified proteins on site. In addition, said pharmacological candidates that affect the activity of a native cancer protein are also identified by exploring drugs with respect to the ability to enhance or reduce the activity of said proteins.

Positive controls and negative controls can be used in the trials. Preferably, control and test samples are made at least in triplicate to obtain statistically significant results. Incubation of all samples occurs for a sufficient time to allow the binding of the agent with the protein. After incubation, non-specific bound material is washed out of the samples and the amount of bound agent, generally labeled, is determined. For example, when a radiolabel is used, samples can be counted in a scintillation counter to determine the amount of bound compound.

Various other reagents may be included in the screening assays. These include reagents such as salts, neutral proteins, for example albumin, detergents, etc. They are used to facilitate optimal protein-protein binding and / or reduce non-specific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may also be used. The mixture of components is added in an order that enables the required binding.

Use of polynucleotides to negatively regulate or inhibit a protein as disclosed herein.

Cancer polynucleotide modulators can be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines or other ligands that bind with cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind with its corresponding molecule or receptor, or block the entry of the sense or antisense oligonucleotide or its conjugated version into the cell. . Alternatively, a polynucleotide cancer modulator can be introduced into a cell containing the target nucleic acid sequence, for example, by formation of a polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models can also be used in screening assays as discussed above, in addition to treatment methods.

Nucleotide inhibitors and antisense

The activity of a cancer-associated protein can be negatively regulated, or completely inhibited, by the use of antisense polynucleotide or small nuclear inhibitor RNA (rRNA), that is, a complementary nucleic acid of, and which can preferably hybridize specifically with a nucleic acid sequence of mRNA encoding, for example, a cancer protein as disclosed herein, mRNA or a sub-sequence thereof. The binding of the antisense polynucleotide with the mRNA reduces the translation and / or stability of the mRNA.

In the context of the present disclosure, antisense polynucleotides may comprise naturally occurring nucleotides or synthetic species formed from naturally occurring subunits or their close counterparts. Antisense polynucleotides may also have altered sugar moieties or bonds between sugars. Examples among these are phosphorothioate and other sulfur-containing species that are known for use in the art. The present invention comprises analogs provided that they act effectively to hybridize with nucleotides as disclosed herein. See, for example, Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA.

Such antisense polynucleotides can be easily synthesized using recombinant means, or they can be synthesized in vitro. The equipment for such synthesis is sold by several suppliers, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those skilled in the art.

Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can be used, for example, to block transcription by binding with the antisense chain. The antisense and sense oligonucleotide comprises a single stranded nucleic acid (RNA or DNA) sequence capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules. The antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally of at least about 12 nucleotides, preferably of about 12 to 30 nucleotides. The ability to derive an antisense or sense oligonucleotide, based on a cDNA sequence encoding a given protein is described, for example, in Stein and Cohen (Cancer Res. 48: 2659 (1988 and van der Krol et al. (BioTechniques 6: 958 (1988)).

Ribozymes

In addition to antisense polynucleotides, ribozymes can be used to direct and inhibit the transcription of nucleotide sequences associated with cancer. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different types of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and ax head ribozymes (see, for example, Castanotto et al., Adv. In Pharmacology 25: 289-317 (1994) for a general review of the properties of different ribozymes).

The general characteristics of hairpin ribozymes are described, for example, in Hampel et al., Nucl. Acids Res.

18: 299-304 (1990); European Patent Publication No. 0360257; U.S. Patent No. 5,254,678. Preparation methods are also known to those skilled in the art (see, for example, WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA 90: 6340-6344 (1993); Yamada et al ., Human Gene Therapy 1: 39-45 (1994); Leavitt et al., Proc. Natl. Acad Sci. USA 92: 699-703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120 ( 1994); and Yamada et al., Virology 205: 121-126 (1994)).

Use of modulators in phenotypic exploration

A test compound can be administered to a population of cancer cells, which have an associated cancer expression profile. By "administration" or "contacting" herein it is understood that the modulator is added to the cells in such a way as to allow the modulator to act on the cell, either by intracellular uptake and action or by surface action mobile. A nucleic acid encoding a protein agent (i.e., a peptide) can be placed in a viral construct such as an adenoviral or retroviral construct, and added to the cell, so that expression of the peptide agent is achieved, for example, document PCT US97 / 01019. Adjustable gene therapy systems can also be used. Once the modulator has been administered to the cells, the cells are washed if desired and allowed to incubate under preferably physiological conditions for some time. The cells are then collected and a new gene expression profile is generated. Therefore, for example, cancer tissue is screened for agents that modulate, for example, induce or suppress, the cancer phenotype. A change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity. Similarly, the alteration of a biological function or signaling pathway is indicative of modulating activity. Defining such identification for the cancer phenotype, explorations for new drugs that alter the phenotype are devised. With this approach, it is not necessary for the pharmacological target to be known and it is not necessary to be represented on the original protein / gene expression scanning platform, nor is it necessary to change the level of transcript for the target protein. The modulator inhibitory function will act as a substitute marker.

As outlined above, scans are performed to evaluate genes or gene products. That is, having identified that a differentially expressed particular gene is important in a particular state, modulation scan of the expression of the gene or the gene product itself is performed.

Use of modulators to affect peptides

Measurements of cancer polypeptide activity, or cancer phenotype are made using various assays. For example, the effects of modulators on the function of a cancer polypeptide or polypeptides are measured by examining parameters described above. A physiological change that affects the activity is used to evaluate the influence of a test compound on the polypeptides as disclosed herein. When functional results are determined using intact or animal cells, various effects can be evaluated such as, in the case of a cancer associated with solid tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcriptional changes to both known and genetic markers. not characterized (for example, by Northern blots), changes in cell metabolism such as changes in cell growth or pH, and changes in second intracellular messengers such as cGNIP.

Methods to identify sequences associated with cancer characterization

The expression of various gene sequences correlates with cancer. Consequently, disorders based on mutant or variant cancer genes are determined. In one example, there are methods for identifying cells that contain variant cancer genes, for example, determining the presence of, in whole or in part, the sequence of at least one endogenous cancer gene in a cell. This is achieved using any variety of sequencing techniques. Methods for identifying the cancer genotype of an individual are disclosed herein, for example, determining all or part of the sequence of at least one gene of the invention in the individual. This is generally done on at least one tissue of the individual, for example, an exposed tissue in Table I, and may include the evaluation of several different tissues or samples of the same tissue. The method may include comparing the sequence of the sequenced gene with a known cancer gene, that is, a wild-type gene to determine the presence of family members, homologies, mutations or variants. The sequence of all or part of the gene can then be compared with the sequence of a known cancer gene to determine if there is any difference. This is done using any variety of known homology programs, such as BLAST, Bestfit, etc. The presence of a difference in the sequence between the patient's cancer gene and the known cancer gene correlates with a pathology or a propensity to a pathology, as outlined herein.

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Examples:

Various aspects of the invention are further described and illustrated by the various examples below, none of which are intended to limit the scope of the invention.

Example 1

Expression analysis of PSCA variants in normal tissues and patient test samples

Previously, PSCA, indicated herein as PSCA v.1, has been identified as an antigen expressed in prostate cancer. Its expression was detected in more than 80% of primary prostate cancers and in most prostate metastases. It has also been shown to be expressed in bladder cancer, ovarian cancer and pancreatic cancer; These cancers are listed in Table I. By immunohistochemical analysis, PSCA has been shown to be overexpressed on the cell surface of most urothelial transition carcinomas, and in 60% of primary pancreatic adenocarcinomas. PSCA expression data has been presented in patent publications (documents PCT / US98 / 04664, PCT / US / 28883, PCT / US00 / 19967) and in peer-reviewed articles (Saffran et al., Proc Natl Acad Sci US A. 27 Feb 2001; 98 (5): 2658-2663; Amara et al., Cancer Res. 15 Jun 2001; 61 (12): 4660-65; Reiter et al., Proc Natl Acad Sci USA. 17 Feb 1998 ; 95 (4): 1735-40; Argani et al., Cancer Res. 1 June 2001; 61 (11): 4320-24).

The specific expression of different variants of PSCA is studied in normal and cancer patient test samples. The primers were designed to differentiate between PSCA v.1 / v.2 / v.4, PSCA v.3 and PSCA v.5. PSCA v.1 / v.2 / v.4 lead to a 425 bp PCR product, PSCA v.3 leads to a 300 bp PCR product, while PSCA v.5 leads to a 910 PCR product size bp (Figure 1I (a)).

First strand cDNA was prepared from bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach, prostate cancer groups, bladder cancer, kidney cancer , colon cancer, lung cancer, ovarian cancer, breast cancer, cancer metastasis and pancreatic cancer (Figure 1I (b)). PCR normalization was performed using actin primers. Semi-quantitative PCR was performed, using specific primers variants at 30 cycles of amplification.

The results show expression of PSCA v.5 mainly in breast cancer, cancer metastasis and pancreatic cancer and to a lesser extent in colon cancer and lung cancer. PSCA v.1 / v.2 / v.4 PCR product was detected in prostate cancer, bladder cancer, kidney cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, cancer metastasis and pancreatic cancer. Among normal tissues, PSCA v.1 / v.2 / v.4 PCR product was detected only in prostate, stomach, and at a lower level in kidney and lung, while PSCA v was not detected. 5 in no normal tissue. No product detected by PSCA v.3 PCR was detected in any of the samples tested.

The primers were designed to differentiate between PSCA v.4 and PSCA v.5 (Figure 1J (a)). PSCA v.4 leads to a 460 bp PCR product, while PSCA v.5 leads to a 945 bp PCR product in size.

First strand cDNA was prepared from bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach, prostate cancer groups, bladder cancer and multi-graft group (xenografts of prostate cancer, kidney cancer and bladder cancer) (Figure 1J (b)). PCR normalization was performed using actin primers. Semi-quantitative PCR was performed, using specific primers variants at 30 cycles of amplification.

The results show the expression of PSCA v.4 in prostate cancer, bladder cancer and group of normal multixenograft, kidney and prostate. PSCA v.5 was detected only in normal prostate and bladder cancer.

The restricted expression of PSCA variants in normal tissues and the expression detected in test samples of cancer patients indicate that PSCA variants are therapeutic, prognostic, laboratory, prophylactic and diagnostic targets for human cancers. Example 2

PSCA splice variants

As used herein, the term "variant" includes transcript variants and single nucleotide polymorphisms (SNPs). Transcript variants are variants of mature mRNA of the same gene that arise by alternative transcription or alternative splicing. Alternative transcripts are transcribed from the same gene but initiate transcription at different points. The splicing variants are mRNA variants cut and spliced differently from the same transcript. In eukaryotes, when a multiexonic gene is transcribed from genomic DNA, the initial RNA is cut and spliced to produce functional mRNA, which has only exons and is used for translation in an amino acid sequence. Consequently, a given gene can have from zero to many alternative transcripts and each transcript can have from zero to multiple variants of

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including diet and drugs between individuals. Therefore, the existence of a SNP and / or combinations of alleles (called haplotypes) have many useful applications, such as diagnosis of inherited diseases, determination of pharmacological reactions and dosage, identification of genes responsible for diseases, and relationship analysis. genetics between individuals (P. Nowotny, JM Kwon and AM Goate, “SNP analysis to dissect human traits”, Curr. Opin. Neurobiol. Oct 2001; 11 (5): 637-641; M. Pirmohamed and BK Park, “Genetic susceptibility to adverse drug reactions ”, Trends Pharmacol. Sci. Jun 2001; 22 (6): 298-305; JH Riley, CJ Allan, E. Lai and A. Roses,“ The use of single nucleotide polymorphisms in the isolation of common disease genes ", Pharmacogenomics. Feb 2000; 1 (1): 39-47; R. Judson, JC Stephens and A. Windemuth," The predictive power of haplotypes in clinical response ", Pharmacogenomics. Feb 2000; 1 (1): 15-26).

SNPs are identified by various methods accepted in the art (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. Oct-Nov 2001; 20 (9): 18-20; KM Weiss, “In search of human variation, ”Genome Res. Jul 1998; 8 (7): 691-697; MM She,“ Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies, ”Clin. Chem. Feb 2001; 47 (2): 164-172). For example, SNPs are identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They have also been discovered by direct sequencing of pooled DNA samples from different individuals or by comparing sequences of different DNA samples. With the rapid accumulation of sequence data in public and private databases, SNPs are also discovered by comparing sequences using computer programs (Z. Gu, L. Hillier and PY Kwok, "Single nucleotide polymorphism hunting in cyberspace," Hum. Mutat. 1998; 12 (4): 221-225). SNPs can be verified and the genotype or haplotype of an individual can be determined by various methods including direct sequencing and high performance microarrays (PY Kwok, "Methods for genotyping single nucleotide polymorphisms," Annu. Rev. Genomics Hum. Genet. 2001; 2 : 235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, “High-throughput SNP genotyping with the Masscode system,” Mol Diag. Dec 2000; 5 (4): 329-340).

Using the methods described above, thirteen SNPs have been identified in the transcript for PSCA v.2. Variant 2 was used, instead of for example variant 1, because it had fewer ambiguous bases than variant 1. Consequently, SNPs were identified in PSCA v.2, at positions 57 (t / c), 367 (c / t), 424 (a / c), 495 (c / g), 499 (c / t), 563 (c / t), 567 (g / a), 627 (g / a), 634 (t / g), 835 (g / a), 847 (g / a), 878 (g / a) and 978 (c / g). Transcripts or proteins with alternative alleles were designated variant PSCA v.6 to v.18, as shown in Figure 1B and Figure 1G.

The nucleotide change in v.6 changed the start codon of v.1 and therefore, translation would not begin until the next ATG (AUG in mRNA), resulting in a 9 AA protein shorter than v protein. one. The nucleotide changes for v.7 and v.8 were silent at the protein level.

Twelve of these 13 SNPs were also present in variant 4. The 12 SNP variants in relation to PSCA v. 4 are designated PSCA v. 19 to v.30. Variants 19 to 27 encode alternative amino acids as shown in Figure 1H.

Reference Example 4

Production of recombinant PSCA in prokaryotic systems

To express recombinant PSCA and PSCA variants in prokaryotic cells, the full or partial length PSCA sequences and PSCA variant cDNA sequences are cloned into any one of several expression vectors known in the art. One or more of the following regions of PSCA variants are expressed: the full length sequence presented in Figure 1, or 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more any contiguous amino acids of PSCA, variants or analogs thereof.

A. Transcription and translation in vitro constructions:

pCRII: To generate sense and antisense RNA probes from PSCA for in situ RNA investigations, pCRII constructs (Invitrogen, Carlsbad CA) are generated that encode all of the PSCA cDNA fragments. The pCRII vector has Sp6 and T7 promoters that flank the insert to drive PSCA RNA transcription for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cellular and tissue expression of PSCA at the RNA level. Transcribed PSCA RNA representing the amino acid coding region of the PSCA gene cDNA is used in in vitro translation systems such as the TnT ™ Coupled Crosslinked System (Promega, Corp., Madison, WI) to synthesize PSCA protein.

B. Bacterial constructions:

PGEX constructs: to generate recombinant PSCA proteins in bacteria that fuse with the Glutathione S-Transferase (GST) protein, all or parts of the PSCA cDNA protein coding sequence are cloned into the pGEX family of GST fusion vectors (Amersham Pharmacia Biotech, Piscataway, NJ). These constructs allow the controlled expression of recombinant PSCA protein sequences with fused GST

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human physiology.

The secondary structure of PSCA protein variants 1, 3, 4 and 6, specifically the predicted presence and location of alpha helices, extended chains and random loops, is predicted from the primary amino acid sequence using the HNN method, Red Hierarchical Neural (NPS @: Network Protein Sequence Analysis TIBS March 2000 Vol. 25, No. 3 [291]: 147-150 Combet C., Blanchet C., Geourjon C. and Deléage G., http: //pbil.ibcp. fr / cgi-bin / npsa_automat.pl? page = npsa_nn.html), which is accessed from the ExPasy molecular biology server located on the web at (.expasy.ch / tools /). The analysis indicates that PSCA variant 1 is composed of 30.89% alpha helix, 21.95% extended chain and 47.15% random loop. Variant 3 of PSCA protein is composed of 14.89% alpha helix, 8.51% extended chain and 76.60% random loop. Variant 4 of PSCA protein is composed of 9.52% alpha helix, 8.99% extended chain, 81.48% random loop. Variant 6 of PSCA protein is composed of 24.56% alpha helix, 21.93% extended chain and 53.51% random loop.

Analysis was carried out regarding the potential presence of transmembrane domains in PSCA variant proteins using various transmembrane prediction algorithms accessed from the ExPasy molecular biology server located on the web at (.expasy.ch / tools /).

Example 7

Generation of polyclonal antibodies to PSCA

Polyclonal antibodies can be induced in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Normally, an immunizing and / or adjuvant agent will be injected into the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with a full-length PSCA protein variant, computer algorithms are used in the design of immunogens that, based on the amino acid sequence analysis, contain characteristics of being antigenic and being available for recognition by the immune system of the immunized host (See the example entitled “Secondary Structure and Antigenicity Profiles”). It would be predicted that said regions are hydrophilic, flexible, in beta spin conformations and are exposed on the surface of the protein.

For example, recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turning regions of PSCA protein variants are used as antigens to generate polyclonal antibodies in New Zealand White rabbits or monoclonal antibodies as described in the example entitled " Generation of Monoclonal Antibodies (MAb) of PSCA ”. For example, in PSCA variant 1, said regions include, but are not limited to, amino acids 28-56 and amino acids 66-94. For variant 3, said regions include, but are not limited to, amino acids 7-39 and amino acids 70-94. For variant 4, said regions include, but are not limited to, amino acids 6-18, amino acids 27-39, amino acids 103-133 and 177-189. For variant 6, said regions include, but are not limited to, amino acids 19-35 and amino acids 57-85. It is useful to conjugate the immunizing agent with a protein known to be immunogenic in the mammal that is immunized. Examples of such immunogenic proteins include, but are not limited to, California limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin and soy trypsin inhibitor. In one embodiment, a peptide encoding amino acids 103-133 of the PSCA variant 4 is conjugated to KLH and used to immunize a rabbit. Alternatively, the immunizing agent may include all or parts of the PSCA variant proteins, analogs or fusion proteins thereof. For example, the amino acid sequences of PSCA variants can be fused using recombinant DNA techniques with any one of several fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and fusion proteins. marked with His. In one embodiment, the PSCA variant 1 sequence, amino acids 18-98 were fused with GST using recombinant techniques in the pGEX expression vector, expressed, purified and used to immunize both rabbits and mice to generate polyclonal and monoclonal antibodies. respectively. Such fusion proteins are purified from bacteria induced using the appropriate affinity matrix.

Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or a constant region of immunoglobulin (see the section entitled "Production of PSCA in Prokaryotic Systems" and Current Protocols In Molecular Biology, Volume 2 , Unit 16, Frederick M. Ausubul et al. Eds., 1995; Linsley, PS, Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566).

In addition to fusion proteins derived from bacteria, protein antigens expressed by mammals are also used. These antigens are expressed from mammalian expression vectors such as the fusion vectors of Tag5 and Fc (see the section entitled "Production of Recombinant PSCA in Eukaryotic Systems") and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, the PSCA variant 1 cDNA, minus the N-terminal leader peptide and GPI C terminal anchor was cloned into the Tag5 mammalian secretion vector, and expressed in 293T cells. The recombinant protein was purified by metal chelation chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified PSCA Tag5 protein was then used as

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electrocellular fusion (BTX, ECM2000).

In one embodiment, the invention provides monoclonal antibodies designated Ha1-1.16, Ha1-5.99, Ha1-4.117, Ha1-4.120, Ha1-4.121 and Ha1-4.37. Antibodies were identified and shown to react and bind with cell surface or immobilized PSCA.

MAb for PSCA were generated using XenoMouse® technology in which the murine kappa and heavy chain light loci have been inactivated and a majority of the human kappa and heavy chain light immunoglobulin loci have been inserted: Hal-1.16 was generated after immunize Xenomice that produced human gamma 1 (13 times with PSCA-GST); Ha1-5.99 was generated after immunizing Xenomice that produced human gamma 2 6 times with Rat1-PSCA cells followed by two injections with PSCA-tag5; Ha1-4.117, HA1-4.37, Ha1-4.120, and Ha1-4121 were generated after immunizing Xenomice that produced human gamma 1 6 times with Rat1-PSCA cells followed by 4 injections with PSCA-tag5. The anti-PSCA MAb, Ha1-1.16, Ha1-5.99, Ha1-4.117, Ha1-4.120 and Ha1

4,121 bind with endogenous cell surface PSCA expressed in prostate cancer xenograft cells.

Antibodies designated Ha1-5.99, Ha1-4.117, Ha1-4.120, Ha1-4.37, Ha1-1.16 and Ha1-4.121 were sent (via Federal Express) to the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108 on May 4, 2004 and assigned Reference numbers PTA-6703, PTA-6699, PTA-6700, PTA-6702, PTA-6698 and PTA-6701 respectively.

DNA coding sequences for anti-PSCA MAb Ha1-1.16, Ha1-5.99, Ha1-4.117, Ha14.120, Ha1-4121 and Ha1-4.37 were determined, after isolating mRNA from the respective hybridoma cells with Trizol reagent ( Life Technologies, Gibco BRL). Total RNA was purified and quantified. Total RNA first strand cDNAs with oligo (dT) 12 18 sensitization were generated using the Superscript Gibco BRL Preamplification system. First strand cDNA was amplified using human immunoglobulin variable heavy chain primers, and human immunoglobulin variable light chain primers. PCR products were cloned into the pCRScript vector (Stratagene, La Jolla). Several clones were sequenced and variable heavy and light chain regions were determined. The amino acid and nucleic acid sequences of the variable heavy and light chain regions are listed in Figure 2 and Figure 3. Alignment of PSCA antibodies for germline V-D-J sequences is shown in Figure 4A-Figure 4M.

Example 9

PSCA antibody scan and identification

Antibodies generated using the procedures set forth in the example entitled "Generation of Monoclonal Antibodies (MAb) of PSCA" were screened and identified using a combination of assays including ELISA, FACS, epitope clustering and affinity for PSCA expressed on the cell surface.

A. Exploration of human MAb of PSCA by FACS.

Primary hybridoma screening for MAb for PSCA was performed by FACS. The protocol was as follows: 50 µl / well of hybridoma supernatant (pure) or purified antibodies (in serial dilutions) were added to 96-well FACS plates and mixed with cells expressing PSCA (endogenous or recombinant,

50,000 cells / well). The mixture was incubated at 4 ° C for two hours. At the end of the incubation, the cells were washed with FACS buffer and incubated with 100 µl of detection antibody (anti hIgG-PE) for 45 minutes at 4 ° C. At the end of the incubation, the cells were washed with FACS buffer, fixed with Formaldehyde and analyzed using FACScan. Data were analyzed using CellQuest Pro software. Filled histograms indicate negative control cell data, and open histograms represent PSCA positive cell data (Figure 9).

Positive hybridomas identified from primary scans were transferred to 24-well plates and supernatants collected for confirmation scans. Confirmation scans included FACS analysis in B300.19-PSCA / 300.19-neo, Rat1-PSCA / Rat1-neo, PC3-PSCA / neo, SW780 (bladder cancer cell line), LAPC9AI (cancer cell line prostate), HPAC (pancreatic cancer cell line) and ELISA assay using Tag5-PSCA, GST-PSCA, GST-PSCA N-term, Med. C-Term, and pET-PSCA.

B. Relative affinity analysis of human PSA MAb

Hybridoma supernatants were tested for their relative binding affinity for cell surface PSCA. The hybridoma supernatants were serially diluted in FACS buffer (FB), from g / ml to sub ng / ml; and were evaluated in a FACS binding assay using LAPC9AI cells. High affinity antibodies provided high IFM values. The IFM values of each point were obtained using CellQuest Pro software and were used for affinity calculation using Graphpad Prism software (Table VII and Table VIII): Sigmoid Dose Response equation (variable slope). The results of the relative affinity analysis are shown in Figure 10.

C. Grouping of epitopes

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Saporin toxin (Advanced Targeting Systems, San Diego, CA) to each well. The cells were allowed to incubate for 5 days at 37 degrees C. At the end of the incubation period, MTS (Promega) was added to each well and incubation was continued for an additional 4 hours. OD was determined at 450 nm. The results in Figure 13 (A) show that the PSCA antibodies HA1-4.121 and HA1-4.117 mediated saporin-dependent cytotoxicity in B300.19-PSCA cells while a control antibody, non-specific human IgG1, did not have no effect The results in Figure 13 (B) show that the addition of a secondary saporin-conjugated antibody that did not recognize human Fc, failed to mediate cytotoxicity (Figure 13 (A) and Figure 13 (B)). These results indicate that drugs or cytotoxic proteins can be selectively delivered to cells expressing PSCA using an appropriate anti PSCA MAb.

Example 13

Cytotoxicity mediated by antibody immunity

PSCA antibodies were evaluated to determine their ability to mediate immunity-dependent cytotoxicity. PSCA antibodies (0-50 µg / ml) were diluted with RHB buffer (RPMI 1640, Gibco Life Technologies, 20 mM HEPES). Cells expressing B300.19-PSCA were washed in RHB buffer and resuspended at a density of 106 cells / ml. In a typical assay, 50 µl of PSCA antibody, 50 µl of diluted rabbit complement serum (Cedarlane, Ontario, Can) and 50 µl of a cell suspension were added together in a 96-well tissue culture plate flat bottom. The mixture was incubated for 2 h at 37 ° C in a 5% CO2 incubator to facilitate complement-mediated cell lysis. Next, 50 µl of Alamar Blue (Biosource Intl. Camarillo, CA) was added to each well and incubation continued for an additional 4-5 hours at 37 ° C. Fluorescence in each well was read using a 96-well fluorimeter with excitation at 530 nm and emission at 590 nm. The results show that PSCA antibodies that had an Ig1 isotype (HA14.121) or an IgG2 isotype (HA1-5.99.1) but not an IgG4 isotype (HA1-6.46) were able to mediate complement-dependent lysis ( Figure 14).

ADCC (Antibody Dependent Cell Cytotoxicity) is a lytic attack mediated by immunity in cells bound with an antibody directed to a specific cell surface antigen. In this case it is PSCA. Immune cells recognize the Fc part of the antibody by binding with Fc receptors on the surface of leukocytes, monocytes and NK lymphocytes that trigger a lytic attack that results in cell death. The ability of PSCA antibodies to mediate this reaction can be assessed by labeling tumor cells in vitro with 51 chromium, europium or a fluorescent molecule and incubating them in the presence of human PSCA MAb together with peripheral blood mononuclear cells. The specific lysis of the tumor cells can be determined by measuring the percentage of lysis of the target tumor cells. Common titration criteria that are determined include the release of radioactivity, europium or fluorescent dye from dead cells using an appropriate detection method. Alternatively, the release of an intracellular enzyme such as lactate dehydrogenase (LDH) can be measured.

Example 14

F fragment generation (Ab ’) 2

The generation of F (Ab ’) 2 fragments of MAb is useful for studying the effects of MAb molecules that retain their bivalent antigen binding site but lack the immune effector Fc domain in therapeutic models in vitro and in vivo. 20 mg of MAb Ha1-4121 were incubated in 20 mM sodium acetate buffer pH 4.5 with and without immobilized pepsin (Perfore. Rockford IL) during the indicated times. Intact MAb and Fc fragments digested by protein A chromatography were removed. Figure 15 shows a Coomasie-stained gel of intact undigested unimaged MAb SDS-PAGE, non-reduced aliquots of digested material taken at the indicated times and a reduced sample of the final digested F (ab ') 2 product. This reagent can be used to treat animals that carry PSCA expression tumors. The antitumor activity observed with this antibody fragment can distinguish intrinsic biological activity from activity mediated by immunity-dependent mechanisms.

Example 15

Expression of human antibodies using recombinant DNA methods

To express anti-PSCA MAb recombinantly in transfected cells, heavy and light variable anti-PSCA chain sequences were cloned upstream of the constant regions of human IgG1 heavy chain and Ig light chain respectively. The complete anti-PSCA human heavy chain and light chain cassettes were cloned downstream of the CMV promoter / enhancer in a cloning vector. A polyadenylation site downstream of the MAb coding sequence was included. Constructs expressing recombinant anti PSCA MAb were transfected into 293T, Cos and CHO cells. HA1-4.121 antibody secreted from recombinant 293-T cells was evaluated for binding to cell surface PSCA in Figure Pia-3A and

it was compared with the same antibody produced from the original hybridoma (Figure 16).

Reference Example 16

HLA class I and class II binding assays

Class I and class II HLA binding assays were performed using purified HLA molecules according to disclosed protocols (eg, PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18.3 .1 (1998); Sidney, et al., J. Immunol. 154: 247 (1995); Sette, et al., Mol. Immunol. 31: 813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and radiolabeled probe peptides with 125I 1-10 nM. After incubation, the MHC-free peptide peptide complexes are separated by gel filtration and the bound peptide fraction is determined. Normally, in preliminary experiments, each MHC preparation is titrated in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind with 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these concentrations of HLA.

Since in these conditions [marker] <[HLA] and IC50 [HLA], the measured IC50 values are reasonable approximations of the true KD values. Peptide inhibitors are normally tested at concentrations ranging from 120 µg / ml to 1.2 ng / ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure for each peptide is calculated by dividing the IC50 of a positive control for inhibition by IC50 for each peptide tested (usually unlabeled versions of the radiolabeled probe peptide). For database purposes, and comparisons between experiments, relative binding values are compiled. These values can then be converted back to IC50 nM values by dividing the IC50 nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data collection is accurate and uniform to compare peptides that have been tested on different days, or with different batches of purified MHC.

Binding assays can be used as outlined above to analyze HLA supermotive carrier peptides and / or HLA motif (see Table IV).

Reference Example 17

Construction of multi-epitopic DNA plasmids of "minigene"

This example analyzes the construction of a minigene expression plasmid. The minigenes plasmids may, of course, contain various epitope configurations or epitope analogs of B, CTL and / or HTL lymphocytes as described herein.

A minigene expression plasmid normally includes multiple peptide epitopes of CTL and HTL. In the present example, peptide epitopes bearing HLA-A2, A3, B7 supermotives and peptide epitopes bearing HLA-A1 and A24 motifs are used in conjunction with DR supermotive epitopes and / or DR3 epitopes. PSCA derived from peptide epitopes bearing supermotives or HLA class I motifs are selected so that multiple motifs / supermotives are represented to ensure a broad population coverage. Similarly, PSCA class II HLA epitopes are selected to provide broad population coverage, that is, both HLA DR-1-4-7 supermotive carrier epitopes and HLA DR-3 motif epitopes are selected for inclusion in the construction of minigén. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.

Such construction may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, protein Ii can be fused with one or more HTL epitopes as described in the art, in which the CLIP sequence of protein Ii is removed and replaced with a class II HLA epitope sequence so that the HLA class II epitope is directed to the endoplasmic reticulum, in which the epitope binds with a class II HLA molecule.

This example illustrates the methods to use for the construction of a minigene-bearing expression plasmid. Other expression vectors that can be used for minigenes compositions are available and are known to those skilled in the art.

The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine Ig kappa light chain signal sequence followed by CTL and / or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading phase fused with the Myc and His antibody epitope marker encoded by the pcDNA 3.1 Myc-His vector.

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Results indicate the magnitude of the HTL response, thereby demonstrating the in vivo immunogenicity of the minigene.

DNA minigenes, constructed as described in the previous example, can also be confirmed as a vaccine in combination with a reinforcing agent using a sensitization and reinforcement protocol. The reinforcing agent may consist of recombinant protein (for example, Barnett et al., Aids Res. And Human Retroviruses 14, Supplement 3: S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, for example, Hanke et al., Vaccine 16: 439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95: 7648-53, 1998; Hanke and McMichael, Immunol Letters 66: 177-181, 1999; and Robinson et al., Nature Med. 5: 526-34, 1999).

For example, the efficacy of the DNA minigen used in a sensitization and reinforcement protocol is initially evaluated in transgenic mice. In this example, A2.1 / Kb IM transgenic mice are immunized with 100 µg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotive carrier peptide. After an incubation period (ranging from 3 to 9 weeks), the mice are reinforced IP with 107 pfu / mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 µg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia booster. After an additional incubation period of two weeks, splenocytes from mice are tested immediately for specific peptide activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then tested for peptide specific activity in an alpha, beta and / or gamma IFN ELISA.

It has been found that the minigene used in a sensitization-enhancement protocol induces greater immune responses to the HLA-A2 supermotive peptides than with DNA alone. Said analysis can also be performed using transgenic mouse models HLA-A11 or HLA-B7 to evaluate the induction of CTL by motif or supermotive epitopes HLA-A3 or HLA-B7. The use of sensitization and reinforcement protocols in humans is described later in the example entitled "Induction of CTL Responses Using a Protocol of Sensitization and Reinforcement)".

Reference Example 19

Multi-antigen polyepitopic vaccine compositions

The PSCA peptide epitopes of the present invention are used together with epitopes of other target tumor associated antigens, to create a vaccine composition that is useful for the prevention or treatment of cancer expressing PSCA and other such antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple PSCA epitopes as well as tumor associated antigens that are often expressed with a target cancer associated with PSCA expression, or it can be administered as a composition comprising a Cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells that have been loaded with peptide epitopes in vitro. Reference Example 20

Use of peptides to evaluate an immune response

Peptides of the invention can be used to analyze an immune response with respect to the presence of specific antibodies, CTL or HTL directed to PSCA. Said analysis can be performed in a manner described in Ogg et al., Science 279: 2103-2106, 1998. In this example, peptides according to the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.

In this example, tetrameric complexes of highly sensitive human leukocyte antigens ("tetramers") are used for a cross-sectional analysis of, for example, PSLA-specific CTL frequencies HLA-A * 0201 of HLA A * 0201 positive individuals in different stages of disease or after immunization comprising a PSCA peptide containing an A * 0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337: 1267, 1997). Briefly, heavy chain of purified HLA (A * 0201 in this example) and glo2 microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane cytosolic tail and terminal COOH addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, glo2 microglobulin, and peptide are replicated by dilution. The 45 kD refolding product is isolated by rapid protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Missouri), adenosine 5 ′ triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1: 4 molar ratio, and the tetrameric product is concentrated at 1 mg / ml. The resulting product is called tetrameric phycoerythrin.

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Example 24

Identification of molecules that interact with PSCA

PSCA, or biologically active fragments thereof, are labeled with 121 1 Bolton-Hunter reagent. (See, for example, Bolton et al. (1973) Biochem. J. 133: 529). Pre-arranged candidate molecules are incubated in the wells of a multiwell plate with the labeled PSCA, washed and any well with labeled PSCA complex is washed. Data obtained using different concentrations of PSCA are used to calculate values with respect to the number, affinity and association of PSCA with the candidate molecules.

Reference Example 25

In vivo assay for the promotion of PSCA tumor growth

The effect of PSCA protein on tumor cell growth is evaluated in vivo by evaluating the tumor growth and development of cells that express or lack PSCA. For example, SCID mice are injected subcutaneously into each 1 x 106 flank of 3T3 cell lines, or prostate cancer (for example PC3 cells) containing tkNeo or PSCA empty vector. At least two strategies can be used: (1) constitutive PSCA expression under the regulation of a promoter such as a constitutive promoter obtained from virus genomes such as polyoma virus, avian smallpox virus (UK document 2,211,504 published on July 5, 1989), adenovirus, (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus and Simian Virus 40 (SV40) or heterologous mammalian promoters , for example, the actin promoter or an immunoglobulin promoter, provided that said promoters are compatible with host cell systems, and (2) regulated expression under the control of an inducible vector system, such as ecdysone, tetracycline, etc. ., provided that said promoters are compatible with host cell systems. The tumor volume is then controlled by measurement per caliper at the time of the appearance of palpable tumors and is monitored over time to determine if the cells expressing PSCA grow at a faster rate and if the tumors produced by cells expressing PSCA demonstrate characteristics of altered aggressiveness (eg enhanced metastasis, vascularization, reduced sensitivity to chemotherapeutic drugs).

In addition, 1 x 105 mice of the same cells can be implanted orthotically to determine if PSCA has an effect on local growth in the prostate, and if PSCA affects the ability of cells to metastasize, specifically to lymph nodes, and bone (Miki T et al, Oncol Res. 2001; 12: 209; Fu X et al, Int J Cancer. 1991, 49: 938). The effect of PSCA on bone tumor formation and growth can be evaluated by injecting prostate tumor cells intratibially.

The assay is also useful for determining the PSCA inhibitory effect of candidate therapeutic compositions, such as for example PSCA intrabodies, PSCA antisense molecules and ribozymes.

Example 26

Inhibition mediated by monoclonal antibody of PSCA of tumors in vivo

The significant expression of PSCA on the cell surface of tumor tissues, together with its restrictive expression in normal tissues makes PSCA a good target for antibody therapy. Similarly, PSCA is a target for immunotherapy based on T lymphocytes. Therefore, the therapeutic efficacy of anti PSCA MAb in mouse models of human prostate cancer xenograft and mouse models of human pancreatic cancer xenograft is evaluated using Recombinant cell lines such as PC3-PSCA and 3T3-PSCA (see, for example, Kaighn, ME, et al., Invest Urol, 1979. 17 (1): 16-23), as well as human prostate xenograft models such as LAPC 9AD (Saffran et al PNAS 1999, 10: 1073-1078).

The efficacy of antibody in tumor growth and metastasis formation is studied, for example, in a xenograft model of pancreatic cancer or mouse orthotopic prostate. The antibodies may not be conjugated, as discussed in this example, or they may be conjugated to a therapeutic modality, as seen in the art. Anti-PSCA MAbs inhibit the formation of both pancreatic and prostate xenografts. Anti-PSCA MAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the usefulness of anti PSCA MAb in the treatment of local and advanced stage prostate cancer, pancreatic cancer and the cancers shown in Table I (see, for example, Saffran, D., et al., PNAS 10: 1073-1078 or the web URL pnas.org/cgi/doi/10.1073/pnas.051624698).

Administration of the anti PSCA MAbs led to the retardation of the growth of established orthotopic tumors and the inhibition of metastasis to distant sites, resulting in a significant prolongation of the survival of tumor-bearing mice. These studies indicate that PSCA is an attractive target for immunotherapy and demonstrate the therapeutic potential of anti PSCA MAb for the treatment of prostate cancer and

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II.) Monotherapy: in relation to the use of anti PSCA antibodies in tumor monotherapy, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, monotherapy is performed clinically in patients with end-stage cancer with extensive metastatic disease. Patients show some disease stabilization. The trials demonstrate an effect in refractory patients with cancerous tumors.

III.) Image capture agent: by binding a radionuclide (for example, iodine or yttrium (I131, Y90) with anti PSCA antibodies, radiolabeled antibodies are used as a diagnostic and / or image capture agent. In said role, the labeled antibodies are located in both solid tumors, as well as metastatic lesions of cells expressing PSCA.In relation to the use of anti PSCA antibodies as image capture agents, the antibodies are used as a complement for the treatment Surgical solid tumors, both as a presurgical examination and postoperative follow-up to determine which tumor remains and / or comes back in. In one embodiment, a PSCA antibody (111In) is used as an image capture agent in a human clinical trial. Phase I in patients who have a carcinoma that expresses PSCA (by analogy see, for example, Divgi et al. J. Natl. Cancer Inst. 83: 97-104 (1991)). They match with conventional anterior and posterior gamma cameras. The results indicate that primary lesions and metastatic lesions are identified.

Dose and route of administration

As will be appreciated by those of ordinary skill in the art, dosage considerations can be determined by comparison with the analogous products that are in the clinic. Therefore, anti-PSCA antibodies can be administered with doses in the range of 5 to 400 mg / m2, with the lowest doses used, for example, in relation to safety studies. The affinity of anti PSCA antibodies in relation to the affinity of an antibody known to its target is a parameter used by those skilled in the art to determine analogous dose regimens. In addition, anti-PSCA antibodies that are completely human antibodies, compared to the chimeric antibody, have slower elimination; consequently, the dosage in patients with such completely human anti-PSCA antibodies may be lower, perhaps in the range of 50 to 300 mg / m2, and still be effective. The dosage in mg / m2, unlike the conventional dose measurement in mg / kg, is a measurement based on surface area and is a convenient dosage measure that is designed to include patients of all sizes from children to adults .

Three different delivery approaches are useful for delivery of anti PSCA antibodies. Conventional intravenous delivery is a conventional delivery technique for many tumors. However, in relation to tumors in the peritoneal cavity, such as tumors of the ovaries, bile duct, other ducts and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody in the tumor and also to minimize the elimination of antibodies Similarly, certain solid tumors have vasculature that is appropriate for regional perfusion. Regional perfusion allows a high dose of antibody at the site of a tumor and minimizes the short-term elimination of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops anti-PSCA antibody treatments in relation to complementary therapy, monotherapy and as an image capture agent. The trials initially demonstrate safety and then confirm the efficacy in repeated doses. The trials are open and compare conventional chemotherapy with conventional therapy plus anti-PSCA antibodies. As will be appreciated, one criterion that can be used in relation to the admission of patients is the levels of PSCA expression in their tumors as determined by biopsy.

As with any protein or therapeutic product based on antibody infusion, safety concerns are primarily related to (i) cytokine release syndrome, that is, hypotension, fever, tremor, chills; (ii) the development of an immunogenic response to the material (ie, the development of human antibodies by the patient for the antibody therapeutic product or HAHA response); and (iii) toxicity to normal cells expressing PSCA. Conventional trials and follow-up are used to control each of these safety concerns. Anti-PSCA antibodies have been found to be safe after administration to humans.

Example 29

Human clinical trial: monotherapy with human anti PSCA antibody

Anti-PSCA antibodies are safe in relation to the complementary assay previously analyzed, a Phase II human clinical trial confirms the efficacy and optimal dosage for monotherapy. Such an assay is achieved and involves the same safety and outcome analysis as the complementary trial described above with the exception that patients do not receive chemotherapy simultaneously with the reception of doses of anti PSCA antibodies.

Example 30

Human clinical trial: diagnostic imaging with anti PSCA antibody

Again, as the complementary therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is conducted regarding the use of anti PSCA antibodies as a diagnostic imaging agent. The protocol is designed in a manner substantially similar to those described in the art, as in Divgi et al. J. Natl. Cancer Inst. 83: 97-104 (1991). Antibodies have been found to be both safe and effective when used as a diagnostic modality.

Example 31

Complementary therapy of human clinical trial with anti-human PSCA antibody and chemotherapeutic therapy, radiotherapy and / or hormonal ablation therapy

A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti PSCA antibody in relation to the treatment of a solid tumor, for example, a tissue cancer listed in Table I. In the study , the safety of individual doses of anti PSCA antibodies is evaluated when used as a complementary therapy to an antineoplastic or chemotherapeutic product or hormonal ablation agent as defined herein, such as, without limitation: cisplatin, topotecan, doxorubicin, Adriamycin, taxol, Lupron, Zoladex, Eulexin, Casodex, Anandron or the like. The assay design includes delivery of approximately six individual doses of an anti-PSCA antibody with antibody dosage that increases from about 25 mg / m2 to about 275 mg / m2 during the course of treatment according to the following schedule or a similar one:

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 MAb dose 25 mg / m2 75 mg / m2 125 mg / m2 175 mg / m2 225 mg / m2 275 mg / m2 Chemotherapy (conventional dose) + + + + + +

Patients are followed closely for one week after each administration of antibody and chemotherapy. In particular, patients are evaluated with respect to the safety concerns mentioned above: (i) cytokine release syndrome, that is, hypotension, fever, tremor, chills; (ii) the development of an immunogenic response to the material (ie, the development of human antibodies by the patient to the therapeutic product of human antibody, or HAHA response); and (iii) toxicity to normal cells expressing PSCA. Conventional trials and follow-up are used to control each of these safety concerns. Patients are also evaluated with respect to the clinical outcome, and particularly the reduction of tumor mass as demonstrated by MRI or other imaging method.

Anti-PSCA antibodies have been shown to be safe and effective. Phase II trials confirm the effectiveness and refine the optimal dosage.

Reference Example 32

RNA interference (RNAi)

RNA interference technology (RNAi) is implemented in various cell assays relevant to oncology. RNAi is a mechanism of posttranscriptional gene silencing activated by double stranded RNA (cRNA). RNAi induces degradation of specific mRNA that leads to changes in protein expression and subsequently in gene function. In mammalian cells, these dsRNAs called short interference RNAs (siRNAs) have the correct composition to activate the route of mRNA that is directed to degradation, specifically some mRNAs. See, Elbashir S. M., et al., Duplexes of 21-nucleotide RNAs Mediate RNA interference in Cultured Mammalian Cells, Nature 411 (6836): 494-8 (2001). Therefore, RNAi technology is successfully used in mammalian cells to silence target genes.

Loss of control of cell proliferation is a hallmark of cancer cells; therefore, the evaluation of the role of PSCA in survival / cell proliferation assays is relevant. Consequently, RNAi is used to investigate the function of the PSCA antigen. To generate siRNA for PSCA, algorithms predicting oligonucleotides that showed critical molecular parameters (G: C content, melting temperature, etc.) were used and has the ability to significantly reduce PSCA protein expression levels when introduced in cells In accordance with this example, PSCA siRNA compositions comprising siRNA (short-acting, double-stranded RNA) corresponding to the nucleic acid ORF sequence of the PSCA protein or sub-sequences thereof are used. Therefore, siRNA sub-sequences are used in this way that are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more than 35 RNA nucleotides in length. These siRNA sequences are complementary and not complementary to at least a part of the mRNA coding sequence. In a preferred embodiment, the sub-sequences are 19-25 nucleotides in length, more preferably 21-23 nucleotides in length. In preferred embodiments, these siRNAs achieve suppression of PSCA antigen in cells that express the protein and have functional effects as described below.

The selected siRNA (oligo PSCA.b) was tested in numerous cell lines in the MTS survival / proliferation assay (which measures cellular metabolic activity). Tetrazolium-based colorimetric assays (ie, MTS) detect viable cells exclusively, since living cells are metabolically active and therefore can reduce tetrazolium salts to colored formazan compounds; dead cells, however, no. In addition, this oligo PSCA.b achieved suppression of the PSCA antigen in cells that expressed the protein and had functional effects as described below using the following protocols.

Mammalian siRNA transfections: the day before siRNA transfection, the different cell lines were plated in media (RPMI 1640 with 10% FBS without antibiotics) at 2 x 103 cells / well in 80 µl (plate format 96 wells) for survival / MTS test. In parallel with the oligo of PSCA-specific siRNA, the following sequences were included in each experiment as controls: a) cells transfected with simulation with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and hybridization buffer (without siRNA); b) Luciferase 4 specific siRNA (target sequence: 5’-AAGGGACGAAGACGAACACUUCTT-3 ’) (SEQ ID NO: 77); and c) Eg5 specific siRNA (target sequence: 5’-AACTGAAGACCTGAAGACAATAA-3 ’) (SEQ ID NO: 78). 10 nM siRNA and 1 ofeg / ml Lipofectamine 2000 final concentration were used.

The procedure was as follows: siRNAs were first diluted in OPTIMEM (serum-free transfection medium, Invitrogen) to 0.1 µM (10-fold concentrate) and incubated at RT 5-10 minutes. Lipofectamine 2000 was diluted to 10 /g / ml (concentrated 10 times) for the total number of transfections and incubated 5-10 minutes at room temperature (RT). Appropriate amounts of Lipofectamine 2000 concentrate 10 times 1: 1 were mixed with 10-fold concentrated siRNA and incubated at RT for 20-30 seconds (5 times concentrated transfection solution). 20 µl of the concentrated transfection solutions were added 5 times to the respective samples and incubated at 37 ° C for 96 hours before analysis.

MTS Assays: The MTS assay is a colorimetric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays based on a tetrazolium compound [3- (4,5-dimethylthiazol-2-yl) -5- (3 -carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, internal salt; MTS (b)] and an electron coupling reagent (phenacin ethosulfate; PES). Assays were performed by adding a small amount of the solution reagent directly to culture wells, incubating for 1-4 hours and then recording the absorbance at 490 nm with a 96-well plate reader. The amount of colored formazan product as measured by the amount of absorbance at 490 nm is directly proportional to the mitochondrial activity and / or the number of living cells in culture.

To address the function of PSCA in cells, PSCA is silenced by transfecting the cell lines of PSCA that express endogenously.

Another embodiment of the invention is a method for analyzing PSCA-related cell proliferation, which is the measurement of DNA synthesis as a marker for proliferation. Labeled DNA precursors (ie 3H-Thymidine) are used and their incorporation into DNA is quantified. The incorporation of the labeled DNA precursor is directly proportional to the amount of cell division that occurs in the culture. Another method used to measure cell proliferation is to perform clonogenic assays. In these assays, a defined number of cells are plated in the appropriate matrix and the number of colonies formed after a growth period after treatment with siRNA is counted.

In PSCA cancer target validation, the complementation of cell survival / proliferation analysis with apoptosis and cell cycle profiling studies are considered. The biochemical hallmark of the apoptotic process is the fragmentation of genomic DNA, an irreversible event that commits the cell to die. One method of observing fragmented DNA in cells is the immunological detection of DNA fragments in histone complex by means of an immunoassay (i.e. cell death detection ELISA) that measures the enrichment of DNA fragments in histone complex (mono and oligonucleosomes) in the cytoplasm of apoptotic cells. This assay does not require pre-labeling of cells and can detect the degradation of DNA in cells that do not proliferate in vitro (i.e. freshly isolated tumor cells).

The most important effector molecules to trigger apoptotic cell death are caspases. Caspases are proteases that, when activated, cleave numerous substrates at the carboxyl terminal site of an aspartate residue that mediates in the very early stages of apoptosis after activation. All caspases are synthesized as proenzymes and their activation involves excision in aspartate residues. In particular, caspase 3 seems to play a central role in the onset of cellular apoptosis events. Trials for determining caspase 3 activation detect early events of apoptosis. After RNAi treatments, detection with Western blotting of the presence of active caspase 3 or proteolytic cleavage of products (ie PARP) found in apoptotic cells further supports an active induction of apoptosis. Because the cellular mechanisms that result in apoptosis are complex, each has its advantages and limitations. Consideration of other criteria / endpoints such as cell morphology, chromatin condensation, membrane blistering, apoptotic bodies help to further support that cell death is apoptotic. Since not all gene targets that regulate growth

5 cell are antiapoptotic, the DNA content of permeabilized cells is measured to obtain the DNA content profile or cell cycle profile. Apoptotic cell nuclei contain less DNA due to leakage outside the cytoplasm (sub G1 population). In addition, the use of DNA stains (ie, propidium iodide) also differentiates between the different phases of the cell cycle in the cell population due to the presence of different amounts of DNA in G0 / G1, S and G2 / M. In these studies subpopulations can be quantified.

10 For the PSCA gene, RNAi studies facilitate the understanding of the contribution of the gene product in cancer pathways. Such active RNAi molecules have use in the identification of assays to explore for MAb that are active antitumor therapeutic products. In addition, siRNAs are administered as therapeutic products to cancer patients to reduce the malignant growth of various types of cancer, including

15 listed in Table 1. When PSCA plays a role in cell survival, cell proliferation, tumorigenesis or apoptosis, it is used as a target for diagnostic, prognostic, preventive and / or therapeutic purposes.

Reference Example 33

20 Detection of PSCA protein in samples from IHC cancer patient trials

PSCA protein expression was detected in tumor test samples of cancer patients using the HA1-4.117 antibody. Tissues included in paraffin, fixed in formalin, were cut into sections of 4

25 micrometers and mounted on glass slides. Sections were lowered, rehydrated and treated with antigen recovery solution (Citra Antigen Recovery Solution; BioGenex, 4600 Norris Canyon Road, San Ramon, CA, 94583) at high temperature. Sections were then incubated in fluorescein-conjugated human monoclonal anti PSCA antibody, Ha1-4,117, for 16 hours at 4 ° C. The slides were washed three times in buffer and further incubated with Rabbit antiflorescein for 1 hour and,

30 after washing in buffer, they were immersed in secondary goat anti-immunoglobulin rabbit antibody conjugated to DAKO EnVision + ™ peroxidase (DAKO Corporation, Carpenteria, CA) for 30 minutes. Sections were then washed in buffer, developed using the DAB kit (SIGMA Chemicals), counterstained using hematoxylin, and analyzed by light field microscopy. The results show the expression of PSCA in tumor cells of prostate adenocarcinoma (A, B), transitional bladder carcinoma (C) and

35 pancreatic ductal adenocarcinoma (D). These results indicate that PSCA is expressed in human cancers and that antibodies directed to this antigen are useful as diagnostic reagents (Figure 17).

These results indicate that PSCA is a target for diagnostic, prognostic and therapeutic applications in cancer.

40 Throughout this application, reference is made to various data contents of websites, publications, patent applications and patents. (Websites are indicated by their Uniform Resource Locator, or URL, web addresses.)

45 Tables

Table I: tissues that express PSCA when they are malignant.

Prostate Pancreas Bladder Kidney Colon Ovarian Lung Breast

TABLE II: Abbreviations of amino acids

ONE LETTER

THREE LETTERS FULL NAME

F

Phe phenylalanine

L

Leu leucine

S

Be serine

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TABLE IV: HLA Class I / II Motives / Supermotives TABLE IV (A): Class I HLA Supermotives / Motives

SUPERMOTIVE

POSITION POSITION POSITION

2 (Primary Anchor)

3 (Primary Anchor) C Terminal (Primary Anchor)

A1

TILVMS FWY

A2

LIVMATQ IVMATL

A3

VSMATLI RK

A24

YFWIVLMT FIYWLM

B7

P VILFMWYA

B27

Rhk FYLWMIVA

B44

ED FWYLIMVA

B58

ATS FWYLIVMA

B62

QLIVMP FWYMIVLA

REASONS

A1

TSM Y

A1

DEAS Y

A2.1

LMVQIAT VLIMAT

A3

LMVISATFCGD KYRHFA

A11

VTMLISAGNCDF KRYH

A24

YFWM FLIW

A * 3101

MVTALIS RK

A * 3301

MVALFIST RK

A * 6801

AVTMSLI RK

B * 0702

P LMFWYAIV

B * 3501

P LMFWYIVA

B51

P LIVFWYAM

B * 5301

P IMFWYALV

B * 5401

P ATIVLMFWY

Bold remains are preferred, italicized items are less preferred. A peptide is considered a motif carrier if it has primary anchors in each primary anchor position for a motif or supermotive as specified in the table above.

TABLE IV (B): HLA Class II supermotive

one

6 9

W, F, Y, V, .I, L

A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y

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TABLE IV (F): Summary of HLA supertypes

Global phenotypic frequencies of HLA supertypes in different ethnic populations Specificity Phenotypic frequency

Supertype Position 2 Extreme C Caucasian Black Japanese Chinese Hispan Average

Terminal N.A. or

B7 P AILMVFWY 43.2 55.1 57.1 43.0 49.3 49.5

A3 AILMVST RK 37.5 42.1 45.8 52.7 43.1 44.2

A2 AILMVT AILMVT 45.8 39.0 42.4 45.9 43.0 42.2

A24 YF FI (YWLM) 23.9 38.9 58.6 40.1 38.3 40.0

(WIVLMT)

B44 E (D) FWYLIMVA 43.0 21.2 42.9 39.1 39.0 37.0

A1 TI (LVMS) FWY 47.1 16.1 21.8 14.7 26.3 25.2

B27 RHK FYL (WMI) 28.4 26.1 13.3 13.9 35.3 23.4

B62 QL (IVMP) FWY (MIV) 12.6 4.8 36.5 25.4 11.1 18.1

B58 ATS FWY (LIV) 10.0 25.1 1.6 9.0 5.9 10.3

TABLE IV (G):

Calculated population coverage provided by different combinations of HLA supertype HLA supertypes Phenotypic frequency

Caucasian Caucasians N.A Japanese Chinese Hispanic Average

83.0 86.1 87.5 88.4 86.3 86.2

A2, A3 and B7 99.5 98.1 100.0 99.5 99.4 99.3

A2, A3, B7, A24, 99.9 99.6 100.0 99.8 99.9 99.8

844 and A1

A2, A3, B7, A24,

B44, A1, 827,

B62, and B 58

The reasons indicate the remains that define supertype specificities. The reasons incorporate remains that have been determined based on published data that are recognized by multiple alleles within the supertype. The remains in parentheses are additional remains that have also been predicted to be tolerated by multiple alleles within the supertype.

Table V: reasons for frequent occurrence

Name

% average identity Description Potential Function

zf-C2H2

3. 4 % Zinc finger, type C2H2 The nucleic acid binding protein acts as a transcription factor, likely nuclear location

cytochrome_b_N

68% Cytochrome b (Nterminal) / b6 / petB membrane bound oxidase, generates superoxide

Ig

19% immunoglobulin domain the domains are one hundred amino acids in length and include a conserved intra-domain disulfide bond.

WD40

18% WD domain, Gbeta repeat tandem repetitions of approximately 40 residues, each containing a Trp-Asp motif. They act in signal transduction and protein interaction

PDZ

2. 3 % PDZ domain can act in the direction of signaling molecules to submembrane sites

LRR

28% Rich repetition in Leucine short sequence motifs involved in protein-protein interactions

Kinase

2. 3 % Protein kinase domain Common conserved catalytic core for both serine / threonine and for tyrosine kinase proteins that contain an ATP binding site and a catalytic site

PH

16% PH domain plextrin homology involved in intracellular signaling or as constituents of the cytoskeleton

EGF

3. 4 % EGF type domain 30-40 amino acids in length found in the extracellular domain of membrane-bound proteins or in secreted proteins

Rvt

49% Reverse transcriptase (RNA-dependent RNA polymerase)

Ank

25% ank repeat Cytoplasmic protein, associates integral membrane proteins with the cytoskeleton

Oxidored_q1

32% NADHUbiquinone / Plastoquinone (complex I), various chains membrane associated. Involved in the translocation of protons across the membrane

Ef hand

24% EF hand calcium binding domain, consists of a loop of 12 residues flanked on both sides by a helical alpha domain of 12 residues

Rvp

79% Aspartyl retroviral protease Aspartyl proteases or acid proteases, centered on a catalytic aspartyl moiety

Collagen

42% Repeating triple collagen helix (20 copies) Extracellular structural proteins involved in connective tissue formation. The sequence consists of the G-X-Y and the polypeptide chains form a triple helix.

Fn3

twenty % Type III fibronectin domain Located in the receptor extracellular ligand binding region and is approximately 200 amino acid residues in length with two pairs of cysteines involved in disulfide bonds

7tm_1

19% 7 transmembrane domains (rhodopsin family) seven hydrophobic transmembrane regions, with the receptor located at the N-terminus extracellularly while the C-terminus is cytoplasmic. Signals through G proteins.

Table VI: Exotic limits of the PSCA transcript v.1

Exon Number

from Start Final Length

one

10 69 60

2

70 177 108

3

178 985 808

Table VII: IFM values of each data point used for affinity calculation.

IFM values

nM

PSCA PSCA PDP3 T = 8 + 4 PDP3 T = 8 + 4

40

869.3 777.06 795.24 661.66

twenty

875.19 835.94 816.34 824.07

10

856.28 847.83 777.85 842.72

5

866.94 817.45 758.15 818.83

2.5

835.47 769.79 742.45 783.5

1.25

813.12 782.84 806.2 792.44

0.625

766.52 689.3 683.64 666.28

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Claims (1)

image 1 image2 image3 image4 image5 Figure 1E-2 image6 Figure 1F. The cDNA sequence (SEQ ID NO: 11) and amino acids (SEQ ID NO: 12) of PSCAv.6 The Kozak sequence is shown in bold, the starting methionine is underlined. image7 150 Figure 1F-2 image8 Figure 1G SNP variants of PSCA v.2, PSCA v.7 to v.18

Variant

Nucleic acid position Nucleic Acid Variation Amino acid variation

PSCA v.7

367 C / T Silent variant

PSCA v.8

424 A / C Silent variant

PSCA v.9

495 C / G Silent variant

PSCAv.10

499 C / T Silent variant

PSCAv.11

563 C / T Silent variant

PSCA v. 12

567 G / A Silent variant

PSCA v.13

627 G / A Silent variant

PSCAv. 14

634 T / G Silent variant

PSCA v.15

835 G / A Silent variant

PSCA v.16

847 G / A Silent variant

PSCAv.17

878 G / A Silent variant

PSCA v.18

978 C / G Silent variant

151 Figure 1H. SNP variants of PSCA v.4, PSCA v.19 to v.30

Variant

Nucleic acid position Nucleic Acid Variation Amino acid position Amino acid variation

PSCA v.19

521 CAT 33 P / L

PSCA v.20

578 A / C 52 Y / S

PSCA v.21

649 C / G 76 H / D

PSCAv. 22

653 CAT 77 P / L

PSCA v.23

717 C / T 98 Silent variant

PSCAv. 24

721 G / A 100 A / T

PSCAv. 25

781 G / A 120 G / S

PSCAv. 26

788 T / G 122 I / O

PSCAv. 27

989 G / A 189 R / Q

PSCA v.28

1001 G / A Silent variant

PSCA v.29

1032 G / A Silent variant

PSCA v.30

1132 C / G Silent variant

152 image9 image10 image11