CN101223191B

CN101223191B - Antibodies and related molecules that bind to PSCA proteins

Info

Publication number: CN101223191B
Application number: CN200680021456XA
Authority: CN
Inventors: 琼·古达斯; 阿亚·雅科博维茨; 贾晓驰; 罗伯特·K·莫里森; 派亚·M·查利塔-艾德; 阿瑟·B·雷塔诺
Original assignee: Agensys Inc
Current assignee: Agensys Inc
Priority date: 2005-04-14
Filing date: 2006-02-15
Publication date: 2012-09-05
Anticipated expiration: 2026-02-15
Also published as: NO345322B1; CN101223191A; NO20075592L

Abstract

Antibodies and molecules derived therefrom that bind to novel PSCA protein, and variants thereof, are described, wherein PSCA exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, PSCA provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The PSCA gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with PSCA can be used in active or passive immunization.

Description

With protein bound antibody of PSCA and associated molecule

Technical field

The molecule that invention as herein described relates to the proteinic antibody that can combine to be known as PSCA and binding fragment thereof and transformed by their.The invention still further relates to diagnosis, prediction, prevention and the treat-ment and the compsn that can be used for treating the cancer of expressing PSCA.

Background technology

Cancer is the human dead second largest cause of disease that causes that is only second to coronary disease.There is every year the millions of people to die from cancer in the world.According to ACS, only in the U.S., cancer causes the healthy subjects more than 500,000 dead every year, and the new case who diagnoses out every year surpasses 1,200,000.Although it is significantly reduce, generally in rising trend by the death that cancer causes by the death due to the heart trouble.Prediction is early stage in the next century, and cancer will become the first cause that causes death.

Worldwide, there are several kinds of cancers to highlight and are main killer.Specifically, lung cancer, prostate cancer, mammary cancer, colorectal carcinoma, carcinoma of the pancreas, ovarian cancer and bladder cancer become the representative of cancer mortality major cause.These cancers and in fact all other cancers all have the common characteristic that causes death.Except the exception of only a few, the cancer metastasis disease also is a lethality.In addition, even if originally some cancer patients survives from preinvasive cancer, but the experience of their common shows that considerable change has taken place in their life.A lot of patients worry that cancer recurs possibly or treatment can be failed, thereby feel very anxiety.A lot of cancer patientss feel in poor health after treatment.In addition, a lot of cancer patientss also can meet with cancer return.

Worldwide, prostate cancer is listed as the 4th in the easiest cancer of the male sex.In North America and Northern Europe, prostate cancer is a modal cancer among the male sex, is to cause the male sex to die from the second largest cause of disease of cancer.Only in the U.S., die from this sick man every year considerably beyond 30,000 people-be only second to lung cancer.Although these digital orders of magnitude are very big, still there is not the efficacious therapy method to the prostate cancer that shifts.Surgery prostatectomy, radiotherapy, hormone break away from therapy, surgery castrating and chemotherapy and are still main form of therapy.Unfortunately these therapies do not play effect to much human, and often relevant with bad consequence.

Aspect diagnosis, the tumor marker that shortage can accurately detect in early days, locate tumor of prostate has significantly limited the diagnosis and the treatment of prostate cancer.Although serum PSA (PSA) assay method has become instrument of great use, people generally believe this method several important aspect lack of specific and universal applicability.

The generation of prostate cancer heterograft that can in the mouse body, recur the different steps of disease has advanced the process of identifying other specific marker of prostate cancer.LAPC (Los Angeles Prostate Cancer) heterograft is the prostate cancer heterograft; It can survive in Reconstruction in Sever Combined Immunodeciency (SCID) mouse extremely; And can show simulation changes the male sex hormone independent form into from male sex hormone dependent form ability (Klein et al.; 1997, Nat.Med.3:402).The prostate cancer markers of up-to-date evaluation comprises PCTA-1 (Su et al.; 1996; Proc.Natl.Acad.Sci.USA 93:7252), Prostato-specific membranes (PSM) antigen (Pinto et al.; Clin Cancer Res 1996 Sep 2 (9): 1445-51), STEAP (Hubert, et al, Proc Natl Acad Sci USA.1999 Dec 7; 96 (25): 14523-8) and PSCA (PSCA) (Reiter et al., 1998, Proc.Natl.Acad.Sci.USA95:1735).

Although the mark of identifying in the past, like PSA, PSM, PCTA and PSCA are played a role to diagnosis and treatment prostate cancer,,, still need identify other mark and treatment target of prostate gland and associated cancer in order further to improve diagnosis and treatment.

Renal cell carcinoma (RCC) has caused grownup's malignant tumour of about 3%.In case the diameter of adenoma reaches 2 to 3cm, just there is the possibility that cancerates.In the grownup, two kinds of main malignant renal neoplasms are renal cell adenocarcinoma and renal plevis or ureteral transitional cell carcinoma.According to estimates, the sickness rate of the renal cell adenocarcinoma of the U.S. surpasses 29,000 examples, 1998, has 11,600 multidigit patients to die from this disease.The less generation of transitional cell carcinoma, the annual sickness rate of the U.S. is about 500 examples.

In decades, surgical operation is the main therapy of renal cell adenocarcinoma.As of late, any general therapy all falls down on the transfer disease.By means of the latest developments of general therapy, particularly immunotherapy, can offensiveness ground near the suitable intravital transfer renal cell carcinoma of patient, and might be with persistent reaction.Yet these patients still need effective therapy.

In all cancer new cases of the U.S., bladder cancer accounts for 5% (in kinds of tumor, ranking the 5th) of male tumor and 3% (in kinds of tumor, ranking the 8th) of female tumor.Sickness rate slowly increases along with the increase gradually of elderly population.According to estimates, 54,500 cases were arranged in 1998, comprise 39,500 male sex and 15,000 women.In the U.S., the sickness rate that regulated by the age is: per 100,000 male sex have 32 patients, and per 100,000 women have 8 patients.Be associated with women's smoking pattern, the ill ratio of historical male/female may reduce at 3: 1.According to estimates, there were 11,000 patients to die from bladder cancer (7,800 male sex and 3,900 women) in 1998.The M & M of bladder cancer sharply increases along with the increase at age, and along with the appearance of population aging, this will become serious day by day problem.

Most of bladder cancer recur in bladder.The treat-ment of bladder cancer is used urinary bladder channel urethrectomy (TUR) and intravesical chemotherapy or immunotherapy for uniting.Many focuses of bladder cancer and recurrence characteristic are showed the limitation of TUR.Only can not cure most muscle-invaded cancer through TUR.Radical cystectomy and diversion of urine are the most effectual ways of eliminating cancer, but undeniable be that they are influential to uropoiesis and sexual function.Therefore, very need the form of therapy that is beneficial to bladder cancer patients.

According to estimates, there were 130,200 routine colorectum oncogenesiss in the U.S. in 2000, comprised 93, the 800 routine colorectal carcinomas and 36, the 400 routine rectum cancer.Colorectal carcinoma is the cancer of masculinity and femininity the 3rd pilosity.1992-1996, sickness rate significantly reduce (annual decline 2.1%).Study the decline that shows sickness rate and possibly give the credit to examination and excision polyp, prevent that polyp from developing into the invasive cancer.According to estimates, 56,300 deaths (die from colorectal carcinoma for 47,700, die from the rectum cancer for 8,600) were arranged, account for 11% of all american cancer death tolls in 2000.

At present, the most general colorectal carcinoma therapy is a surgical operation, for the cancer that does not spread as yet, often is recoverable.Penetrate the intestines wall or diffused to for the Most patients of lymphoglandula for the cancer degree of depth, should give chemotherapy before operation or after the operation or chemotherapy adds radiotherapy.Colorectal carcinoma patient needs permanent colostomy (opening an osculum to discharge refuse in the body at belly) sometimes, and the less needs of rectal cancer patient should operation.The colorectal carcinoma patient still needs effective diagnosis and form of therapy.

According to estimates, new 164,100 lungs and the bronchogenic carcinoma case of occurring in 2000 accounts for 14% of all american cancer diagnosed SARS cases.The sickness rate of male sex's lung and bronchogenic carcinoma significantly reduces, and per 100,000 philtrums were ill up to ill 70.0 people to 1996 of 86.5 people from 1984.In the eighties of last century the nineties, gathering way of women's sickness rate begins to slow down.1996, women's sickness rate was that per 100,000 philtrums, 42.3 people are ill.

According to estimates, lung in 2000 and bronchogenic carcinoma cause 156,900 people dead, account for 28% of all cancer mortality numbers.1992-1996, the lung cancer mortality among the male sex significantly descend (annual decline 1.7%), and the mortality ratio among the women still increases (annual increase by 0.9%) gradually.Since 1987, compare with mammary cancer, the women who dies from lung cancer every year is more, for the women more than 40 years old, lung cancer be women's cancer mortality main diseases because of.The ratio that reduced number of smokers in preceding 30 years most possibly causes the reduction of lung cancer morbidity rate and mortality ratio; Yet, for the male sex, reduce some hysteresis of work of women's smoking pattern.It is worth noting that although the lowering speed of adult tobacco rate of utilization is tending towards slowly, the tobacco rate of utilization among the youngster is the trend that increases gradually once more.

Can confirm that the treatment of lung and bronchogenic carcinoma is selected according to the type and the stage of cancer, it comprises surgical operation, radiation and chemotherapy.In partial cancer, the therapy of selecting usually is a surgical operation for various fixed.Because before finding cancer, cancer spreads usually, unite the use radiation and chemotherapy when often needing the orthopaedic surgical operations operation.For small cell lung cancer, the therapy that can select is only with chemotherapy or with chemotherapy and radiotherapy associating; Through using this scheme, high per-cent patient's the state of an illness alleviates, and in some cases, this good curative effect can keep for a long time.Yet lung and bronchogenic carcinoma constantly need efficacious therapy and diagnostic method.

Estimate to occur in the American Women in 2000 the new mammary cancer infringement cases of about 182,800 examples.In addition, expectation can be diagnosed out the new male breast carcinoma case of about 1,400 example in 2000.Increase approximately after 4% every year from the eighties of last century the eighties, the sickness rate of women breast cancer has stopped increasing in the nineties, maintains about 110.6 cases of per 100,000 philtrums.

Only, there were 41,200 people (40,800 women, 400 male sex) to die from mammary cancer according to estimates in 2000 in the U.S..Mammary cancer ranks second in women's cancer mortality.According to up-to-date data, at 1992-1996, mortality ratio significantly reduces, and the reduction of mortality ratio is the most remarkable in young white man and Black women.The reduction of mortality ratio possibly be early detection and the result who improves therapy.

Consider seek medical advice environment and patient's selection, the mammary cancer therapy comprises lumpectomy (local excision's tumour) and extracts the lymphoglandula of arm below; The lymphoglandula of mastectomy (excision breast) and excision arm below; Radiotherapy; Chemotherapy; Or hormonotherapy.Often unite and use two or more therapies.Multinomial research shows that for breast carcinoma of early stage the long-term surviving rate that lumpectomy adds after the radiotherapy is similar to modified radical mastectomy survival rate afterwards.The marked improvement of Reconstruction provides several kinds of selections for postmastectomy breasst reconstruction.Recently, in mastectomy, carried out breasst reconstruction.

To around have the local excision of the DCIS (DCIS) of the normal galactophore tissue of q.s can prevent the local recurrence of DCIS.Breast is carried out radiotherapy and/or tamoxifen can reduce the chance that DCIS take place all the other mammary tissues.This point is most important, if because DCIS is not added treatment, can develop into invasive mammary cancer.Yet these therapies have severe side effect or sequela.Therefore, need effective mammary cancer therapy.

According to estimates, there is the new ovarian cancer case of 23,100 examples in the U.S. in 2000.Account for 4% of all cancers of women, and in the Obstetric and Gynecologic Department cancer, rank second.At 1992-1996, the ovarian cancer sickness rate significantly reduces.According to estimates, there were 14,000 people to die from ovarian cancer in 2000.Compare with any other female reproductive system cancer, ovarian cancer causes more deaths.

Surgical operation, radiation and chemotherapy are selectable ovarian cancer therapies.Surgical operation generally includes extracts one or two ovary, uterine tube (salpingo oophorectomy) and uterus (uterectomy).To some tumours of phase very early, the young woman of particularly those hope being born child only need be extractd the ovary of catching an illness.As far as advanced ovarian cancer, should attempt extracing all abdomen inner disease focis to strengthen the effect of chemotherapy.Ovarian cancer still needs effective treat-ment.

According to estimates, there is the new carcinoma of the pancreas case of 28,300 examples in the U.S. in 2000.In in the past 20 years, the sickness rate of male sex's carcinoma of the pancreas descends.The sickness rate of women's carcinoma of the pancreas remains unchanged basically, but number has begun to descend.The U.S. had 28,200 people to die from carcinoma of the pancreas approximately in 2000.In in the past 20 years, the deaths in men rate has faint but significant downtrending (descend 0.9% every year approximately), and women's mortality ratio slightly increases.

Surgical operation, radiation and chemotherapy are selectable carcinoma of the pancreas therapies.These therapies can prolong a lot of patients' survival time and/or alleviate their symptom, but can not cure Most patients.Obviously, also need other to be directed against treatment for cancer and diagnostic method.Comprise antibody, vaccine and small molecules as therapeutic modality.In addition, also need these therapeutic modalities be used as research tool to diagnose, to detect, to monitor and to promote the state of the art of all cancer therapy and research field.

The therepic use of monoclonal antibody (MAb) is implemented (G.Kohler and C.Milstein, Nature 256:495-497 (1975)).At present, monoclonal antibody has been approved for treatment transplanting, cancer, transmissible disease, cardiovascular diseases and inflammation.Different isoforms has different effector functions.For panimmunity sphaeroprotein isotype, in the present different three-dimensional structure of the difference table of this function (P.M.Alzari et al., Annual Rev.Immunol., 6:555-580 (1988)).

Because mouse is convenient to immunization, and can be identified as exotic with most of human antigens, therefore, generally all derive from mouse to the MAb of people's target with potential treatment function.Yet during as the human therapy agent, mouse MAb has the inherent shortcoming.Their need dosed administration more frequently, because compare with people's antibody, mouse MAb is shorter in the intravital circulation of people half life.More bad is: can cause human immune system to be reacted for human immune system repetitive administration murine antibody, murine protein matter is identified as exotic, and produce human anti-mouse antibody (HAMA) reaction.This HAMA reaction can cause transformation reactions, and murine antibody is removed from system fast, thereby the murine antibody therapy was lost efficacy.For fear of this influence, people have attempted in the mouse body, setting up human immune system.

Initial trial is hoped to set up can be with antibody with human sequence and antigen reactive transgenic mice (referring to Bruggemann et al.; Proc.Nat ' l.Acad.Sci.USA 86:6709-6713 (1989)); But the DNA amount of available cloning vector ability stable maintenance is limited; Therefore, this trial is restricted.Using yeast artificial chromosome (YAC) cloning vector can be that fragment imports transgene mammal with the macrospecies of people Ig locus.Use people V that YAC can will find, that arrange with uniform distances in the people's gene group, the major part of D and J district gene and human constant region importing mouse in fact.Known this type of transgenic mice strain is XenoMouse

mouse; It can be available from Abgenix, Inc. (Fremont CA).

The invention summary

The invention provides can with PSCA albumen and polypeptide fragment bonded antibody thereof and binding fragment thereof and the molecule of transforming by their.But the present invention includes polyclone and monoclonal antibody, mouse and other Mammals antibody, chimeric antibody, humanization and human antibody and with the antibody of mark note or therapeutical agent mark.In some embodiments, the precondition that need to satisfy be: the complete nucleotide sequence of code pattern 3 and/or do not prepare the complete amino acid sequence of Fig. 2 not.In some embodiments, the complete nucleotide sequence of the Fig. 3 that encoded and/or prepared the complete amino acid sequence of Fig. 2, any in them is all its human unit dosage form separately.

The present invention also provides the method that detects PSCA polynucleotide and proteinic existence and situation in the multiple biological sample, and the method for identifying the cell of expressing PSCA.Embodiment of the present invention provide monitoring to have or suspect that the growth regulating with some forms is unusual, as whether having the method for psca gene product in the tissue of cancer or the blood sample.

The present invention also provides the multiple cancer of expressing PSCA that is used to treat, and like immunogenicity or the curative compsn and the strategy of the cancer of the listed tissue of Table I, comprises that purpose is to suppress PSCA and transcribes, translates, the therapy and the cancer vaccine of processing or function.On the one hand; The invention provides be used for treating people experimenter express PSCA cancer compsn with comprise the method for using said composition; Wherein compsn contains the carrier that is applicable to the people, and one or more suppressed PSCA of people's unitary dose produce or the medicament of function.Preferred vector is unique people's carrier.In another aspect of this invention, medicament is immunoreactive moity to take place with PSCA albumen.The non-limitative example of said moity includes but not limited to antibody (like strand, mono-clonal, polyclone, humanization, chimeric or people's antibody), its function equivalent (can be natural, also can be synthetic) and combination thereof.Antibody can or be treated the moity coupling with diagnosis.On the other hand, medicament is the small molecules that this paper defines.

Description of drawings

Fig. 1. Figure 1A has shown the cDNA and the aminoacid sequence of PSCA (being also referred to as " PSCAv.1 " or " PSCA variant 1 ").Underscore be initial methionine.ORFs extends to 389 from nucleic acid 18, comprising terminator codon.

Figure 1B has shown the cDNA and the aminoacid sequence of PSCA variant 2 (being also referred to as " PSCAv.2 ").Underscore be the initial methionine codon.ORFs extends to 427 from nucleic acid 56, comprising terminator codon.

Fig. 1 C has shown the cDNA and the aminoacid sequence of PSCA variant 3 (being also referred to as " PSCAv.3 ").Underscore be the initial methionine codon.ORFs extends to 707 from nucleic acid 423, comprising terminator codon.

Fig. 1 D has shown the cDNA and the aminoacid sequence of PSCA variant 4 (being also referred to as " PSCAv.4 ").Underscore be the initial methionine codon.ORFs extends to 993 from nucleic acid 424, comprising terminator codon.

Fig. 1 E has shown the cDNA and the aminoacid sequence of PSCA variant 5 (being also referred to as " PSCAv.5 ").Underscore be the initial methionine codon.ORFs extends to 1479 from nucleic acid 910, comprising terminator codon.

Fig. 1 F has shown the cDNA and the aminoacid sequence of PSCA variant 6 (being also referred to as " PSCAv.6 ").Underscore be the initial methionine codon.ORFs extends to 427 from nucleic acid 83, comprising terminator codon.

Fig. 1 G.PSCAv.2, PSCAv.7 SNP variant extremely v.18.PSCAv.7 extremely v.18 albumen has 123 amino acid.V.18, variant PSCAv.7 is to being to compare the variant that single nucleotide difference is arranged with PSCAv.2, and their encoded protein matter is identical with PSCAv.2.Although separately show these SNP variants, they also can occur with arbitrary combination, and can come across listed any the transcribing in the variant of above-mentioned Figure 1A to 1F.

Fig. 1 H.PSCAv.4, PSCAv.19 SNP variant extremely v.30.PSCAv.19 extremely v.30 albumen has 189 amino acid.V.30, variant PSCAv.19 is to compare the variant that single nucleotide difference is arranged with PSCAv.4 extremely.V.24, v.10 PSCAv.9, v.11, only has an amino acid different with albumen v.25 with PSCAv.1.PSCAv.23, v.28, v.29 with v.30 encoded protein matter is identical with PSCAv.4.Although separately represent these SNP variants, they also can occur with any combination, and can appear at and transcribe in v.3 and v.4 any of variant.

The expression of Fig. 1 I.PSCA variant.(1I (a)) design primer is to distinguish variant PSCAv.1/v.2/v.4, PSCAv.3 and PSCAv.5.Primer A and B represent that they cause producing the 425bp product of following PCR product: PSCAv.1/v.2/v.4, the 300bp product of PSCAv.3, the 910bp product of PSCAv.5 by the small arrow on the exon among the figure.(1I (b)) prepares the first chain cDNA from healthy tissues and cancer sample library; Said healthy tissues is bladder, brain, heart, kidney, liver, lung, prostate gland, spleen, Skelettmuskel, testis, pancreas, colon, stomach, and said cancer is prostate cancer, bladder cancer, kidney, colorectal carcinoma, lung cancer, ovarian cancer, mammary cancer, metastasis of cancer (cancer metastasis) and carcinoma of the pancreas.Carry out PCR with the Actin muscle primer, to realize normalization method.Use the variant Auele Specific Primer, take turns amplification cycles with 30 and carry out half-quantitative PCR.The result shows that PSCAv.5 mainly expresses in mammary cancer, metastasis of cancer and carcinoma of the pancreas, and the expression level in colorectal carcinoma and lung cancer is lower.PSCAv.1/v.2/v.4 PCR product detects in prostate cancer, bladder cancer, kidney, colorectal carcinoma, lung cancer, ovarian cancer, mammary cancer, metastasis of cancer and carcinoma of the pancreas.In healthy tissues, PSCAv.1/v.2/v.4 PCR product only detects in prostate gland, stomach, and the expression level of this product in kidney and lung is lower, and PSCAv.5 in any healthy tissues, all detect less than.The PCR product of PSCAv.3 in any sample, all detect less than.

The expression of Fig. 1 J.PSCAv.4 and PSCAv.5.(1J (a)) design primer is to distinguish variant PSCAv.4 and PSCAv.5, and said primer is represented by the arrow that is designated as B and C among the figure.The primer that is specific to PSCAv.4 causes producing the PCR product of 460bp, and the primer that is specific to PSCAv.5 causes producing size and is the PCR product of 945bp.(1J (b)) prepares the first chain cDNA from healthy tissues and cancer sample library; Said healthy tissues is bladder, brain, heart, kidney, liver, lung, prostate gland, spleen, Skelettmuskel, testis, pancreas, colon, stomach, and said cancer is prostate cancer, bladder cancer and multiple-heterograft storehouse (multi-xenograft pool) (prostate cancer, kidney and bladder cancer heterograft).Carry out PCR with the Actin muscle primer, to realize normalization method.Use the variant Auele Specific Primer, take turns amplification cycles with 30 and carry out half-quantitative PCR.The result shows that PSCAv.4 expresses in prostate cancer, bladder cancer and multiple-heterograft storehouse, normal kidney and prostate gland.PSCAv.5 only detects in normal prostatic and bladder cancer.

The aminoacid sequence of Fig. 2 .PSCA antibody.The aminoacid sequence of Fig. 2 A.H1-1.10VH.Underscore be CH.The aminoacid sequence of Fig. 2 B.H1-1.10VL.Underscore be constant region of light chain.

The Nucleotide and the aminoacid sequence of Fig. 3 .PSCA antibody.The cDNA of Fig. 3 A.H1-1.10VH and aminoacid sequence.Underscore be the CH part.The cDNA of Fig. 3 B.H1-1.10VL and aminoacid sequence.Underscore be the constant region of light chain part.

Fig. 4 .PSCA antibody and the sequence alignment of planting (germline) V-D-J of system sequence.The sequence alignment of Fig. 4 A.H1-1.10VH and people VH4-31.The sequence alignment of Fig. 4 B.H1-1.10VL and people O2.

The expression of Fig. 5 .PSCA albumen in reorganization mouse (murine), rat and human cell line.With carrying the retrovirus of people PSCA cDNA and neomycin resistance gene or only carrying mouse, rat and the human cell line shown in the contrast virus infection of neomycin resistance gene.Stable recombinant cell lines when being chosen in G418.With 1G8 anti--PSCA MAb (5 μ g/ml) carries out FACS dyeing, measures psca expression.The FACS distribution plan of each clone shown in the figure only has the fluorescence of expression cell surface psca expression to change in the clone that is infected by PSCA.These clones can be used as immunogen and the MAb screening reagent is used for the MAb exploitation, and can be used for function test.

Fig. 6. purifying PSCA albumen from intestinal bacteria.PET-21b carrier transformed into escherichia coli bacterial strain BL21 pLysS with the PSCAcDNA that carries coded amino acid 21-94.Induce the logarithmic phase culture to express PSCA albumen, through affinity chromatography purifying PSCA albumen from the solvable or soluble level of bacterial lysate is divided with IPTG.What show among the figure is that elutriated fraction is through the SDS-PAGE of Coomassie blue stain gel.This protein can be used as MAb and pAb immunogen and can be used as antibody (Ab) screening reagent.

Fig. 7. purifying is by the recombinant glycosylated PSCA albumen of 293T cell expressing.PsecTag2 carrier transfection 293T cell with the PSCA cDNA that carries coded amino acid 28-100.Through obtain the clone of stable secretion reorganization PSCA with the HYG screening.Carry out affinity chromatography with 1G8 MAb, with the PSCA albumen in the culture (conditioned culture) of purifying process cultivation.Low pH elutriated fraction is through the SDS-PAGE of Coomassie blue stain gel shown in the figure.This albumen has the conditions of streaking (smear) of wide range of molecular weights, shows reorganization PSCA protein glycosylation, and this also is found in the PSCA of endogenous expression.

Fig. 8. purifying GST-PSCA albumen from intestinal bacteria.With the pGEX-2T transformed into escherichia coli bacterial strain BL21 DE3 of coding with the PSCA amino acid/11 8-98 of glutathione-S-transferase (GST) fusion.GST-PSCA albumen is induced from the logarithmic phase culture with sec.-propyl-β-D-sulfo-gala pyranoside (IPTG), carries out affinity chromatography with purifying from bacterial lysate with glutathione agarose matrix.What show among the figure is to contain the gsh elutriated fraction of GST-PSCA through the SDS-PAGE of Coomassie blue stain gel.Complete GST-PSCA fusion rotein and less important (minor) degraded product that contains GST have been shown among the figure.This protein can be used as MAb and pAb immunogen and can be used as the Ab screening reagent.

Fig. 9. through FACS screening people psca antibody.Measure the AC in the supernatant through ELISA.In 96 hole FACS plates, add 50 μ l/ holes ((neat) of not diluted) antibody, and carry out serial dilution.Add the cell (endogenic or reorganization, 50,000 cells/well) of expressing PSCA, mixture is incubated 2 hours in 4 ℃.After the insulation, cell detects antibody with 100 μ l again in 4 ℃ and (resists-hIgG-PE) be incubated 45 minutes with the washing of FACS damping fluid.When insulation finished, cell washed with the FACS damping fluid, with formaldehyde fixed and use FACScan to analyze.Use CellQuest Pro software analysis data.Solid histogram is represented the data of negative control antibody, and hollow histogram is represented the data of PSCA-positive cell.

Figure 10. through the H1-1.10 internalization on the detected PC3-PSCA cell of facs analysis.Use the internalization of PC3-PSCA cell research H1-1.10.At first cell is incubated 90 minutes, so that antibody combines with cell surface in 4 ℃.Then cell is divided into two groups, one group places 37 ℃ of insulations so that the antibody internalization, and one group places 4 ℃ with comparing (not internalization).After 37 ℃/4 ℃ insulations, use pickling to peel off and cell surface bonded H1-1.10.Use and change termination (permeablization stop) thoroughly so that detection antibody combines with the H1-1.10 of internalization.After the insulation of detection antibody, cell is analyzed with FACS, calculates average fluorescent strength.The result shows: 37 ℃ the insulation 2 hours after, 40%H1-1.10 is by internalization.

Figure 11. the PC-3 cell that will after transforming, express neomycin resistance gene (neo) or PSCA is laid on 96 hole tissue culture wares in triplicate.Cell attachment is spent the night; Remove substratum then, replace with fresh culture, anti--PSCA MAb H1-1.10 of concentration shown in wherein containing or H1-1.10 and 3 times excessive with (the Advanced Targeting Systems of SA shown in the sapotoxin albumen coupling; San Diego, CA).With cell insulation 4 days, measure survival per-cent with MTT test (Promega Corp).Psca antibody H1-1.10 is specific, does not kill and wound the cell (A figure) of not expressing PSCA because it does not mediate.When being incubated with the SA in identification people Fc district rather than with the SA of discerning goat antibody Fc district, the H1-1.10 mediated cell kills and wounds (B figure).

Figure 12. the LNCap cell that will after transforming, express neomycin resistance gene (neo) or PSCA is laid on 96 hole tissue culture wares in triplicate.Cell attachment is spent the night; Remove substratum then; Replacement is with fresh culture; Anti--PSCA MAb H1-1.10 of concentration shown in wherein containing or H1-1.10 and 3 times of excessive anti-people's SAs or with anti-people's SA of sapotoxin albumen coupling (Advanced TargetingSystems, San Diego, CA).With cell insulation 4 days, measure survival per-cent with MTT test (Promega Corp).Psca antibody H1-1.10 is specific, does not kill and wound the LNCap cell of not expressing PSCA because it does not mediate.

Figure 13. the complement-mediated type cytotoxicity of anti--H1-1.10 antibody.H1-1.10 (0-50 μ g/ml) dilutes with RHB damping fluid (RPMI 1640, Gibco Life Technologies, 20mM HEPES).The cell of expressing B300.19-PSCA is with the washing of RHB damping fluid, with 10 ⁶The density of individual cell/ml is resuspended.In typical test, (Cedarlane, Ontario Can) add flat tissue culture 96 orifice plates with 50 μ l cell suspending liquids together with 50 μ l psca antibodies, the diluted rabbit complement of 50 μ l serum.In 37 ℃, at 5%CO ₂In the incubator mixture is incubated 2 hours so that the lysis of complement-mediation.(Biosource Intl.Camarillo CA), in 37 ℃, continues insulation 4-5 hour in each hole, to add 50 μ l Alamar Blue.Use 96 hole photofluorometers, read the fluorescence in each hole with the exciting light of 530nm and the emission light of 590nm.The result shows: H1-1.10 (people Ig1) can mediate the target cell cracking that depends on complement, and contrast human IgG1 (H3-1.4) can not.

The subcutaneous growth of people's male sex hormone-independent form prostate cancer heterograft of SCID mouse is transplanted in Figure 14 .PSCA MAb H1-1.10 inhibition.With people's male sex hormone-independent form prostate tumor cells UGB-1 (2.0 * 10 ⁶Individual cell/mouse) subcutaneous injection to male SCID mouse.With mouse random packet (every group of 10 mouse), as shown in the figure at the 0th day begin treatment, intraperitoneal (i.p.) injection H1-1.10, PBS or contrast human IgG1 MAb.Give the animal two treatments weekly, till the 16th research day, 5 dosage of administration altogether.As shown in the figure, every at a distance from 3 to 4 days, use calipers to measure tumor growth.Gross tumor volume is pressed width ²Calculate * length/2, and wherein width is minimum size (dimension), and length is maximum size.The result shows: and the people is anti--and PSCA monoclonal antibody H1-1.10 can significantly suppress to be transplanted to the growth (p＜0.01 is proposed through the Kruskal-Wallis check and being confirmed) of the subcutaneous human prostata cancer heterograft of SCID mouse.

Figure 15 .PSCA MAb H1-1.10 is suppressed at the subcutaneous growth of the people's male sex hormone-independent form prostate cancer heterograft of setting up in the SCID mouse.With people's male sex hormone-independent form human prostate tumour cell UGB-1 (1.5 * 10 ⁶Individual cell/mouse) subcutaneous injection to male SCID mouse.When tumour reaches about 70mm ³The time, mouse random packet (every group of 10 mouse) are as shown in the figure at the 0th day begin treatment, intraperitoneal (i.p.) injection H1-1.10 or vehicle Control.Give the animal two treatments weekly, till the 21st research day, 7 dosage of administration altogether.As shown in the figure, every at a distance from 3 to 4 days, use calipers to measure tumor growth.Gross tumor volume is pressed width ²Calculate * length/2, and wherein width is minimum size, and length is maximum size.The result shows: and the people is anti--and PSCA monoclonal antibody H1-1.10 can significantly suppress to be transplanted to the growth (p＜0.05 is confirmed through Student ' st-check) of the subcutaneous human prostata cancer heterograft of having set up of SCID mouse.

The subcutaneous growth of people's male sex hormone-dependent form prostate cancer heterograft of SCID mouse is transplanted in Figure 16 .PSCA MAb H1-1.10 inhibition.With people's male sex hormone-dependent form tumour cell LAPC9-AD (2.0 * 10 ⁶Individual cell/mouse) subcutaneous injection to male SCID mouse.With mouse random packet (every group of 10 mouse), as shown in the figure at the 0th day begin treatment, intraperitoneal (i.p.) injection H1-1.10 or contrast.Give the animal two treatments weekly, till the 18th research day, 6 dosage of administration altogether.As shown in the figure, every at a distance from 3 to 4 days, use calipers to measure tumor growth.Gross tumor volume is pressed width ²Calculate * length/2, and wherein width is minimum size, and length is maximum size.The result shows: and the people is anti--and PSCA monoclonal antibody H1-1.10 can significantly suppress to be transplanted to the growth (p＜0.01 is confirmed through the Kruskal-Wallis check) of the subcutaneous human prostata cancer heterograft of SCID mouse.

The subcutaneous growth of the human pancreas cancer heterograft of SCID mouse is transplanted in Figure 17 .PSCA MAb H1-1.10 inhibition.With human pancreas's tumour cell HPAC (3.0 * 10 ⁶Individual cell/mouse) subcutaneous injection to male SCID mouse.With mouse random packet (every group of 10 mouse), as shown in the figure at the 0th day begin treatment, intraperitoneal (i.p.) injection H1-1.10 or contrast human IgG1.Give the animal two treatments weekly, till the 18th research day, 5 dosage of administration altogether.As shown in the figure, every at a distance from 3 to 4 days, use calipers to measure tumor growth.Gross tumor volume is pressed width ²Calculate * length/2, and wherein width is minimum size, and length is maximum size.The result shows: and the people is anti--and PSCA monoclonal antibody H1-1.10 can suppress to be transplanted to the growth (p＜0.05, check through Student ' s t-definite) of the subcutaneous human pancreas cancer heterograft of SCID mouse.

The subcutaneous growth of the human bladder cancer heterograft of SCID mouse is transplanted in Figure 18 .PSCA MAb H1-1.10 inhibition.With human bladder cancer cell SW780 (2.0 * 10 ⁶Individual cell/mouse) subcutaneous injection to male SCID mouse.With mouse random packet (every group of 10 mouse), as shown in the figure at the 0th day begin treatment, intraperitoneal (i.p.) injection H1-1.10, PBS or contrast human IgG1 MAb.Give the animal two treatments weekly, till the 18th research day, 6 dosage of administration altogether.As shown in the figure, every at a distance from 3 to 4 days, use calipers to measure tumor growth.Gross tumor volume is pressed width ²Calculate * length/2, and wherein width is minimum size, and length is maximum size.The result shows: and the people is anti--and PSCA monoclonal antibody H1-1.10 can suppress to be transplanted to the growth (p＜0.05, check through Dunnett definite) of the subcutaneous SW780 human bladder cancer heterograft of SCID mouse.

Detailed Description Of The Invention

The chapters and sections general introduction

I.) definition

II.) PSCA polynucleotide

II.A.) purposes of PSCA polynucleotide

II.A.1) monitoring gene is unusual

II.A.2) antisense embodiment

II.A.3.) primer and primer are right

II.A.4.) nucleic acid molecule of separation coding PSCA

II.A.5.) recombinant nucleic acid molecules and host-vector system

III.) PSCA-related protein

III.A.) carry the proteinic embodiment of primitive

III.B.) expression of PSCA-related protein

III.C.) modification of PSCA-related protein

III.D.) purposes of PSCA-related protein

IV.) psca antibody

V.) PSCA cell immune response

VI.) PSCA transgenic animal

VII.) method of detection PSCA

VIII.) method of the state of monitoring PSCA-genes involved and product thereof

IX.) identify and the interactional molecule of PSCA

X.) Treatment and composition for

X.A.) anti-cancer vaccine

X.B.) PSCA is used as the target based on the therapy of antibody

X.C.) PSCA is as the target of cell immune response

X.C.1. minigene vaccine

The combination of X.C.2.CTL peptide and auxiliary peptide

The combination of X.C.3.CTL peptide and T cell initiator

X.C.4. the vaccine composition that contains the DC of useful CTL and/or htl peptide pulse

X.D.) adoptive immunotherapy

X.E.) use vaccine for the purpose of treating or prevent

X.I.) diagnosis of PSCA and prediction embodiment

XII.) function of inhibition psca protein

XII.A.) suppress PSCA with intracellular antibody

XII.B.) suppress PSCA with recombinant protein

XII.C.) suppressing PSCA transcribes or translates

XII.D.) generality of relevant therapeutic strategy is considered

XIII.) evaluation of PSCA modulator, sign and application

XIV.) therepic use of RNAi and siRNA (siRNA)

XV.) test kit/goods

I.) definition

Except as otherwise noted, all T.T.s used herein, note and other scientific terminology or specific term and person of ordinary skill in the field of the present invention general understand equivalent in meaning.In some cases, quote in order to clearly demonstrate and/or to be easy to, this paper has defined the term with known implication, and the content of definition described herein does not have the different of what essence with the general meaning of understanding in this area.A lot of technology and method that this paper described or quoted are understandable; Those skilled in the art use conventional methodology; Sambrook et al. for example, Molecular Cloning:A Laboratory Manual 2nd.edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor; N.Y. the molecular cloning method that is widely used described in is learned, and can use these technology and method prevailingly.Except as otherwise noted, can comprise prevailingly using being purchased test kit and compositions and methods according to the method and/or the parameter of manufacturer's definition in the time of suitably.

Term " advanced prostate cancer ", " local advanced prostate cancer ", " terminal illness " and " local terminal illness " refer to the prostate cancer that has been spread by prostatic utriculus, comprise T3-T4 and N+ phase disease under C phase disease, the C1-C2 phase disease under the Whitmore-Jewett system and TNM (tumour, lymphoglandula, the transfer) system under the system of U.S. uropoiesis association (AUA).Generally speaking, do not recommend local terminal illness patient and carry out surgical operation, the patient of the prostate cancer of location (being confined to organ) compares with suffering from clinically, and these patients' result is far short of what is expected.Clinically, the characteristics of local terminal illness are on the prostate gland outer ledge, can touch hard thing, and perhaps prostate gland substrate top is asymmetric or have hard thing to exist.At present,, diffuse to the operation edge or invade seminal vesicle, can after radical prostatectomy, carry out pathological diagnosis local advanced prostate cancer if tumour is invaded or infiltrated prostatic utriculus.

" changing Natively glycosylated pattern " among this paper refers to the one or more sugared moity that exists in (glycosylation site through removing the basis that constitutes glycosylation or eliminate glycosylation through chemistry and/or Enzymology method) disappearance natural PS CA sequence, and/or adds the non-existent glycosylation site of script in one or more natural PS CA sequences.In addition, this term comprises the qualitative variation of natural protein glycosylation, comprises the variation of existing different sugar moity character and ratio.

Term " analogue " refers to molecule (like the PSCA-related protein) similar with another kind of molecular structure or that have similar or individual features.For example, the proteic analogue of PSCA can be combined the antibody of PSCA or T cell-specific to combine by specificity.

Only if clearly demonstrate in addition, the implication of term " antibody " is the most wide in range.Therefore, " antibody " can be natural or artificial preparation, the monoclonal antibody that for example produces through conventional hybridization knurl technology.Anti--fragment that psca antibody comprises mono-clonal and polyclonal antibody and contains antigen-binding domains and/or one or more complementary determining regions of these antibody.Term used herein " antibody " refers to and can specificity combine PSCA and/or show required bioactive any type of antibody or its fragment; It covers monoclonal antibody (comprising full length monoclonal antibodies), polyclonal antibody, multi-specificity antibody (like bi-specific antibody) and antibody fragment especially, as long as they can specificity combine PSCA and/or show required biological activity.Any specific antibody all can be used for the method and composition that this paper provides.Therefore; In one embodiment; Term " antibody " comprises a kind of molecule, and it contains at least one variable region that derives from light chain immunoglobulin molecule and derives from the variable region of heavy chain molecule with at least one, and they form the specific binding site of target antigen altogether.In one embodiment, antibody is IgG antibody.For example, antibody is IgG1, IgG2, IgG3 or IgG4 antibody.Can in cell culture, phage or include but not limited to produce in the multiple animal of milk cow, rabbit, goat, mouse, rat, hamster, cavy, sheep, dog, cat, monkey, chimpanzee, ape can be used for the antibody in the inventive method and the compsn.Therefore, in one embodiment, antibody of the present invention is Mammals antibody.Can use initial antibody of phage technology separation or generation to have through the specificity of change or the variant of avidity characteristic.This technology is conventional and a well-known technology in this area.In one embodiment, produce antibody through recombinant technology known in the art.For example, through producing recombinant antibodies with the carrier transfection host cell of the dna sequence dna that contains encoding antibody.Can use dna sequence dna transfection that one or more carriers will express at least one VL and a VH district to host cell.The technology that produces and produce antibody about reorganization can be referring to for example: Delves, ANTIBODY PRODUCTION:ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, et al., MONOCLONAL ANTIBODIES (OxfordUniversity Press, 2000); Goding, MONOCLONAL ANTIBODIES:PRINCIPLES AND PRACTICE (Academic Press, 1993); CURRENTPROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, latest edition).Can modify antibody of the present invention so that the effectiveness of antibody-mediated required function becomes stronger through recombinant technology.Therefore, use recombinant technology also to fall within the scope of the present invention through replacing the antibody of modifying.Replace normally conservative the replacement.For example, at least one amino acid in the antibody constant region can be substituted by different residues.Referring to for example USP 5,624,821, USP 6,194,551, WO 9958572; And Angal, etal., Mol.Immunol.30:105-08 (1993).Modification in the amino acid comprises amino acid whose disappearance, interpolation, replacement.In some cases, carry out this change and be in order to reduce unnecessary activity, for example the cytotoxicity of complement-dependent form.Often can provide the material that to survey signal to come traget antibody through covalently or non-covalently connecting.Multiple mark and coupling technology are known, existing wide coverage in science and technology and the patent documentation.Can screen these antibody to combine normal or defective PSCA.Referring to for example ANTIBODY ENGINEERING:A PRACTICAL APPROACH (Oxford UniversityPress, 1996).Following external can identifying with in vivo tests has required bioactive suitable antibody; Said in vitro tests includes but not limited to: propagation, migration, adhesion, soft agar growth, blood vessel generation, cell-cell communication, apoptosis, transhipment, signal transduction, in vivo tests is as suppressing tumor growth.The antibody that this paper provides also can be used for diagnostic uses.As catching or non--neutralizing antibody, can screen them and combine with specific antigens but can not suppress antigenic acceptor-combination or bioactive ability.As neutralizing antibody, antibody can be used for CBA.They also can be used for quantitative PSCA or its acceptor.

" antibody fragment " be defined as can with its target bonded partial immunity globulin molecule variable region, i.e. antigen-land at least.In one embodiment, it cover especially single anti--psca antibody and clone's (comprising agonist, antagonist and neutralizing antibody) thereof with have that multi-epitope is specific to be resisted-the psca antibody compsn.Antibody in the inventive method and the compsn can be mono-clonal or polyclonal antibody.Antibody can be and antigen bonded antibody fragment form, comprises Fab fragment, F (ab ') ₂Fragment, strand variable region etc.Can use method well-known in the art to produce the fragment of complete molecule, comprise enzymatic digestion and recombinant technology.

Any type of " antigen " used herein all can be used for producing the antibody that is specific to PSCA.Therefore, causing antigen can be single epi-position, a plurality of epi-position or the combination that is merely complete protein or whole protein and one or more immunogenicity tougheners known in the art.Causing antigen can be isolating full length protein, cell surface proteins (carrying out immunity as using by at least a portion antigen cells transfected) or soluble protein (as only partly carrying out immunity with proteinic ectodomain).Can in the cell of genetic modification, produce antigen.The DNA of coding for antigens can be genome or non--genomic (like cDNA), and coding at least a portion ectodomain.Term used herein " part " refers to the minimized number amino acid or the nucleic acid of the required immunogenicity of antigens epi-position of lucky formation.Any genophore that is suitable for transforming required cell be can use, adenovirus carrier, plasmid and non--virus vector included but not limited to, like cation lipid.In one embodiment, the antibodies specific in the method and composition of the present invention combines at least a portion of the ectodomain of required PSCA.

The antibody that this paper provides or its Fab can with " biologically active agent " coupling.Term used herein " biologically active agent " refers to any ability conjugated antigen and/or enhancing or mediates the synthetic or natural compounds of the biological action of desirable enhancing cell killing toxin.

In one embodiment, can be used for binding fragment of the present invention is that bioactive fragment is arranged.Term used herein " has biological activity " and refers to antibody or antibody fragment capable to combine required epitope and can directly or indirectly bring into play biological action.Directly effect includes but not limited to modulation, stimulates and/or suppresses growth signals; Modulation, stimulation and/or inhibition resist-the apoptosis signal; Modulation, stimulate and/or suppress apoptosis or downright bad signal, modulation, stimulate and/or suppress ADCC cascade and modulation, stimulation and/or suppress the CDC cascade.

" dual specific " antibody also can be used for method and composition of the present invention.Term used herein " bi-specific antibody " refers to the antibody that at least two different antigens epi-positions is had binding specificity, generally is monoclonal antibody.In one embodiment, epi-position derives from identical antigen.In another embodiment, epi-position derives from two different antigens.The method for preparing bi-specific antibody is known in the art, for example, and through being total to-expressing two heavy chain immunoglobulin/light chains to the generation bi-specific antibody of can recombinating.Referring to for example Milstein et al., Nature 305:537-39 (1983).Perhaps, use chemical bond to prepare bi-specific antibody.Referring to for example Brennan, et al., Science 229:81 (1985).Bi-specific antibody comprises bispecific antibody fragment.Referring to for example Hollinger, et al., Proc.Natl.Acad.Sci.U.S.A.90:6444-48 (1993), Gruber, et al., J.Immunol.152:5368 (1994).

The monoclonal antibody of this paper comprises " chimeric " antibody especially; Wherein a part of heavy chain and/or light chain with derive from specific species or belong to the identical or homology of corresponding sequence in the antibody of antibodies specific class or subclass; And all the other chains with derive from another species or belong to the identical or homology of corresponding sequence in the antibody of another antibody class or subclass; The fragment that also comprises said antibody is as long as they can specificity combine target antigen and/or show required biological activity (USP 4,816; 567 with Morrison et al., Proc.Natl.Acad.Sci.USA 81:6851-6855 (1984)).

Term " chemotherapeutics " refers to all compounds that can effectively suppress tumor growth.The non-limitative example of chemotherapeutics comprises alkylating agent, for example mustargen (nitrogen mustards), ethylenimine compound and alkyl sulfonic ester; Metabolic antagonist, for example folic acid, purine or pyrimidine antagonist; Mitotic inhibitor, the for example compound and the growth factor receptor antagonist of vinca alkaloids (vinca alkaloids) and podophyllotoxin (podophyllotoxin) verivate, cell toxicant microbiotic, destruction or interference DNA expression.In addition, chemotherapeutics comprises cytotoxic agent (as defined herein), antibody, biomolecules and small molecules.

Term " the optimized sequence of codon " refers to through replacing any frequency of utilization and is lower than about 20% codon, to the specific host species and optimized nucleotide sequence.This paper will be except the codon optimization; Through eliminating wrong polyadenylation sequence; Eliminate the exon splicing signal, eliminate transposon-appearance Tumor-necrosis factor glycoproteins and/or optimization GC content and be optimised with the nucleotide sequence of in given host species, expressing and be called " expression enhanced sequence ".

" combinatorial library " is through mixing the set that multiple compound " member " (like reagent) carries out the multiple compound that chemosynthesis or biosynthesizing produce.For example; The linear combination library of compounds is to form through mix the amino acid that a cover is called as the given compound length of having of compound member (being the amino acid number in the polypeptide compound) with each possible mode like polypeptide (like mutain) library.Can synthesize multiple compound (Gallop et al., J.Med.Chem.37 (9): 1233-1251 (1994)) through combined hybrid compound member.

The preparation of combinatorial library and screening are the well-known technology of those skilled in the art.Said combination of compounds library includes but not limited to that peptide library is (referring to for example USP 5,010,175, Furka; Pept.Prot.Res.37:487-493 (1991), Houghton et al., Nature; 354:84-88 (1991)), peptoid (PCT publication number WO91/19735), encoded peptide (PCT publication number WO 93/20242); Biology-oligomer (PCT publication number WO92/00091) at random, benzodiazepine (benzodiazepine) (USP 5,288; 514), diversomer is like NSC 9226, benzodiazepine and dipeptides (Hobbs et al.; Proc.Nat.Acad.Sci.USA 90:6909-6913 (1993)), vinylogous polypeptide (vinylogous peptides) (Hagihara et al., J.Amer.Chem.Soc.114:6568 (1992)); Non-peptide class peptide mimics (Hirschmann et al., J.Amer.Chem.Soc.114:9217-9218 (1992)) with β-D-glucose support, the similar organic synthesis of little library of compounds (Chen et al.; J.Amer.Chem.Soc.116:2661 (1994)), few carbamate (Cho, et al.; And/or peptidyl phosphonic acid ester (Campbell et al., J.Org.Chem.59:658 (1994)) Science 261:1303 (1993)).Generally can be referring to Gordonet al., J.Med.Chem.37:1385 (1994), nucleic acid library is (referring to for example Stratagene, Corp.); PNAG3 library (referring to for example USP 5,539,083), antibody library is (referring to for example Vaughn et al.; Nature Biotechnology 14 (3): 309-314 (1996) and PCT/US96/10287), the carbohydrate library (referring to for example Liang et al., Science 274:1520-1522 (1996) and USP 5,593; 853), and the organic molecule library is (referring to for example benzodiazepine, Baum; C & EN, Jan 18, and page 33 (1993); Isoprenoid (isoprenoid), USP 5,569,588; U 25560 (thiazolidinone) and metathiazole alkane ketone (metathiazanone), USP 5,549,974; Tetramethyleneimine (pyrrolidine), USP 5,525,735 and 5,519,134; Morpholino compounds (morpholinocompound), USP 5,506,337; Benzodiazepine, USP 5,288,514 etc.).

The instrument of preparation combinatorial library can be purchased (referring to for example 357 NIPS, 390 NIPS, AdvancedChem Tech, Louisville KY; Symphony, Rainin, Woburn, MA; 433A, AppliedBiosystems, Foster City, CA; 9050, Plus, Millipore, Bedford, NIA).Develop multiple well-known robot system and be used for the solution phase chemistry.These systems comprise the automatically working station, as by Takeda Chemical Industries, LTD. (Osaka; Japan) the robotization synthesizer of exploitation, and multiple robot system (Zymate H, the Zymark Corporation that utilizes robot arm; Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), the synthetic operation that the simulated scholar of said robot system ability carries out.Above-mentioned any instrument all is applicable to the present invention.(in case of necessity) these instruments are reequiped so that they can be operated by mode as herein described, this point is conspicuous as far as the those of skill in the art of association area.In addition, multiple combinatorial library itself also be can be purchased (referring to for example ComGenex, Princeton, NJ; Asinex, Moscow, RU; Tripos, Inc., St.Louis, MO; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, PA; MartekBiosciences, Columbia, MD etc.).

Term used herein " the conservative replacement " refers to that aminoacid replacement is well known by persons skilled in the art, generally can not change the biological activity of gained molecule.Those skilled in the art it is generally acknowledged that the non--required area at polypeptide carries out single amino acids and replaces and can significantly not change biological activity (referring to for example Watson; Et al.; MOLECULAR BIOLOGY OF THE GENE, The Benjamin/Cummings Pub.Co., p.224 (the 4th edition 1987)).Those preferably listed replacements of giving an example according to Table III (a-b).For example, said change comprises with any other the hydrophobic amino acid of any replacement in Isoleucine (I), Xie Ansuan (V) and the leucine (L); With aspartic acid (D) replace L-glutamic acid (E) otherwise and; With Stimulina (Q) replace l-asparagine (N) otherwise and; And with Serine (S) replace Threonine (T) otherwise and.Other replacement also can be considered to guard, and this depends on environment and its effect in protein three-dimensional structure of specific amino acids.For example, glycocoll (G) and L-Ala (A) often can exchange use, and L-Ala (A) and Xie Ansuan (V) also are like this.Hydrophobic relatively methionine(Met) (M) often can exchange with leucine and Isoleucine and use, and can exchange with Xie Ansuan sometimes and use.Notable feature at amino-acid residue is the position of its electric charge, and Methionin (K) and l-arginine (R) often can exchange use, and the difference of these two amino-acid residue pK is not remarkable.In special environment, other change also can be considered to " guarding " (referring to the Table III (a) of for example this paper; Page 13-15 " Biochemistry " 2 ^NdED.Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei etal., J Biol Chem 1995 May 19; 270 (20): 11882-6).Other replacement also allows, and can confirm these replacements by rule of thumb or according to known conservative replacement.

Term " cytotoxic agent " refers to and can suppress or prevent cell expressing activity, cell function and/or cause the ruined material of cell.Ri, chemotherapeutics and toxin desired to comprise in this term, and the toxin that enzymatic activity is arranged like small molecules toxin or bacterium, fungi, plant or animal-origin comprises its fragment and/or variant.The example of cytotoxic agent includes but not limited to Ao Ruisitading (auristatin), Ao Ruisitading E, duomycin (auromycin), maytansinoid (maytansinoids), yttrium (yttrium), bismuth (bismuth), ricin (ricin), ricin A-chain, Kang Bailaisitading (combrestatin), times ganmycin (duocarmycin), many Luo Sitading (dolostatin), Zorubicin (doxorubicin), daunorubicin (daunorubicin), taxol (taxol), cis-platinum (cisplatin), cc1065, ethidium bromide, MTC (mitomycin), VP (etoposide), teniposide (tenoposide), vincristin (vincristine), vincaleucoblastine (vinblastine), NST-757 (colchicine), dihydroxyl anthracin diketone (dihydroxy anthracin dione), NSC-3053 (actionmycin), diphtheria toxin, PE (PE) A, PE40, toxalbumin (abrin), abrin A chain, modeccin (modeccin) A chain, α-Zhou Qujunsu (alpha-sarcin), sets toxalbumin (gelonin) in vain, silk splits lucky mycin (mitogellin), Restrictocin (retstrictocin), phenomycin (phenomycin), enomycin (enomycin), curcin (curicin), crotin (crotin), calicheamicin (calicheamicin), Sapaonaria offcinalis suppressor factor, glucocorticosteroid, other chemotherapeutics and ri, like At ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹²Or ²¹³, P ³²With ri Lu, comprise Lu ¹⁷⁷Antibody also can with anti--cancer prodrug activating enzyme coupling, said enzyme can change prodrug into its activity form.

Term used herein " bivalent antibody (diabody) " refers to the little antibody fragment with two antigen binding sites, and said fragment is at identical polypeptied chain (V _H-V _L) in contain and light chain variable structural domain (V _L) continuous weight chain variable structural domain (V _H).To such an extent as to can not the paired joint through using between two structural domains on the very short same chain of length, said structural domain can only and produce two antigen binding sites with the complementary structure territory pairing of another chain.Bivalent antibody more fully is described in for example EP 404,097; WO93/11161; With Hollinger et al., Proc.Natl.Acad.Sci.USA 90:6444-48 (1993).

" gene product " is used to represent peptide/protein or mRNA in this article.For example, " gene product of the present invention " is called as " cancer aminoacid sequence ", " cancer protein ", " protein of the listed cancer of Table I ", " cancer mRNA ", " mRNA of the listed cancer of Table I " etc. in this article sometimes.In one embodiment, cancer protein is by the nucleic acid encoding of Fig. 1.Cancer protein can be fragment, or the coded full length protein of the nucleic acid of Fig. 1.In one embodiment, use cancer determined amino acid sequence sequence identity or similarity.In another embodiment, sequence is the coded proteinic natural allele variant of nucleic acid of Fig. 1.In another embodiment, sequence is the sequence variants that this paper describes in detail.

" different conjugate " antibody can be used for method and composition of the present invention.Term used herein " different conjugate antibody " refers to two covalently bound antibody.Can use the currently known methods of synthetic protein chemical field to prepare this antibody, comprise the use linking agent.Referring to for example USP 4,676,980.

" high flux screening " test of measuring existence, shortage, content or other characteristic of specific nucleic acid or protein is that those skilled in the art are well-known.Similarly, also be well-known in conjunction with test and reporter gene assay method.Therefore, for example, USP 5,559,410 disclose proteinic high flux screening method; USP 5,585,639 disclose nucleic acid bonded high flux screening method (being array); And USP 5,576,220 and 5,541,061 discloses the method for high flux screening part/antibodies.

In addition, high throughput screening system can be purchased (referring to for example Amersham Biosciences, Piscataway, NJ; Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc.Fullerton, CA; Precision Systems, Inc., Natick, MA etc.).The entire operation flow process of these systems is generally carried out automatically, comprises the pipetting of all samples and reagent, separatory, regularly insulation the and finally microtest plate in the detector that is applicable to assay method is carried out reading.The system of these moulding provides high throughput and quick start and high-caliber handiness and customizability.The manufacturers of these systems provides the detailed method of operation to multiple high throughput system.Therefore, for example, Zymark company provides technical bulletin, has wherein described the screening system that is used to detect genetic transcription modulation, part combination etc.

Term " homologue " refers to the molecule that homology is arranged with another kind of molecule, for example, has the molecule of same or similar compound residue sequence in the corresponding position.

In one embodiment, the antibody that provides of this paper is " people's antibody ".Term used herein " people's antibody " refers to a kind of like this antibody, and wherein whole in fact light chain and sequence of heavy chain comprise that complementary determining region (CDR) all derives from people's gene.In one embodiment; Through trioma technology, people B-cell technology (referring to for example Kozbor; Et al.; Immunol.Today 4:72 (1983)), EBV transformation technology (referring to for example Cole et al.MONOCLONAL ANTIBODIES AND CANCERTHERAPY 77-96 (1985)) or use phage display (referring to for example Marks et al., J.Mol.Biol.222:581 (1991)) preparation human monoclonal antibodies.In specific embodiments, in transgenic mice, produce people's antibody.The technology of preparation part to whole person's antibody is known in the art, can use any this type of technology.According to a particularly preferred embodiment, but at the complete human antibody sequence of preparation in the transgenic mice of transforming expressing human heavy chain and light chain antibody gene.The description that can produce the transgenic mice of people's antibody about preparation can be referring to for example WO 02/43478 and USP 6,657,103 (Abgenix) and patent familieses thereof.The B cytogamy that makes self energy produce the transgenic mice of required antibody then can produce the hybridoma cell line of antibody continuously with preparation.Referring to for example USP 5,569,825; 5,625,126; 5,633,425; 5,661,016; With 5,545,806; And Jakobovits, Adv.Drug Del.Rev.31:33-42 (1998); Green, et al., J.Exp.Med.188:483-95 (1998).

" human leucocyte antigen (HLA) " or " HLA " be people I class or the main histocompatibility complex of II class (MHC) albumen (referring to for example Stites, et al., IMMUNOLOGY, 8 ^THED., Lange Publishing, LosAltos, CA (1994)).

Term used herein " humanized antibody " refers to the antibody formation that contains non--people (like mouse) antibody sequence and human antibody sequence.Said antibody is chimeric antibody, and it contains the minimum sequence that derives from non--human normal immunoglobulin.Generally speaking; Humanized antibody contains at least one basically; Be generally two entire infrastructure territories in the variable domains, wherein all or whole basically hypermutation rings and non--human normal immunoglobulin corresponding, all or whole basically FR districts be the FR district of human normal immunoglobulin sequence.Humanized antibody is also optional to contain at least a portion constant region for immunoglobulin (Fc), generally contains at least a portion human normal immunoglobulin constant region.Referring to for example Cabilly, U.S. Patent number 4,816,567; Queen et al. (1989) Proc.Nat ' l Acad.Sci.USA 86:10029-10033; With ANTIBODYENGINEERING:A PRACTICAL APPROACH (Oxford University Press 1996).

The term that uses in the context of relevant polynucleotide " hybridization " refers to conventional hybridization conditions; Preference is like the hybridization in 50% methane amide/6 * SSC/0.1%SDS/100 μ g/ml ssDNA; Wherein hybridization temperature is higher than 37 ℃, and the wash temperature in 0.1 * SSC/0.1%SDS is higher than 55 ℃.

Term " isolating " or " biological pure " refer to basically or do not contain in fact usually and the material of the native state material of the component of existence together.Therefore, isolating peptide of the present invention preferably do not contain usually be in situ environment in the relevant material of peptide.For example; When a kind of polynucleotide are separated with the polynucleotide that pollute basically; Can be referred to as " isolating ", the polynucleotide of said pollution are corresponding to the gene except that psca gene or complementary with it, and perhaps coding is except that psca gene product or its polypeptide fragment.Those skilled in the art can easily use the separate nucleic acid method to obtain isolating PSCA polynucleotide.When utilizing physics, machinery or chemical process, when from relevant with PSCA albumen usually cellular component, extracting PSCA albumen, can think that this protein is " isolating ".Those skilled in the art can easily utilize the purification process of standard to obtain isolating PSCA albumen.Perhaps, can prepare isolating protein through chemical process.

Suitable " mark " comprises radionuclide, enzyme, substrate, cofactor, suppressor factor, fluorescence moity, chemoluminescence moity, magnetic-particle etc.Instruct the patent of the use of said mark to comprise USP 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241.In addition, the antibody that provides of this paper can be used as the antigen binding constituents of fluor.Referring to for example Zeytun et al., Nat.Biotechnol. 21: 1473-79 (2003).

Term " Mammals " refers to and is classified as mammiferous any biology, comprises mouse, rat, rabbit, dog, cat, milk cow, horse and people.In one embodiment of the invention, Mammals is a mouse.In another embodiment of the invention, Mammals is the people.

Term " prostate cancer of transfer " and " transfer disease " refer to the prostate cancer that diffuses to regional nodes or far-end site, desire to comprise D phase disease and the TxNxM+ phase disease under the TNM system under the AUA system.Identical with the situation of local advanced prostate cancer, carry out surgical operation generally for the patient who shifts disease, hormone (male sex hormone disengaging) therapy is preferred form of therapy.The patients with prostate cancer that has shifted finally developed into the nullvalent state of male sex hormone in 12 to 18 months that treat after beginning.In 6 months after developing into state for this reason, there is half male sex hormone not to be had the death of reaction approximately.The most common site that prostate cancer shifts is a bone.It often is scleroblast rather than osteolysis (promptly causing reticulated bone to form) that the bone of prostate cancer shifts.Bone shifts and is most commonly in backbone, secondly is femur, pelvis, rib cage, skull and humerus.Other common transfer site comprises lymphoglandula, lung, liver and brain.Generally diagnose the prostate cancer of transfer through the pelvic lymphadenectomy under opening or the laparoscopy, body radioactivity radioisotope scanning, bone roentgenograph X art and/or bone infringement biopsy.

Term used herein " modulator (modulator) " or " test-compound " or " drug candidate " or the semantic term description that is equal to be that any desire is measured expression that it directly or indirectly changes cancerous phenotype or cancer sequence (like nucleic acid or protein sequence); Or the molecule of the ability of the effect of cancer sequence (like signal transduction, genetic expression, protein interaction etc.), for example protein, oligopeptides, little organic molecule, polysaccharide, polynucleotide etc.On the one hand, modulator can in the proteinic effect of cancer of the present invention." neutralization " refers to protein active and is suppressed or blocks, and the effect of pair cell subsequently.On the other hand, modulator is through can the neutralize effect of gene of the present invention and respective egg white matter thereof of the said proteinic level that neutralizes.In preferred embodiments, modulator changes the effector pathway in nucleic acid that this paper provides or protein expression distribution plan or downstream.In one embodiment, modulator suppresses cancerous phenotype, for example makes it to become the healthy tissues fingerprint.In another embodiment, modulator is induced cancerous phenotype.Generally speaking, the multiple mixture that tried of parallel electrophoresis with different reagent concentrations, thus obtain difference reaction to different concns.Generally with one of them concentration as negative control, promptly as zero-dose or be lower than detection level.

Modulator, drug candidate or test-compound comprise a plurality of compounds categories, but are generally organic molecule, be preferably molecular weight greater than 100 to less than about 2,500 daltonian little organic cpds.Preferred small molecules is less than 2000, or less than 1500 or less than 1000 or less than 500 dalton.Drug candidate contains with protein and carries out the necessary functional group of structural interaction, particularly hydrogen bond, generally comprises at least and amine, carbonyl, hydroxyl or carboxyl preferably contains at least two chemical functional groups.Drug candidate often contains by the substituted carbocyclic ring of one or more above-mentioned functional groups or heterocycle structure and/or aromatics or gathers aromatic structure.Modulator also contains biomolecules, for example peptide, carbohydrate, lipid acid, steroid, purine, pyrimidine, verivate, analog and combination thereof.Particularly preferably be peptide.One type of modulator is a peptide, and for example about 5 to about 35 amino acid whose peptides, and preferred about 5 to about 20 amino acid whose peptides, and preferred about 7 to about 15 amino acid whose peptides especially.Preferred cancer modulation protein is a solubility, comprise non--stride the film district, and/or have the terminal Cys of N-to help solvability.In one embodiment, segmental C-end remains free acid, and the N-end is that free amine is to help and the halfcystine coupling.In one embodiment, cancer protein of the present invention and immunogenic drug coupling as herein described.In one embodiment, cancer protein and BSA coupling.Peptide of the present invention, for example having the peptide of preferred length can be interconnection or be connected to produce long peptide/protein with other amino acid.Modulator peptide can be the digest of natural protein mentioned above, peptide or " bias " peptide at random at random.In preferred embodiments, be antibody or its fragment defined herein based on peptide/proteinic modulator.

The cancer modulator also can be a nucleic acid.The nucleic acid modulator can be natural acid, random nucleic acid or " bias " random nucleic acid.For example, can with the digest that uses protokaryon or eukaryotic gene group in to the similar method of method of protein mentioned above.

Term used herein " monoclonal antibody " refers to by a group antibody that obtains of the antibody of homogeneous basically, and promptly except the natural sudden change that possibly have minute quantity, each antibody in the antibody population is identical.Monoclonal antibody is the antibody of the anti-single epitope of high degree of specificity.On the contrary, (polyclone) antibody preparation of routine generally comprises the multiple antibody of anti-(or being specific to) different epi-positions.In one embodiment, in containing the single antigen of a plurality of epitopes, polyclonal antibody contains a plurality of monoclonal antibodies with different epitope specificities, affinity or avidity.Qualifier " mono-clonal " expression derives from the characteristic of the antibody of the antibody population of homogeneous basically, and should not be construed as needs to produce antibody through any specific method.For example, can pass through Kohler et al., the hybridoma method that Nature 256:495 (1975) at first describes, or recombinant DNA method (referring to for example USP 4,816, the 567) preparation of passing through is used for monoclonal antibody of the present invention.Also can use for example Clackson et al., Nature 352:624-628 (1991) and Marks et al., the described technology of J.Mol.Biol.222:581-597 (1991) is separated from phage antibody library " monoclonal antibody ".Measure the combination K of these monoclonal antibodies through ELISA _dUsually be about 1M, more generally be at least about 300nM, generally be at least about 30nM, preferably be at least about 10nM, more preferably be at least about 3nM or higher.

" primitive " in the biological primitive of PSCA-related protein, mentioned refers to any aminoacid profile that forms the protein portion primary sequence; It and particular functionality (protein-protein interaction for example; Protein-DNA interaction etc.) or modify (for example phosphorylation, glycosylation or amidation); Or location (for example secretion sequence, nuclear localization sequence etc.) is relevant, or the sequence that is associated with body fluid or cell immunogenicity.Primitive can be maybe arranging to general some positions relevant with some functions or characteristic of adjacency.In the context of HLA primitive; " primitive " refers to can be by the residue pattern in the peptide of the qualification length of specific HLA molecular recognition; Usually as far as I class HLA primitive; The length of said peptide is about 8 to about 13 amino acid, and as far as II class HLA primitive, the length of said peptide is about 6 to about 25 amino acid.For for each protein of everyone HLA allelotrope coding, generally be different with HLA bonded peptide primitive, difference is the pattern of firsts and seconds anchor residue.Common primitive is listed in the Table V.

" drug excipient " comprises the material that is described below: adjuvant, carrier, pH-regulator and buffer reagent, osmotic pressure regulator, wetting agent, sanitas etc.

" medicine can be accepted " refer to nontoxic, inertia and/or with the compsn of people or other Mammals physical compatibility.

Term " polynucleotide " refers to the nucleotide polymer form that length is at least 10 bases or base pair, and it can be the modified forms of ribonucleotide or deoxynucleotide or any Nucleotide, and it comprises the DNA and/or the RNA of strand and double chain form.In the art, this term often exchanges with " oligonucleotide " and uses.Polynucleotide can contain nucleotide sequence as herein described, and thymidine wherein shown in Figure 1 (T) also can be a uridylic (U); This definition is relevant with difference between DNA and the RNA chemical structure, is that uridylic (U) rather than this observations of thymidine (T) are relevant especially with one of 4 main bases among the RNA.

Term " polypeptide " refers to and is at least about 4,5,6,7 or 8 polymer of amino acid.In whole specification sheets, used the trigram or the single-letter amino acid title of standard.In the art, this term often exchanges with " peptide " or " protein " and uses.

HLA " one-level anchor residue " is the amino acid of peptide sequence specific location, and it can provide the point of contact between immunogenic peptide and the HLA molecule.Generally will limit inner 1 to 3 of the peptide of length, be generally " primitive " that 2 one-level anchor residues are defined as immunogenic peptide.According to thinking that these residues are fit to contact closely with the peptide binding groove of HLA molecule, its side chain is buried in the special bag that combines ditch.For example, in one embodiment, be positioned at of the present invention 8,9,10,11 or the 2nd (from the N-terminal position) and the position of C-terminal of 12 residue peptide epi-positions to the one-level anchor residue of I class HLA molecule.Perhaps, in another embodiment, with II class HLA molecule bonded peptide one-level anchor residue each interval, rather than be positioned at the end of peptide, wherein the length of peptide generally is at least 9 amino acid.Having listed the one-level anchor position of each primitive and hyper-base unit in the Table IV (a) puts.For example, through changing the existence or the shortage of the specific residue in one-level shown in the Table IV and/or the secondary anchor position, can produce similar peptide.Can use said similar peptide to modulate to contain binding affinity and/or colony's coverage of the peptide of specific HLA primitive or hyper-base unit.

" ri " includes but not limited to following example (Table IV (I) has listed file names with the example of non-limiting purposes).

Used " randomization " or the semantic speech that is equal to referred to when this paper mentioned nucleic acid and protein: every kind of nucleic acid and protein are made up of in fact at random Nucleotide and amino acid respectively.These peptides (or nucleic acid as herein described) at random can mix any Nucleotide or amino acid in any position.Can design compound method producing randomized protein or nucleic acid, thereby on sequence length, form all or most of possible combination, and then form randomized candidate's biological activity protein agent library.

In one embodiment, the library is " completely randomization ", does not all have sequence preference or constant sequence in any position.In another embodiment, the library is " bias at random " library.That is, some positions in the sequence remain unchanged, or have only a limited number of possibility.For example; Nucleotide or amino-acid residue are only in limited classification, and for example randomization in the residue of hydrophobic amino acid, hydrophilic residue, the bias of having living space (little or big) is used to produce the nucleic acid binding domains; Produce the halfcystine of crosslinked usefulness; The proline(Pro) that the SH-3 structural domain is used, Serine, Threonine, tyrosine or Histidine etc. that phosphorylation site is used perhaps have preference etc. to purine.

" reorganization " DNA or RNA molecule are at external DNA or the RNA molecule that carried out molecule manipulation.

Term used herein " strand Fv " or " scFv " or " strand " antibody refer to the V that contains antibody _HAnd V _LThe antibody fragment of structural domain, wherein these structural domains are present on the single polypeptied chain.The Fv polypeptide is generally at V _HAnd V _LAlso contain peptide linker between the structural domain, thus make sFv form required can with antigen bonded structure.The summary of relevant sFv, referring to Pluckthun, THE PHARMACOLOGY OFMONOCLONAL ANTIBODIES, vol.113, Rosenburg and Moore eds.Springer-Verlag, New York, pp.269-315 (1994).

The non-limitative example of " small molecules " comprises with PSCA and combining or interactional compound that part comprises hormone, neuropeptide; Chemokine; Taste-additive (odorant), phosphatide, and their function equivalent that can combine and preferably can suppress the psca protein function.Said non-limiting micromolecular molecular weight is preferably less than about 10kDa, is more preferably less than about 9, about 8, about 7, about 6, about 5 or about 4kDa.In some embodiments, small molecules and the relevant or combination with it on physiology of PSCA albumen; There is not said small molecules in the natural pathways metabolism; And/or for non-aqueous solution, the easier water-soluble solution of said small molecules.

Term used herein " specific " refers to antibody and optionally combines with target epitope.Under a given cover condition, through relatively with suitable antigenic combine and with the binding specificity that combines to measure antibody of uncorrelated antigen or antigen mixture.If compare with uncorrelated antigen or antigen mixture, many 2,5,7 times at least of the suitable antigens of antibodies are preferably 10 times, can think that so antibody is specific.In one embodiment, specific antibody be only combine with PSCA antigen and not with uncorrelated antigen bonded antibody.In another embodiment, specific antibody is to combine people PSCA antigen, and debond and PSCA antigen have 70%, 75%, 80%, 85%; 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99% or non--antigenic the antibody of people PSCA of homoamino acid homology more.In another embodiment, specific antibody is to combine people PSCA antigen, in conjunction with mouse PSCA antigen, but combination human antigen's the higher antibody of degree.In another embodiment, specific antibody is to combine people PSCA antigen, in conjunction with primate PSCA antigen, but combination human antigen's the higher antibody of degree.In another embodiment, specific antibody be combine people's PSCA antigen and any non--people PSCA antigen, but combine the higher antibody of degree of human antigen or its any combination.

Those skilled in the art are easy to measure " the tight degree " of hybridization conditions, generally calculate tight degree by rule of thumb according to probe length, wash temperature and salt concn.Generally speaking, long probe needs higher temperature maintenance suitably to anneal, and short probe needs lower temperature.When having complementary strand in the environment that is lower than its melting temperature(Tm), denaturing nucleic acid sequence annealed ability is again generally depended in hybridization.But homology required between probe and the hybridization sequences is high more, and spendable relative temperature is high more.As a result, higher relative temperature tends to make reaction conditions more tight, and lower temperature can make reaction conditions more not tight.About other details and the explanation of the tight degree of hybridization, can be referring to Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

The characteristic of " stringent condition " defined herein or " high stringent condition " is following, but is not limited thereto: (1) utilizes LIS and high-temperature wash, 0.015M sodium-chlor/0.0015M Trisodium Citrate/0.1% sodium lauryl sulphate for example, 50 ℃; (2) in crossover process, use denaturing agent; Like methane amide, for example contain 50% (v/v) methane amide/0.1%Ficoll/0.1% Vinylpyrrolidone polymer of 0.1% bovine serum albumin/contain 750mM sodium-chlor, the 50mM sodium phosphate buffer of 75mM Trisodium Citrate; PH6.5,42 ℃; Or (3) utilize 50% methane amide, 5 * SSC (0.75M NaCl; 0.075M Trisodium Citrate), 50mM sodium phosphate (pH6.8), 0.1% trisodium phosphate, 5 * Denhardt solution, through salmon sperm DNA (50 μ g/ml), 0.1%SDS and 10% dextran sulfate of supersound process; 42 ℃, during washing with 0.2 * SSC (sodium chloride/sodium citrate), 42 ℃ and 50% methane amide; 55 ℃, then in 55 ℃ of 0.1 * SSC that containing EDTA, carry out the washing of height-tight degree." moderate stringent condition " is described in; But be not limited to Sambrook et al.; MolecularCloning:A Laboratory Manual; New York:Cold Spring Harbor Press, 1989, comprise and use tight degree washing soln and the hybridization conditions (for example temperature, ionic strength and %SDS) lower than above-mentioned condition.The example of moderate stringent condition is: containing 1% bovine serum albumin, 0.5M sodium phosphate (pH7.5), 1.25mM EDTA and 7%SDS 5 * SSC (150mM NaCl in 65 ℃; The 15mM trisodium citrate) incubated overnight in the solution; Then in 50 ℃, with 2 * SSC/1%SDS and 0.2 * SSC/0.1%SDS washing filter membrane.Those skilled in the art understand how to adjust temperature, ionic strength etc. to comply with the factor such as probe length etc. in case of necessity.

HLA " hyper-base unit " is by the coded total peptide binding specificity of HLA molecule of two or more HLA allelotrope.Table IV (f) has been listed the whole phenotypic frequency of HLA-super categories in the different crowd.The non-limiting component of a plurality of super categories is following:

A2：A ^*0201，A ^*0202，A ^*0203，A ^*0204，A ^*0205，A ^*0206，A ^*6802，A ^*6901，A ^*0207

A3：A3，A11，A31，A ^*3301，A ^*6801，A ^*0301，A ^*1101，A ^*3101

B7：B7，B ^*3501-03，B ^*51，B ^*5301，B ^*5401，B ^*5501，B ^*5502，B ^*5601，B ^*6701，B ^*7801，B ^*0702，B ^*5101，B ^*5602

B44：B ^*3701，B ^*4402，B ^*4403，B ^*60(B ^*4001)，B61(B ^*4006)

A1：A ^*0102，A ^*2604，A ^*3601，A ^*4301，A ^*8001

A24：A ^*24，A ^*30，A ^*2403，A ^*2404，A ^*3002，A ^*3003

B27：B ^*1401-02，B ^*1503，B ^*1509，B ^*1510，B ^*1518，B ^*3801-02，B ^*3901，B ^*3902，B ^*3903-04，B ^*4801-02，B ^*7301，B ^*2701-08

B58：B ^*1516，B ^*1517，B ^*5701，B ^*5702，B58

B62：B ^*4601，B52，B ^*1501(B62)，B ^*1502(B75)，B ^*1513(B77)

Table IV (g) has been listed the colony's coverage through calculating that different HLA-super categories combinations provide.

" treatment " used herein speech refers to any improvement to any disease consequence with semantic relevant term, and for example survival time prolongs, sickness rate reduces, and/or alleviates the spinoff of accompanying with selected form of therapy; This area is understandable to be: though eradicate disease fully is not that treatment behavior is necessary, but still is preferred.

" transgenic animal " (for example mouse or rat) is to contain genetically modified animal in the cell, and said transgenic is imported into animal or ancestors of (for example embryo stage) animal in utero." transgenic " is the DNA that is integrated in the cellular genome, becomes transgenic animal by said cell development.

HLA used herein or cell immune response " vaccine " are to contain or the compsn of one or more peptides of the present invention of encoding.Said vaccine has a plurality of embodiments, for example the mixture of one or more single peptides; By one or more peptides that multi-epitope peptide comprised; Or the nucleic acid of encode said single peptide or polypeptide, the minigene of the multi-epitope peptide of for example encoding." one or more peptides " comprises from 1 to 150 kind or more kinds of any integer kind peptides, for example is at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18; 19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35; 36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,55,60; 65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145 or 150 kind or more kinds of peptide of the present invention.

Choose wantonly through fatization, interpolation target or other sequence and come modified peptides or polypeptide.I class hla peptide of the present invention can mix or be connected with II class hla peptide, so that Kiwi poison T lymphocyte and helper T cell.The HLA vaccine also can contain the antigen presenting cell of peptide-pulse, for example dendritic cell.

Term " variant " refers to and said type or the discrepant molecule of standard, for example is equipped with the protein of one or more different aminoacids residues in the corresponding positions of specifically described protein (PSCA albumen as shown in Figure 1).Analogue is a routine variant proteins.Montage isotype and SNP (SNP) are another routine variants.

" PSCA-related protein " of the present invention comprises concrete those protein identified of this paper and allele variant, guards replacement variant, analogue and homologue; According to the method that is easy to obtain in the method for this paper general introduction or this area, need not to carry out the over-drastic experiment promptly separable/produce and identify them.Also comprise the fusion rotein that the proteic part of different PSCA or its fragment combination form, and the fusion rotein of PSCA albumen and heterologous polypeptide.Said PSCA albumen is commonly referred to as the PSCA-related protein, protein of the present invention or PSCA.Term " PSCA-related protein " refers to 4,5,6,7,8,9,10,11, and 12,13,14,15,16,17,18,19,20,21,22,23,24,25 or 25 more than the amino acid; Or be 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 a plurality of amino acid whose polypeptide fragment or psca protein sequence.

II.) PSCA polynucleotide

On the one hand; The invention provides and the corresponding or complementary polynucleotide of all or part of psca gene, mRNA and/or encoding sequence (preferably they are unpack format); Comprise coding PSCA-related protein and segmental polynucleotide thereof, DNA, RNA; DNA/RNA heterozygote and associated molecule; With psca gene or mRNA sequence or its part complementary polynucleotide or oligonucleotide, and ability and psca gene, the polynucleotide or the oligonucleotide (being commonly referred to as " PSCA polynucleotide ") of the multi-nucleotide hybrid of mRNA or coding PSCA.Under all situations of in this joint, mentioning, the T among Fig. 1 also can be U.

The embodiment of PSCA polynucleotide comprises: have the PSCA polynucleotide of sequence shown in Figure 1, have PSCA nucleotide sequence shown in Figure 1, wherein T is the PSCA polynucleotide of U; At least 10 PSCA polynucleotide with polynucleotide sequence shown in Figure 1 in abutting connection with Nucleotide; Or have at least 10 of polynucleotide sequence shown in Figure 1 in abutting connection with Nucleotide, wherein T is the PSCA polynucleotide of U.

The coding relatively polynucleotide of long PSCA protein part also falls into scope of the present invention.For example, can produce the polynucleotide of about amino acid/11 (or 20 or 30 or 40 etc.) to about amino acid 20 (or 30, or 40 or 50 etc.) of code pattern 1 or PSCA albumen shown in Figure 3 or " variant " through multiple technologies well-known in the art.These polynucleotide passages can comprise any part of PSCA sequence shown in Figure 1.

II.A.) purposes of PSCA polynucleotide

II.A.1. monitoring gene is unusual

Polynucleotide mentioned above have multiple different concrete purposes.People's psca gene is plotted on the chromosome position shown in the embodiment that is entitled as " chromosome mapping of PSCA ".For example,, can use the polynucleotide of coding PSCA albumen different zones to identify that the cytogenetics of this chromosomal foci is unusual because psca gene is plotted on this karyomit(e), for example be accredited as relevant with multiple cancer unusually.In some genes, the multiple chromosome abnormalty that comprises rearrangement be accredited as common cytogenetics in the multiple various cancers unusual (referring to for example Krajinovic et al., Mutat.Res.382 (3-4): 81-83 (1998); Johansson et al., Blood 86 (10): 3905-3914 (1995) and Finger et al., P.N.A.S.85 (23): 9158-9162 (1988)).Therefore, the polynucleotide of coding PSCA albumen specific region provide new instrument, and it can be used for describing in the chromosomal region of coding PSCA and may causing malignant phenotype's cytogenetics unusual to be superior to accuracy in the past.In this context; These polynucleotide can satisfy the susceptibility of this area expansion karyomit(e) screening; Thereby (referring to for example Evans et al, Am.J.Obstet.Gynecol 171 (4): 1055-1057 (1994)) to identify the demand of trickleer and rarer chromosome abnormalty.

In addition, owing to confirmed PSCA high expression level in prostate cancer and other cancer, psca gene product use PSCA polynucleotide in can estimated normally to the method for the situation in the cancerous tissue.The general polynucleotide of coding PSCA albumen specific region that use are estimated the psca gene specific region, as contain the existence of perturbation in the zone of one or more primitives disappearance, insertion, point mutation or the change of the forfeiture of antigen etc. (as cause).The test of giving an example comprises that RT-PCR test and single strand conformation polymorphism (SSCP) analysis are (referring to for example Marrogiet al.; J.Cutan.Pathol.26 (8): 369-378 (1999)), they all utilize the polynucleotide of coded protein specific region to detect these zones in the protein.

II.A.2. antisense embodiment

Other concrete nucleic acid-related embodiment that the disclosed the present invention of this paper desires to comprise is genomic dna, cDNA, ribozyme and antisense molecule and based on selecting main chain or comprising the nucleic acid molecule that can select base; They can be natural origin or synthetic, and comprise the RNA that can suppress PSCA or the molecule of protein expression.For example, antisense molecule can be RNA or other molecule, comprises PNAG3 (PNA) or non--nucleic acid molecule, for example mode and DNA or the RNA specificity bonded phosphorothioate derivative to depend on base pair.Those skilled in the art use disclosed PSCA polynucleotide of this paper and polynucleotide sequence can easily obtain the nucleic acid molecule of these classifications.

Antisense technology must use can be positioned at the exogenous oligonucleotide of intracellular target polynucleotide bonded.Term " antisense " refers to oligonucleotide target intracellular with it, like complementary this fact of PSCA.Referring to for example Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of GeneExpression, CRC Press, 1989; And Synthesis 1:1-5 (1988).PSCA antisense oligonucleotide of the present invention comprises and shows enhanced cancer cell Growth Inhibition verivate, like S-oligonucleotide (phosphorothioate derivative or S-oligo, referring to Jack Cohen, document is the same).S-oligo (nucleosides thiophosphatephosphorothioate) is the electric analogue such as grade of oligonucleotide (O-oligo), and wherein the non-bridged Sauerstoffatom of phosphate group is replaced by sulphur atom.Through with sulphur transhipment reagent 3H-1,2-benzo dithio-3-ketone-1, the 1-dioxide is handled corresponding O-oligo, can prepare S-oligo of the present invention.Referring to 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).Other PSCA antisense oligonucleotide of the present invention comprises morpholino antisense oligonucleotide known in the art (referring to for example Partridge et al., 1996, Anbsense&NucleicAcid Drug Development 6:169-175).

PSCA antisense oligonucleotide of the present invention generally can be and preceding 100 the 5 ' codons of psca gene group sequence or the RNA or the DNA of back 100 3 ' codons or corresponding mRNA complementation and stable hybridization.Do not need absolute complementation, but preferred high-caliber complementation.Use with the oligonucleotide of this regional complementarity and can optionally hybridize, and do not hybridize with the peculiar mRNA of other regulator subunit of protein kinase with PSCA mRNA.In one embodiment, PSCA antisense oligonucleotide of the present invention is 15 to the 30-segment fraction of polymer that have with the antisense DNA molecule of the sequence of PSCA mRNA hybridization.Optional PSCA antisense oligonucleotide is and preceding 10 the 5 ' codons of PSCA or the 30-aggressiveness oligonucleotide of the regional complementarity in back 10 the 3 ' codons.Perhaps, antisense molecule is modified to utilize ribozyme to suppress the expression of PSCA, referring to for example L. A.Couture D.T.Stinchcomb; Trends Genet12:510-515 (1996).

II.A.3. primer and primer are right

Other specific embodiments of these Nucleotide of the present invention comprises that primer and primer are right; Their can increase specifically polynucleotide of the present invention or its any specific parts; Also comprise probe, they can optionally or specifically be hybridized with nucleic acid molecule of the present invention or its any part.But can use the mark note, for example ri, fluorescent chemicals, bioluminescent compound, chemiluminescence compound, metal chelator or enzyme labelled probe.Said probe and primer can be used for the existence of PSCA polynucleotide in the test sample, and can be used as the instrument that detects the cell of expressing PSCA albumen.

The example of said probe comprises the polynucleotide that contain all or part of people PSCA cDNA sequence shown in Figure 1.The right example of primer of ability specific amplification PSCAmRNA has also been described among the embodiment.Those skilled in the art understand that the sequence that provides based on this paper can prepare various primer and probe, and they are used for amplification effectively and/or detect PSCA mRNA.

PSCA polynucleotide of the present invention can be used for multiple purpose, include but not limited to be used as probe and primer with amplification and/or detection psca gene, mRNA or its fragment; Be used as reagent with diagnosis and/or prediction prostate cancer and other cancer; As the encoding sequence that can mediate the PSCA expression of polypeptides; Instrument as modulation or expression of inhibition psca gene and/or the translation of PSCA transcript; With as therapeutical agent.

The present invention includes and use any probe as herein described from natural origin; As identifying and isolate PSCA or PSCA associated nucleic acid sequences in people or other Mammals; The present invention also comprises isolated nucleic acid sequences itself, and said sequence can contain whole or most of sequences of used probe.

II.A.4. the nucleic acid molecule that separates coding PSCA

PSCA cDNA as herein described can separate the polynucleotide of other coding psca gene product; And separation coding psca gene product homologue; The perhaps polynucleotide of montage isotype, allele variant and the mutant forms of psca gene product, and the polynucleotide that separate the analogue of coding PSCA-related protein.Be used to separate encode the multiple molecular cloning method of full-length cDNA of psca gene be well-known (referring to for example Sambrook, J.et al., Molecular Cloning:ALaboratory Manual; 2d edition; Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology.Ausubel et al., Eds., Wiley and Sons, 1995).For example, can use easily to use and be purchased cloning system (like Lambda ZAP Express, Stratagene) lambda particles phage cloning process is learned.Survey through using, can identify the phage clone that contains psca gene cDNA through the PSCA of mark cDNA or its fragment.For example, in one embodiment, can synthesize PSCA cDNA (like Fig. 1) or its part, and they are used as probe to obtain the overlapping and full-length cDNA corresponding to psca gene.Through with PSCA dna probe or primer screening genome dna library, Bacterial Artificial Chromosome Library (BAC), yeast artificial chromosome library (YAC) etc. can isolate psca gene itself.

II.A.5. recombinant nucleic acid molecules and host-vector system

The present invention also provides and has contained the PSCA polynucleotide; The recombinant DNA of its fragment, analogue or homologue or RNA molecule; Include but not limited to phage, plasmid, phasmid, clay, YAC, BAC and multiple virus well-known in the art and non--virus vector, the present invention also provides by said recombinant DNA or RNA molecule and has transformed or cells transfected.The method that produces said molecule is well-known (referring to for example Sambrook et al, 1989, document is the same).

The present invention also provides the host-vector system that contains recombinant DNA molecules at suitable protokaryon or eukaryotic host cell, and said recombinant DNA molecules contains PSCA polynucleotide, its fragment, analogue or homologue.Suitably the example of eukaryotic host cell comprises yeast cell, vegetable cell or zooblast, like mammalian cell or insect cell (as can be by the cell of baculovirus infection, like Sf9 or HighFive cell).Suitably the example of mammalian cell comprises multiple prostate cancer cell line; Like DU145 and TsuPr1; But the prostate cancer cell line of other transfection or transduction; Primary cell (PrEC), and multiple routine is used for the mammalian cell (like COS, CHO, 293,293T cell) of express recombinant protein matter.More specifically, can use the conventional use in this area and known any host-vector system, use the polynucleotide that contain PSCA encoding sequence or its fragment, analogue or homologue to produce PSCA albumen or its fragment.

Be suitable for expressing PSCA albumen or its segmental multiple host-vector system and be can obtain (referring to for example Sambrook et al, 1989, document is the same; Current Protocols in Molecular Biology, 1995, document is the same).The preferred vector that is used for the Mammals expression includes but not limited to pcDNA 3.1myc-His-tag (Invitrogen) and retroviral vector pSRotkneo (Muller et al., 1991, MCB 11:1785).Use these expression vectors, can for example comprise 293,293T, rat-1 expresses PSCA in several kinds of prostate cancers of NIH3T3 and TsuPrl and the non--prostate cell line.Host-vector system of the present invention can be used for producing PSCA albumen or its fragment.Can use the functional performance of said host-vector system research PSCA and PSCA sudden change or analogue.

Through the construct mammalian cells transfected of the PSCA-associated nucleotide that is encoded, can produce recombinant human PSCA albumen or its analogue or homologue or fragment.For example; Expression plasmid transfection 293T cell with coding PSCA or its fragment, analogue or homologue; In the 293T cell, express the PSCA-related protein, use the purification process (for example using anti--psca antibody to carry out affinity purification) of standard to separate reorganization PSCA albumen.In another embodiment, PSCA encoding sequence subclone to retroviral vector pSR α MSVtkneo, and is used to infect multiple mammal cell line, like NIH 3T3, TsuPrl, 293 and rat-1, thus set up the clone of expressing PSCA.Also can use multiple other expression system well-known in the art.Encode and can be used for producing the reorganization PSCA albumen of secreted form with the expression construct of the interior leading peptide that is connected of PSCA encoding sequence frame.

Like what this paper discussed, variation appears in the Feng Yu property permission psca gene sequence of genetic code.Especially, specific host type known in the art often has special codon preference, can be the sequence of required host's preference with disclosed sequence modification therefore.For example, preferred analogue codon sequence is generally to use the higher codon of frequency to substitute rare codon (promptly frequency of utilization is lower than about 20% codon in required host's known array).For example, utilization can derive from the internet, uses table like the codon on the URL dna.affrc.go.jp/-nakamura/codon.html, calculates the codon preference of specific species.

Known other sequence modification can strengthen the protein expression in the cell host.Said modification comprises removes code error polyadenylation signal, exon splice site signal, transposon-appearance repeating sequences and/or other for people knew to the deleterious sequence of genetic expression.Known with reference to expressing in the host cell calculates, and the GC content of sequence is adjusted into the M.L. of given cell host.If possible, sequence is modified the hair clip secondary mRNA structure to avoid inferring.Other useful modification comprises like Kozak, Mol.Cell Biol., and 9:5073-5080 (1989) is said, adds the translation initiation consensus sequence at the starting point place of ORFs.Those skilled in the art understand the eukaryote rrna only only under rare condition, abolished in this general principle of the initial translation in AUG codon place of contiguous 5 ' (referring to for example Kozak PNAS 92 (7): 2662-2666, (1995) and Kozak NAR 15 (20): 8125-8148 (1987)).

III.) PSCA-related protein

Another aspect of the present invention provides PSCA-related protein.The proteic specific embodiments of PSCA comprises having Fig. 1, the polypeptide of all or part of aminoacid sequence of preferred PSCA that Figure 1A lets others have a look at.Perhaps, the proteic embodiment of PSCA is included in variant, homologue or the analogue polypeptide that has change in the PSCA aminoacid sequence shown in Figure 1.

The embodiment of PSCA polypeptide comprises: the PSCA polypeptide with sequence shown in Figure 1; As shown in Figure 1, wherein T is the PSCA peptide sequence of U; At least 10 with polypeptide of sequence shown in Figure 1 in abutting connection with Nucleotide; Or have as shown in Figure 1ly, wherein T is that at least 10 of polypeptide of sequence of U are in abutting connection with peptide.

Amino acid abbreviations is provided in the Table II.Frequent conserved amino acid replacement can be in protein, carried out and proteinic conformation or function can be do not changed.Protein of the present invention can contain 1,2,3,4, and 5,6,7,8,9,10,11,12,13,14,15 conservative replacements.

The disclosed embodiment of the present invention of this paper comprise multiple PSCA protein variant or the analogue of accepting for this area, for example have aminoacid insertion, disappearance and substituted polypeptide.Use methods known in the art, can prepare the PSCA variant like site-directed mutagenesis, L-Ala scanning and PCR mutagenesis.Can carry out site-directed mutagenesis (Carter et al., Nucl.Acids Res., 13:4331 (1986) to clone's DNA; Zoller et al.; Nucl.Acids Res.; 10:6487 (1987)), box mutagenesis (Wells et al., Gene, 34:315 (1985)), restricted selection mutagenesis (Wells et al.; Philos.Trans.R.Soc.London SerA, 317:415 (1986)) or other known technology to produce PSCA modification D NA.

Also can use scanning amino acid analysis to identify along active, like one or more amino acid of the relevant contiguous sequence of protein-protein interaction with particular organisms.Preferred scanning amino acid is less relatively, neutral amino acid.Said amino acid comprises L-Ala, glycocoll, Serine and halfcystine.Wherein preferred L-Ala because it has eliminated the side chain on the β carbon, unlikely changes the main chain conformation of variant as scanning amino acid.The Another reason of preferred L-Ala is that it is modal amino acid.In addition, the position discovery L-Ala of being covered and exposing through being everlasting (Creighton, The Proteins, (W.H.Freeman Co., N.Y.); Chothia, J.Mol.Biol., 150:1 (1976)).If L-Ala replaces the variant that can not produce q.s, then can use with joining amino acid.

PSCA variant, analogue or homologue defined herein have distinctive feature, they have at least one can with the epi-position of PSCA albumen " cross reaction ", said PSCA albumen has the aminoacid sequence of Fig. 1.Said " cross reaction " refers to PSCA variant specificity bonded antibody or T cell and also can combine with the PSCA protein-specific with aminoacid sequence shown in Figure 1.When polypeptide no longer contained any epi-position that can be discerned with original PSCA protein-specific bonded antibody or T cell, this polypeptide no longer was a proteinic variant shown in Figure 1.Those skilled in the art understand that the antibody of identification of protein combines with the epi-position of different sizes, and rank is about 4 or 5 amino acid whose one group of adjacency or is not considered to the typical amino acid number of minimum epi-position in abutting connection with amino acid.Referring to 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.

The aminoacid sequence of other type PSCA-related protein variant and Fig. 1 or its fragment share 70%, 75%, 80%, 85% or 90% or higher similarity.The PSCA protein variant of another special category or analogue contain the biological primitive of one or more PSCA described herein or known in the art.Therefore, the present invention includes the PSCA fragment analogue (nucleic acid or amino acid) that for original segments, has through function (like the immunogenicity) characteristic that changes.Should understand that primitive described herein or known in the art is applicable to nucleic acid or the aminoacid sequence of Fig. 1.

Like what this paper discussed, embodiment of the present invention comprise polypeptide, and it contains the partial sequence of PSCA albumen full length amino acid sequence shown in Figure 1.For example, representative embodiment of the present invention comprises having PSCA shown in Figure 1 proteic any 4,5,6,7,8,9,10,11,12,13,14,15 or the amino acid whose peptide/protein of more a plurality of adjacency.

The peptide synthetic technology of use standard or use chemical cracking method well-known in the art can produce the PSCA-related protein.Perhaps, can use recombination method to produce the nucleic acid molecule of coding PSCA-related protein.In one embodiment, nucleic acid molecule provides the segmental instrument of qualification of generation PSCA albumen (or its variant, homologue or analogue).

III.A.) carry the proteinic embodiment of primitive

Disclosed other illustrative embodiment of the present invention of this paper comprises the PSCA polypeptide, and it contains the amino-acid residue of contained one or more biological primitives in the PSCA peptide sequence shown in Figure 1.Whether multiple primitive is known in the art, can exist said primitive (referring to for example URL network address: pfam.wustl.edu/ in the assess proteins through a plurality of known internet sites; Searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html; Psort.ims.u-tokyo.ac.jp/; Cbs.dtu.dk/; Ebi.ac.uk/interpro/scan.html; Expasy.ch/tools/scnpsitl.html; Epimatrix ^TMAnd Epimer ^TM, Brown University, brown.edu/Research/TB-HIV_Lab/epimafix/epimatrix.html; And BIMAS, bimas.dcrt.nih.gov/.).

List and identified the subsequence that carries primitive of all PSCA variant proteins among Table V-XVIII and the XXII-LI.

Table IV (h) has been listed several kinds of common primitives based on pfam retrieval (referring to URL network address pfam.wustl.edu/).The abbreviation of (1) primitive title has been listed on each hurdle in the Table IV (h), the identity per-cent that (2) are found in primitive family different members, (3) primitive title or description and (4) modal function; If primitive is relevant with the position, also comprise positional information.

The PSCA primitive that preceding text are discussed is regulated relevant with growth failure; And PSCA crosses expression (referring to for example Table I) in certain cancers; Given this observations can use the polypeptide that contains one or more PSCA primitives that preceding text discuss to illustrate malignant phenotype's particular feature.For example, known casein kinase i I, cAMP and camp dependent form protein kinase and protein kinase C be with the malignant phenotype develop relevant enzyme (referring to for example Chen et al., Lab Invest., 78 (2): 165-174 (1998); Gaiddon etal., Endocrinology 136 (10): 4331-4338 (1995); Hall et al., Nucleic AcidsResearch 24 (6): 1119-1126 (1996); Peterziel et al., Oncogene18 (46): 6322-6329 (1999) and O ' Brian, Oncol.Rep.5 (2): 305-309 (1998)).In addition, glycosylation and myristoylation also are to modify with cancer and cancer progression proteins associated matter that (referring to 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 kind of and cancer and cancer progression proteins associated matter modification (referring to for example Treston et al., J.Nab.CancerInst.Monogr. (13): 169-175 (1992)).

In another embodiment, protein of the present invention contains one or more immunocompetence epi-positions of identifying according to the method for accepting for this area, like the peptide of listing among Table V-XVIII and the XXII-LI.Use special algorithm can confirm the CTL epi-position, can be best and specific HLA allelotrope bonded peptide (Table IV for example thereby identify in the PSCA albumen; Epimatrix ^TMAnd Epimer ^TM, BrownUniversity, URL brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; And BIMAS, URL bimas.dcrt.nih.gov/.).In addition, identifying has enough binding affinities with the HLA molecule and the method for relevant peptide is well-known in the art with becoming the immunogenicity epi-position, need not to test and can implement through over-drastic.In addition, identify that the method for the peptide that can be used as the immunogenicity epi-position is well-known in the art, need not the experiment of process over-drastic and can in external or body, implement this method.

In addition, the principle that produces the analogue of said epi-position in order to modulate immunogenicity known in the art.For example, can begin with the epi-position that carries CTL or HTL primitive (referring to the HLAI class and the HLAII class primitive/hyper-base unit of for example Table IV).Through changing the amino acid of next specific location, with distinctive another aminoacid replacement in this position it, can prepare similar epi-position.For example, the residue that defines with Table IV is the basis, with any other residue, replaces deleterious residue like preferred residue; Replace more not preferred residue with preferred residue; Or with the original preferred residue that occurs of another preferred residue replacement.Put or other position the one-level anchor position that replacement can occur in the peptide; Referring to for example Table IV.

Many pieces of reference have reflected the technology of the epi-position in relevant evaluation and generation desired protein and the analogue thereof.Referring to the for example WO97/33602 of Chesnut et al.; Sette, Immunogenetics 199950 (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 1,997 45 (4): 249-258; Sidney et al., J.Immunol.1996 157 (8): 3480-90; And Falk et al., Nature351:290-6 (1991); Hunt et al, Science 255:1261-3 (1992); Parker et al., J.Immunol.149:3580-7 (1992); Parker et al., J.Immunol.1 52:163-75 (1994); Kastet al, 1,994 152 (8): 3904-12; Borras-Cuesta et al., Hum.Immunol.200061 (3): 266-278; Alexander et al., J.Immunol.2000 164 (3): 1625-1633; Alexanderet al., PMID:7895164, UI:952025 82; O ' Sullivan et al., J.Immunol.1991147 (8): 2663-2669; Alexander et al., Immunity 19941 (9): 751-761 and Alexanderet al., Immunol.Res.1998 18 (2): 79-92.

Related embodiment of the present invention comprises and contains Table IV (a); IV (b); IV (c), IV (d) and the different primitives shown in the IV (h), and/or one or more suppositions CTL epi-position of Table V-XVIII and XXII-LI; And/or one or more suppositions HTL epi-position of Table X LVIII-LI, and/or one or more T cells known in the art combine the polypeptide of the combination of primitives.Preferred embodiment does not contain insertion, disappearance or replaces in primitive or in the intervening sequence of polypeptide.In addition, the either side at these primitives comprises that the embodiment of a plurality of N-ends and/or C-terminal amino acid residue is desirable (for example, comprises the major part of polypeptide structure, wherein include primitive).Usually, be positioned at the N-end of primitive either side and/or the number of C-terminal amino acid residue and be about 1 to 100 amino-acid residue, be preferably 5 to about 50 amino-acid residues.

The present invention includes the PSCA-related protein of various ways (being preferably unpack format).The PSCA protein molecular of purifying is substantially free of other bonded protein or molecule of impairing PSCA and antibody, T cell or other part.Separate and the character of purifying and the purposes that level depends on the desire realization.The embodiment of PSCA-related protein comprises the PSCA-related protein and functional, the soluble PSCA-related protein of purifying.In one embodiment, functional, soluble PSCA albumen or its fragment have kept and antibody, T cell or other part bonded ability.

The present invention also provides the PSCA albumen of the bioactive fragment that contains PSCA aminoacid sequence shown in Figure 1.Said protein shows the proteic characteristic of original PSCA, for example causes the ability that the ability specificity combines original PSCA albumen associated epitope that produces; Can be by antibodies; Can cause HTL or CTL to activate; And/or also can be discerned with urporotein specificity bonded HTL or CTL.

Use multiple analytical technology well-known in the art; Comprise for example Chou-Fasman, Gamier-Robson, Kyte-Doolittle; Eisenberg; Karplus-Schultz or Jameson-Wolf analytical method, or based on immunogenicity can be inferred and/or identify the PSCA-related polypeptide that contains interesting especially structure.The fragment that contains said structure can be used to produce subunit-specific anti-psca antibody or T cell especially, or is used for identifying and PSCA bonded cytokine.For example, use Hopp, T.P.and Woods, K.R., 1981, the method for Proc.Natl.Acad.Sci.U.S.A.78:3824-3828 can produce hydropathy profile and identify immunogenic peptide fragment.Use Kyte, J.and Doolittle, R.F., 1982, the method for J.Mol.Biol.157:105-132 can produce parent/hydrophobicity profile and identify immunogenic peptide fragment.Use Janin J., 1979, the method for Nature 277:491-492 can produce and can reach residue per-cent (%) distribution plan and identify immunogenic peptide fragment.Use Bhaskaran R., Ponnuswamy P.K., 1988, the method for Int.J.Pept.Protein Res.32:242-255 can produce average flexible distribution plan and identify immunogenic peptide fragment.Use Deleage, G., Roux B., 1987, the method for Protein Engineering 1:289-294 can produce β-corner distribution plan and identify immunogenic peptide fragment.

Use special algorithm can confirm the CTL epi-position, can (for example pass through to use SYFPEITHI website www.URLsyfpeithi.bmi-heidelberg.com/ with specific HLA allelotrope bonded peptide best thereby identify in the PSCA albumen; Table IV (A)-(E); Epimatrix ^TMAnd Epimer ^TM, BrownUniversity, URL (brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); And BIMAS, URL bimas.dcrt.nih.gov/.).Illustrated these epi-positions, can infer people MHCI quasi-molecule, like HLA-A1, A2, A3, A11, A24, the PSCA peptide epitopes of presenting in B7 and the B35 environment is (referring to for example Table V-XVIII, XXII-LI).Specifically; Except the SYFPEITHI website that is positioned at URL syfpeithi.bmi-heidelberg.com/; Also with the relevant portion of the proteic complete amino acid sequence of PSCA and other variant (promptly as far as the HLAI quasi-molecule; Supposition has 9 flank residues at the either side of point mutation or in the junction of exon; As far as the HLAII quasi-molecule, inferring has 14 flank residues at the either side of point mutation or in the exon junction corresponding to this variant) import in the hla peptide primitive searching algorithm in Bioinformatics and Molecular Analysis Section mentioned above (BIMAS) website.

Ken doctor Parker according to the combination of particular peptide sequence in the ditch of HLAI quasi-molecule, particularly HLA-A2 work out hla peptide primitive searching algorithm (referring to for example Falk et al., Nature351:290-6 (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)).This algorithm can be located 8-aggressiveness, 9-aggressiveness and the 10-mer peptides that derives from the whole protein sequence, and combines by level arrangement what they and HLA-A2 and multiple other HLAI quasi-molecule were inferred.A variety of HLAI class binding peptides are 8-, 9-, 10 or the 11-aggressiveness.For example, as far as I class HLA-A2, epi-position preferably contains leucine (L) or methionine(Met) (M) at the 2nd, and contains Xie Ansuan (V) or leucine (L) (referring to for example Parker et al., J.Immunol.149:3580-7 (1992)) at the C-end.PSCA infers that the selected result of binding peptide is shown in Table V-XVIII and the XXII-LI of this paper.In Table V-XVIII and XXII-XLVIII, selected candidate 9-aggressiveness and 10-aggressiveness and position, the aminoacid sequence of each particular peptide and the combination scoring of estimation have been shown to each family member.In Table X LVIII-LI, selected candidate 15-aggressiveness and position, the aminoacid sequence of each particular peptide and the combination scoring of estimation have been shown to each family member.Be equivalent to 37 ℃ in conjunction with scoring, estimated peptide complex the half the of required time that dissociate that contain during pH6.5.That infers peptide with the highest combination scoring and cell surface HLAI quasi-molecule combines the closelyst, and the bonded time is also the longest, so is the best immunogenicity target of T-cell recognition.

Through making the HLA on the defective clone T2 of antigen-processing express stabilization; Can estimate peptide and the allelic actual situation that combines of HLA (referring to for example Xue et al.; Prostate 30:73-8 (1997) and Peshwa et al., Prostate 36:129-38 (1998)).Through at antigen presenting cell, as stimulating CD8+ CTL (CTL) under the existence of dendritic cell, can be in the immunogenicity of in-vitro evaluation particular peptide.

Should understand through BIMAS site, Eimer ^TMAnd Epimatrix ^TMThe site is inferred; Or through known in the art or become the HLAI class or the II class primitive of the part of this area; Primitive specialization shown in Table IV (or is used www website URL syfpeithi.bmi-heidelberg.com/; Or BIMAS, bimas.dcrt.nih.gov/ measures) each epi-position all " be applicable to " PSCA albumen of the present invention." being applicable to " used herein refers to vision or through computer based mode discovery method and estimates PSCA albumen, and these technology are that those skilled in the art can grasp.Each carries HLAI class primitive, length is the PSCA albumen subsequence of 8,9,10 or 11 amino-acid residues, or carry HLAII class primitive, length be 9 or the subsequence of more a plurality of amino-acid residues also fall into scope of the present invention.

III.B.) express the PSCA-related protein

In the embodiment that embodiment describes hereinafter; Can in by commercially available expression vector cells transfected (like the 293T cell), express PSCA easily; Said expression vector like coding PSCA, have the CMV-driving expression vector (pcDNA3.1/mycHIS of terminal 6 * His of C-and MYC mark; Invitrogen or Tag5, GenHunter Corporation, Nashville TN).The Tag5 carrier provides IgG κ secretion signal, and this signal helps in transfectional cell, producing excretory PSCA albumen.For example, use the nickel post, can from substratum, be purified into the PSCA of excretory HIS-mark according to standard technique.

III.C.) modify the PSCA-related protein

The modification of PSCA-related protein is included within the scope of the present invention like covalent modification.One type covalent modification comprises the purpose amino-acid residue that makes the PSCA polypeptide and organic derivatizing agent reaction, said reagent can with the proteic selected side chain of PSCA or N-or the reaction of C-terminal residue.The another kind of PSCA polypeptid covalence that the scope of the invention comprised is modified and is comprised the proteinic Natively glycosylated pattern of change the present invention.Another kind of PSCA covalent modification comprises with USP 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179, the mode described in 337 is with PSCA polypeptide and multiple nonprotein base polymer, like a kind of connection in polyoxyethylene glycol (PEG), W 166 or the polyoxyalkylene.

Also can modify PSCA-related protein of the present invention contains the PSCA that merges with another kind of heterologous polypeptide or aminoacid sequence with formation chimeric molecule.Can be through the synthetic said chimeric molecule of the method for chemistry or reorganization.Chimeric molecule can have the protein of the present invention that merges with another kind of tumour-related antigen or its fragment.Perhaps, protein of the present invention can contain the segmental fusions of PSCA sequence (amino acid or nucleic acid), to be created on its whole length not and amino acid shown in Figure 1 or the direct homologous molecule of nucleotide sequence.Said chimeric molecule can contain a plurality of identical PSCA subsequences.Chimeric molecule can contain the fusions of PSCA-related protein and poly Histidine epi-position mark; Said fusions can provide epi-position and immobilized nickel selective binding, and chimeric molecule also can contain the fusions of PSCA-related protein and cytokine or growth factor.The epi-position mark generally is positioned at proteic amino of PSCA or C-terminal.In another embodiment, chimeric molecule can contain the fusions of PSCA-related protein and Tegeline or Tegeline specific region.For the chimeric molecule (being also referred to as " immunoadhesin ") of bivalent form, said fusions can be and the regional fusions of IgG molecule Fc.The PSCA polypeptide that the Ig fusions preferably includes with solvable (membrane spaning domain disappearance or inactivation) form replaces intramolecular at least one variable region of Ig.In preferred embodiments, the Tegeline fusions comprises the hinge region of IgG1 molecule, CH2 and CH3, or hinge region, CH1, CH2 and CH3 district.About producing the Tegeline fusions, can be referring to the for example USP 5,428,130 of promulgation on June 27 nineteen ninety-five.

III.D.) purposes of PSCA-related protein

Protein of the present invention has multiple different concrete purposes.Because PSCA is high expression level in prostate cancer and other cancer, can normally use the PSCA-related protein in the method for psca gene product situation in to cancerous tissue estimating, thereby illustrate the malignant phenotype.The general existence of using perturbation (like disappearance, insertion, point mutation etc.) in the polypeptide evaluation region that the derives from PSCA albumen specific region zone of one or more primitives (as contain).The test of giving an example utilizes the antibody or the T cell of target PSCA-related protein; To estimate normally characteristic that should the zone in the cancerous tissue or to cause the immunne response to epi-position, said protein contains the amino-acid residue of contained one or more the biological primitives of PSCA peptide sequence.Perhaps, use the PSCA-related protein to screen the factor with this regional interaction of PSCA, said protein contains the amino-acid residue of proteic one or more the biological primitives of PSCA.

Proteic fragment/the subsequence of PSCA can be used for especially producing and identify structural domain-specific antibody (for example discerning the antibody of epi-position in the outer or born of the same parents of the proteic born of the same parents of PSCA); Be used for evaluation and PSCA or its special construction territory bonded reagent or cytokine; Be used for multiple treatment and diagnostic purpose, include but not limited to the method for diagnostic test, cancer vaccine and the said vaccine of preparation.

Protein by psca gene or its analogue, homologue or segment encoding serves many purposes, and includes but not limited to produce antibody and identifies and the method for psca gene product bonded part with other reagent and cellular component with being used for.Antibody to PSCA albumen or the generation of its fragment can be used for diagnosis and prediction experiment, and is used for developing method that processing feature is to express the proteic human cancer of PSCA (cancer listed like Table I).Said antibody can be at cell inner expression and is used for treating the patient's of said cancer method.Also can use PSCA-associated nucleic acid or protein to produce HTL or ctl response.

The multiple proteic immunological testing of PSCA that can be used for detecting be can use, polytype radioimmunoassay, EUSA (ELISA), enzyme linked immunological fluorescent test (ELIFA), immunocytochemistry etc. included but not limited to.Can traget antibody, and with its immune video picture reagent (for example being used to radiate the scitiphotograph visualization method) as the cell that can detect expression PSCA.Describe further like this paper, PSCA albumen also can be used to produce cancer vaccine especially.

IV.) psca antibody

On the other hand, the invention provides and PSCA-related protein bonded antibody.Under physiological condition, the preferred antibodies specificity combines the PSCA-related protein, but also debond (or faintly combining) is not the peptide or the protein of PSCA-related protein.In this article, the example of physiological condition comprises: 1) PBS; 2) contain the Tris-BS of 25mM Tris and 150mM NaCl; Or 3) common salt solution (0.9%NaCl); 4) animal serum is like human serum; Or 5) 1) to 4) arbitrary combination; Preferred these take place when being reflected at pH7.5, perhaps in the scope of pH7.0 to 8.0, take place, and perhaps in the scope of pH6.5 to 8.5, take place; These are reflected under 4 ℃ to 37 ℃ the temperature and take place.For example, can combine the PSCA-related protein with PSCA bonded antibody, for example its homologue or analogue.

Psca antibody of the present invention can be used for cancer (referring to for example Table I) diagnosis and prediction experiment and developing method especially and learn.Similarly, said antibody can be used for treating, diagnosing and/or predict that prostate cancer and other also can be expressed or crosses and express the cancer of PSCA.In addition, the antibody of cell inner expression (like single-chain antibody) can be used to treat the cancer relevant with psca expression, like late period or metastasized prostate cancer or other late period or metastatic cancer.

The present invention also provides multiple and can be used for detecting and the quantitative immunological testing of the PSCA-related protein of PSCA and sudden change.In the time of suitably, said test comprises the psca antibody that one or more could discern and combine the PSCA-related protein.These tests include but not limited to carry out in polytype radioimmunoassay, EUSA (ELISA), the enzyme linked immunological fluorescent test (ELIFA) etc. in panimmunity test well-known in the art.

Immunology of the present invention is non--and antibody test comprises that also T cell immunogenicity test (suppress or stimulate) and main histocompatibility complex (MHC) combine test.

In addition, the present invention also provide can detect prostate cancer with other can express the immune developing method of the cancer of PSCA, include but not limited to use radiation scitiphotograph visualization method through the mark psca antibody.Said test can be used for detecting, monitor and predict the cancer of expressing PSCA clinically, like prostate cancer.

Psca antibody also can be used in purifying PSCA-related protein and the method for separating PSCA homologue and associated molecule.For example, the method for purifying PSCA-related protein comprises: allowing under psca antibody and the PSCA-related protein bonded condition, with being incubated with solid substrate link coupled psca antibody and the lysate that contains the PSCA-related protein or other solution; Wash solids matrix is with removal of contamination; Wash-out PSCA-related protein from the link coupled antibody.Other purposes of psca antibody of the present invention comprises proteic resisting-idiotype antibody of generation simulation PSCA.

The multiple method for preparing antibody is well-known in the art.For example, PSCA-related protein, peptide or the suitable mammalian hosts of fragment immunity through with separation or immune coupling form can prepare antibody (Antibodies:A Laboratory Manual; CSH Press; Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)).In addition, also can use the fusion rotein of PSCA, like PSCA GST-fusion rotein.In concrete embodiment, produce the gst fusion protein that contains the whole of Fig. 1 or Most amino-acids sequence earlier, again it is used as immunogen to produce suitable antibody.In another embodiment, synthetic PSCA-related protein and with it as immunogen.

In addition; (relevant commentary can be referring to Donnelly et al. to the immunogenic immunne response of encoding to produce to use naked DNA immunological technique known in the art (containing or do not contain the PSCA-related protein of purifying or the cell of expression PSCA); 1997, Ann.Rev.Immunol.15:617-648).

Can analyze PSCA Argine Monohydrochloride sequence shown in Figure 1, be used to produce antibody to select the proteic specific region of PSCA.For example, use the hydrophobicity and the hydrophilicity analysis of PSCA aminoacid sequence, to identify the hydrophilic region in the PSCA structure.Use multiple other method known in the art; Can easily identify PSCA albumen zone and other regional and structural domain of demonstrating the immunogen structure; Said method such as Chou-Fasman, Gamier-Robson, Kyte-Doolittle; Eisenberg, Karplus-Schultz or Jameson-Wolf analyze.Use Hopp, T.P.and Woods, K.R., 1981, the method for Proc.Natl.Acad.Sci.U.S.A.78:3824-3828 can produce hydropathy profile.Use Kyte, J.and Doolittle, R.F., 1982, the method for J.Mol.Biol.157:105-132 can produce parent/hydrophobicity profile.Use Janin J., 1979, the method for Nature 277:491-492 can produce and can reach residue per-cent (%) distribution plan.Use Bhaskaran R., Ponnuswamy P.K., 1988, the method for Int.J.Pept.Protein Res.32:242-255 can produce average flexible distribution plan.Use Deleage, G., Roux B., 1987, the method for Protein Engineering 1:289-294 can produce β-corner distribution plan.Therefore, all fall in the scope of the present invention by any each zone that identifies in these programs or the method.The embodiment that this paper provides will further illustrate the preferred method that produces psca antibody.Preparation is well-known in the art as the method for immunogenic protein or polypeptide.Preparation protein and carrier also are well-known in the art like the method for the immunogenic conjugate of BSA, KLH or other carrier protein.In some cases, for example using, carbodiimide reagent carries out direct coupling; In other cases, such as Pierce Chemical Co., the connection reagent that Rockford, IL provide is effective.Often through in the suitably long time, injecting and using suitable adjuvant to use the PSCA immunogen, this point it will be appreciated by those skilled in the art that.In immune programme for children, can detect antibody titers to measure the antibody that whether has formed q.s.

Can produce the PSCA monoclonal antibody through multiple method well-known in the art.For example, use the standard hybridoma technology of Kohler and Milstein or the modification technique that known immortalization produces the B cell of antibody, preparation can be secreted the immortal cell line of required monoclonal antibody.Through antigen is that the immunoassay of PSCA-related protein filter out the immortal cell line that can secrete required antibody.When identifying suitable infinite multiplication cell culture, can expand cell, and from vitro culture thing or ascites, produce antibody.

Also can produce antibody of the present invention or fragment through recombination method.Also can in chimeric or complementary determining region (CDR) grafted antibody in multiple source, produce and the proteic desired zone specificity of PSCA bonded zone.Also can produce humanization or people's psca antibody, and preferably they are used for therapeutic purpose.Through replacing corresponding human antibody sequence, be well-known (referring to for example Jones et al, 1986, Nature321:522-525 thereby make mouse and the humanized method of other non-human antibody with one or more non-human antibody CDR; Riechmann et al., 1988, Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536).Also referring to Carter et al., 1993, Proc.Natl.Acad.Sci.USA 89:4285 and Sims et al., 1993, J.Immunol.151:2296.

Produce whole person's monoclonal antibody method and comprise phage display and transgenic method (relevant commentary can be referring to Vaughan et al., 1998, Nature Biotechnology 16:535-539).Use has utilized the clone technology in large-scale people Ig assortment of genes library (being phage display); Can produce complete people PSCA monoclonal antibody (Griffiths and Hoogenboom; Building an in vitro immune system:human antibodies from phage display libraries.In:Protein Engineering ofAntibody 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.Id., pp 65-82).The PCT patented claim W098/24893 of disclosed Kucherlapati and Jakobovits etc. was said according on December 3rd, 1997; The transgenic mice that use contains human immunoglobulin gene's seat after genetic engineering modified also can prepare complete people PSCA monoclonal antibody (also can be referring to Jakobovits; 1998, Exp.Opin.Invest.Drugs7 (4): 607-614; The USP 6,162,963 of promulgation on December 19th, 2000; 6,150,584 of promulgation on November 12nd, 2000; With on September 5th, 2000 promulgation 6,114,598).This method has been avoided the required manipulation in vitro of display technique of bacteriophage, and can effectively produce real people's antibody of high-affinity.

In the time of suitably, use the PSCA-related protein, express cell or its extract of PSCA,, comprise that Western trace, immunoprecipitation, ELISA and facs analysis confirm the reactivity of psca antibody and PSCA-related protein through multiple well-known method.But psca antibody or its fragment can by mark note mark or with second kind of molecule coupling.But suitable mark note includes but not limited to ri, fluorescent chemicals, bioluminescent compound, chemiluminescence compound, metal chelator or enzyme.In addition, use method generation well known in the art to be specific to two-specific antibody of two or more PSCA epi-positions.Also can produce homodimer antibody (for example Wolff et al., Cancer Res.53:2560-2565) through crosslinking technological known in the art.

In one embodiment; The invention provides the monoclonal antibody that is accredited as H1-1.10; This monoclonal antibody is sent to American type culture collection (ATCC) on May 4th, 2005 (via Federal Express), and P.O.Box 1549, Manassas; VA 20108, and specified preserving number is PTA-6697.

V.) PSCA cell immune response

The antigenic mechanism of T cell recognition is described.Effective peptide epitopes vaccine composition of the present invention can inductive treatment or preventative immunoreation in a lot of races in world's population scope.In order to understand immunoreactive numerical value of compsn inducing cell of the present invention and effectiveness, this paper provides the simple commentary with immunologically-mediated technology.

HLA molecule and the antigenic mixture of peptide are used as by the part of the limited T cell recognition of HLA-(Buus, S.et al., Cell 47:1071,1986; Babbitt, B.P.et al, Nature 317:359,1985; Townsend, A.and Bodmer, H., Annu.Rev.Immunol.7:601,1989; Germain, R.N., Annu.Rev.Immunol.11:403,1993).The substituted antigen analogues of single amino acids and the endogenous that checks order combine through studying, the peptide of natural process; Identify the Key residues that combines the required primitive of HLA antigen molecule corresponding to specificity; And be shown in Table IV (referring to for example Southwood; Et al, J.Immunol.160:3363,1998; Rammensee, et al, Immunogenetics 41:178,1995; Rammensee et al, SYFPEITHI, access via World Wide Web at URL (134.2.96.221/scripts.hlaserver.dll/home.htm); Sette, A.and Sidney, J.Curr.Opin.Immunol.10:478,1998; Engelhard, V.H., Curr.Opin.Immunol.6:13,1994; Sette, A.and Grey, H.M., Curr.Opin.Immunol.4:79,1992; Sinigaglia, F.andHammer, J.Curr.Biol.6:52,1994; Ruppert et al., Cell 74:929-937,1993; Kondoet 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 1999 Nov, 50 (3-4): 201-12, Review).

In addition; The peptide that the X-radiocrystallography analysis of HLA-peptide complex is disclosed the HLA molecule combines the bag in crack/ditch; It holds the residue that the peptide part carries with allelotrope-specific mode; These residues have determined to contain the HLA binding ability of peptide of these residues conversely (referring to for example Madden, D.R.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, E.Y.Curr.Opin.Immunol9:75,1997; Brown, J.H.et al., Nature 364:33,1993; Guo, H.C.et al, Proc.Natl.Acad.Sci.USA 90:8053,1993; Guo, H.C.et al, Nature360:364,1992; 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.).

Therefore, I class and II class allelotrope-specificity HLA combines the definition of primitive or I class or II class hyper-base unit can identify in the protein and the relevant zone of combination specific HLA antigen.

Therefore, be tested and appraised the process of HLA primitive, identified candidate vaccine based on epi-position; Combine further evaluate candidate vaccine of test through the HLA-peptide, to measure the binding affinity and/or the binding time of epi-position and corresponding HLA molecule thereof.Can carry out other conclusive evidence work, thereby in these candidate vaccines, select in the epi-position that has preferred characteristics aspect population covering and/or the immunogenicity.

Multiple tactical comment cell immunogenicity capable of using comprises:

1) estimate the former generation T cell culture derive from normal individual (referring to for example Wentworth, P.A.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., HumanImmunol 59:1,1998).This method is included in external, when having antigen presenting cell, is stimulated the peripheral blood lymphocyte that derives from the normal subjects to reach several time-of-weeks with trying peptide.The T cell that is specific to said peptide at this moment between in be activated, use the lymphokine that for example relates to peptide sensitization target cell-or ⁵¹The Cr-release test can detect said T cell.

2) immune HLA transgenic mice (referring to for example Wentworth, P.A.et al., J.Immunol.26:97,1996; Wentworth, P.A.et al, Int.Immunol 8:651,1996; Alexander, J.et al, J.Immunol.159:4753,1997).For example, in said method, will be in peptide subcutaneous administration in the incomplete Freund's adjuvant in the HLA transgenic mice.After several weeks of immunity, take out splenocyte, tried peptide in the presence of about 1 week of external cultivation splenocyte.Use for example relates to peptide sensitization target cell and the antigenic target cell of expressing endogenous generation ⁵¹Cr-release test detection of peptides-specific T-cells.

3) illustrate through the immune body of effective immunization and/or chronic's memory t cell responses (referring to for example Rehermann, B.et al., J.Exp.Med.181:1047,1995; Doolan, D.L.et al, Immunity 7:97,1997; Bertoni, R.et al., J.Clin.Invest.100:503,1997; Threlkeld, S.C.et al., J.Immunol.159:1648,1997; Diepolder, H.M.et al., J.Virol.71:6011,1997).Therefore, detect anamnestic reaction through cultivating PBL, said PBL derives from because of disease is exposed to antigen, thereby has produced the experimenter of " nature " immunne response, maybe must control oneself and carry out the patient to this antigenic immunization.Tried peptide and antigen presenting cell (APC) in the presence of, cultivate 1-2 week to activate " memory " T cell (comparing) at external PBL with the experimenter with " originally " T cell.When cultivate finishing, use for example to relate to that peptide sensitization target, T cell proliferation or lymphokine discharge ⁵¹The Cr-release test detects the T cytoactive.

VI.) PSCA transgenic animal

Also can use the nucleic acid of coding PSCA-related protein to produce transgenic animal or " knocking out " animal, said animal can be used for exploitation and screening useful reagent in treatment conversely.According to the technology of having set up, can use the genomic dna of the cDNA clones coding PSCA of coding PSCA.Then cloned genes group sequence is used to produce transgenic animal, said animal contains the cell of the DNA that expresses coding PSCA.Produce transgenic animal, particularly the method for the animal such as mouse or rat is the ordinary skill in the art, and it is described in the USP 4,870,009 of the USP promulgation on September 26th, 4,736,866 and 1989 of for example promulgation on April 12nd, 1988.Usually, specific cell becomes the genetically modified target of the PSCA that mixes tissue-specific enhancer.

Can use the genetically modified transgenic animal that comprise a copy coding PSCA to check that the DNA of coding PSCA expresses the increase role.Said animal can be used as test animal, is used to detect according to thinking to prevent that animal from suffering to cross the reagent of expressing relevant pathology disease with said DNA.According to this aspect of the invention, treat animal with reagent, genetically modifiedly do not treat animal and compare with carrying, the reduction of pathology disease incidence can be represented the potential therapeutic intervention to the pathology disease.

Perhaps; Can use non--people's homologue of PSCA to make up PSCA and " knock out " animal; As the endogenous gene of coding PSCA and import in the animal embryo cell, coding PSCA through changing the result of homologous recombination between the genomic dna, said animal has damaged or through the gene of the coding PSCA that changes.For example, can be according to the technology of having set up, the cDNA of use coding PSCA comes the genomic dna of clones coding PSCA.The part of the genomic dna of coding PSCA can be by disappearance or by another kind of gene, can be used for monitoring like coding integration selective marker gene replacement.Usually, carrier (5 ' with 3 ' end) comprises the not change flanking DNA (description of relevant homologous recombination vector can be referring to for example Thomas and Capecchi, Cell, 51:503 (1987)) of several thousand bases.Carrier is imported embryonic stem cell line (for example passing through electroporation), select wherein importing DNA with the cell (Li et al., Cell, 69:915 (1992)) of endogenous dna generation homologous recombination.Then with in the blastocyst of selected injection cell to animal (like mouse or rat) to form aggregation chimera (referring to for example Bradley; InTeratocarcinomas and Embryonic Stem Cells:A Practical Approach; E.J.Robertson, ed. (IRL, Oxford; 1987), pp.113-152).Then chimeric embryo is implanted in the female raising animal body of suitable false pregnancy, the embryo finally can only grow up to " knocking out " animal.Can identify the filial generation that carries homologous recombination DNA in the sexual cell through standard technique, use it for breeding animals, wherein all cells of animal all contains the DNA through homologous recombination.For example, knock-out animal is characterised in that can defend some pathology diseases, or PSCA polypeptide and produce the pathology disease for want of.

VII.) method of detection PSCA

Another aspect of the present invention relates to the method that detects PSCA polynucleotide and PSCA-related protein, and the method for identifying the cell of expressing PSCA.The expression and distribution figure of PSCA makes it become the diagnostic flag that shifts disease.Therefore, the information that situation provided of psca gene product can be used for predicting multiple factor, comprises susceptibility, development speed and/or tumor challenge power to terminal illness.Like what this paper went through; Can analyze the psca gene product situation in patient's sample through several different methods well-known in the art, said method comprises immunohistochemical analysis, comprises the multiple Northern engram technology of in situ hybridization, (for example laser-catch little-dissect and carry out on the sample) RT-PCR analyzes, Western engram analysis and tissue array analysis.

More particularly, the invention provides the detection of biological sample, like the test of the PSCA polynucleotide in serum, bone, prostate gland and other tissue, urine, seminal fluid, the cell product etc.The PSCA polynucleotide that can survey comprise for example psca gene or its fragment, PSCA mRNA, selectable splice variant PSCAmRNA and contain the recombinant DNA or the RNA molecule of PSCA polynucleotide.Multiple amplification PSCA polynucleotide and/or the method that detects its existence are well-known in the art, can be used in the present invention's practice in this respect.

In one embodiment, the method for the PSCA mRNA in the detection of biological sample comprises: use at least one primer to produce the cDNA of said sample through rt; Use the PSCA polynucleotide as the cDNA that has the amplification of justice and antisense primer to be produced, to amplify PSCA cDNA; Detect the existence of the PSCA cDNA that is increased.The optional sequence of measuring the PSCA cDNA of amplification.

In another embodiment, the method for psca gene comprises in the detection of biological sample: the genomic dna of sample separation at first; Use the PSCA polynucleotide as justice and the antisense primer isolating genomic dna that increases is arranged; And the existence of the psca gene of detection amplification.Can design the adopted and antisense probe combination of suitably having of any number by PSCA nucleotide sequence (referring to for example Fig. 1), and they are used for this purpose.

The present invention also provides and has detected tissue or other biological sample, as whether having the proteic test of PSCA in serum, seminal fluid, bone, prostate gland, urine, the cell product etc.The method that detects the PSCA-related protein also is well-known, comprises that for example immunoprecipitation, immunohistochemical analysis, Western engram analysis, molecule combine test, ELISA, ELIFA etc.For example, the method for the existence of PSCA-related protein comprises in the detection of biological sample: the recombinant protein that at first makes sample and psca antibody, its PSCA-fragment reaction or contain the antigen-calmodulin binding domain CaM of psca antibody contacts; The combination of PSCA-related protein in the test sample then.

The method of identifying the cell of expressing PSCA also falls into scope of the present invention.In one embodiment, the test of the cell of evaluation expression psca gene comprises the existence that detects PSCA mRNA in the cell.The method that detects the specific mRNA in the cell is well-known; Comprise the cross experiment (as using in situ hybridization, Northern trace and correlation technique) that for example uses complementary DNA probe through mark PSCA ribose probe; And multiple nucleic acid amplification test (as using the RT-PCR and other amplification type detection method of PSCA specificity complementary primer, like branched DNA, SISBA, TMA etc.).Perhaps, test that identify to express the cell of psca gene comprises and detecting in the cell or by the existence of the PSCA-related protein of emiocytosis.Multiple detection method of protein is well-known in the art, can be used for detecting PSCA-related protein and the cell of expressing the PSCA-related protein.

The psca expression analysis also can be used as the instrument of identifying and estimating the medicament of modulation psca gene expression.For example, psca expression significantly raises in prostate cancer, and PSCA expresses in the cancer of the listed tissue of Table I.Evaluation can suppress the psca expression in the cancer cell or cross the molecule or the biological reagent of expressing to have therapeutic value.For example, through using screening, can identify said medicament through RT-PCR, nucleic acid hybridization or the quantitative psca expression of antibodies.

Known cancer is a process that multistep is rapid; Wherein the cell growth is gradually by unusual adjusting; Cancer state before cell is developed into by normal physiological status; Develop into cancerous state (referring to for example Alers et al., Lab Invest.77 (5): 437-438 (1997) and Isaacset al., Cancer Surv.23:19-32 (1995)) then.In this context, in biological sample, search by the evidence (like the unusual psca expression in the cancer) of the cell of unusual adjusting growth can develop in pathologic state such as cancer treatment select more limitation with or predict the relatively poor stage before early detection go out said abnormal physiology state.In said search procedure, can be with the PSCA compared in the PSCA state in the targeted biological specimen for example and the corresponding normal specimens (for example derive from this individuality or another does not receive the sample of the individuality of pathological effect).The change of PSCA state in the biological sample (comparing with normal specimens) provides the cell growth by the unusual evidence of regulating.Except using the biological sample do not receive pathological effect as the normal specimens, also can use predetermined standard value, like predetermined normal mRNA expression level (referring to for example Grever etal., J.Comp.Neurol.1996 Dec 9; 376 (2): 306-14 and USP 5,837,501) come the PSCA state in the comparative sample.

Use the term " state " in this context according to the implication of accepting for its field, this term refers to the circumstance or state of gene and product thereof.Usually, those skilled in the art use the circumstance or state of a plurality of parameter evaluation genes and product thereof.Said parameter includes but not limited to the position (position that comprises the cell of expressing PSCA) and the level of gene product expression, and the biological activity of the gene product of expressing (like PSCAmRNA, polynucleotide and polypeptide).Generally speaking, the change of PSCA state comprises PSCA and/or expresses the change of position of the cell of PSCA, and/or the increase of PSCA mRNA and/or protein expression.

Through multiple method well-known in the art, include but not limited to immunohistochemical analysis, in situ hybridization, laser capture little-dissect that the RT-PCR that carries out on the sample analyzes, Western engram analysis and the tissue array analysis PSCA state in can analytic sample.The general method of estimating the state of psca gene and gene product can be referring to people such as for example Ausubel in nineteen ninety-five editor's Current Protocols InMolecular Biology the 2nd (Northern trace), 4 (Southern traces), 15 (immunoblottings) and 18 (pcr analysis) unit.Therefore; Available by one of skill in the art several different methods is estimated the PSCA state in the biological sample; Said method includes but not limited to that genome Southern analyzes Northern analysis and/or the pcr analysis (being used to check for example PSCA mRNA polynucleotide sequence or changes of expression level) of (being used for checking the for example perturbation of psca gene), PSCA mRNA, and Western and/or immunohistochemical analysis (being used for checking that for example variation, the localized variation of sample polypeptide, the variation of PSCA protein expression level and/or PSCA albumen combines counterpart with polypeptide the bonded of peptide sequence change).Can survey recombinant DNA or RNA molecule that the PSCA polynucleotide comprise for example psca gene or its fragment, PSCA mRNA, selectable splice variant PSCA mRNA and contain the PSCA polynucleotide.

The expression and distribution figure of PSCA makes its diagnostic flag that becomes the part and/or shift disease, and the information of relevant biological sample growth or oncogenic potential is provided.Particularly, the information that provides of the state of PSCA can be used for predicting the susceptibility to special disease period, development and/or tumor challenge property.The invention provides the cancer of measuring PSCA state and diagnosis expression PSCA, like the method for cancer and the test of the listed tissue of Table I.For example; Because for normal prostate tissue; PSCAmRNA expression level in prostate cancer and other cancer is very high; The test of PSCA mRNA transcript or protein level is diagnosed with PSCA and is regulated diseases associated unusually in can the in-service evaluation biological sample, and said test can be provided for the information of forecasting of confirming that suitably treatment is selected.

The information that the psca expression state provides comprises existence, period and the position of heteroplasia cell, preceding cancer and cancer cell, can predict the susceptibility in each disease period and/or estimates the aggressiveness of tumour.In addition, the psca expression distribution plan makes it can be used as the video picture reagent that shifts disease.Therefore; One aspect of the present invention relates to multiple molecule prediction and diagnostic method; Said method can be used for checking the PSCA state in the biological sample, and said sample for example derives to be suffered from or suspect the pathology of suffering from the cell growth that is characterised in that unusual adjusting, like the individuality of cancer.

As stated, can check the PSCA state in the biological sample through several different methods well-known in the art.For example, through whether there being the cell (for example expressing PSCAmRNA or proteinic cell) of expressing PSCA in the assess sample, can check the PSCA state in the biological sample of taking from the health specific position.For example; Because the change of PSCA state is often relevant with the unusual cell growth of regulating in the biological sample; Therefore, when in the biological sample (like lymphoglandula) that does not contain the cell of expressing PSCA usually, finding said cell, this inspection can provide the cell growth to receive the unusual evidence of regulating.Specifically, a sign of the unusual cell growth of regulating is cancer cell is transferred to health from initial organ (like prostate gland) a different zones (like lymphoglandula).In this context; It is important receiving the evidence of the unusual cell growth of regulating; Because in most of patients with prostate cancer, can detect hiding nodus lymphoideus transferring rate; Said transfer relevant with known PD predictor (referring to for example Murphy et al., Prostate42 (4): 315-317 (2000); Su et al., Semin.Surg.Oncol.18 (1): 17-28 (2000), Freemanet al., J Urol 1995 Aug 154 (2 Pt1): 474-8).

On the one hand; The invention provides the state that derives from the expressed psca gene product of cell of suspecting the individuality of suffering from the disease (like hyperplasia or cancer) relevant through mensuration with the growth of unusual adjusting cell; Then the psca gene product state in state of being measured and the corresponding normal specimens is compared, thus the method for monitoring psca gene product.For normal specimens, exist unusual psca gene product to provide in the given the test agent and have the indication that receives the unusual cell growth of regulating in the individual cells.

On the other hand, the invention provides and can be used for measuring the test that whether has cancer in the individuality, it comprises and detects PSCA mRNA or the remarkable increase of protein expression for corresponding normal cell or tissue expression level in subject cell or the tissue sample.For example, can estimate to include but not limited to whether there is PSCA mRNA in the listed tissue of Table I.The existence of remarkable psca expression can be used for representing appearance, existence and/or the seriousness of cancer in any of these tissue, because the PSCA mRNA of lower level is not expressed or only expressed to corresponding healthy tissues.

In related embodiment, on protein level rather than nucleic acid level, measure the PSCA state.For example, said method comprises measures the PSCA protein level tried in the tissue sample by cell expressing, and the PSCA level of expressing in the level of being measured and the corresponding normal specimens is compared.In one embodiment, use the for example proteic existence of immunohistochemical methods method evaluation PSCA.Can detect the psca antibody of PSCA protein expression to use in the well-known in the art multiple test form of this purpose or combine counterpart.

In another embodiment, the state that can estimate PSCA Nucleotide and aminoacid sequence in the biological sample is to identify the perturbation in these molecular structures.Said perturbation comprises insertion, disappearance, replacement etc.Said evaluation is useful because can a large amount of and growth observe in by unusual phenotype proteins associated matter of regulating perturbation in Nucleotide and the aminoacid sequence (referring to for example Marrogi et al., 1999, J.Cutan.Pathoi.26 (8): 369-378).For example, the sudden change in the PSCA sequence can be represented the existence or the reinforcement of tumour.Therefore, said test has the value of diagnosis and prediction, the wherein potential loss of the sudden change presentation function of PSCA or the increase of tumor growth.

The multiple test that can be used for observing the perturbation (perturbation) in Nucleotide and the aminoacid sequence is well-known in the art.For example, can be observed the size and the structure of the nucleic acid or the aminoacid sequence of psca gene product through Northern, Southern, Western, PCR and dna sequencing method that this paper discussed.In addition, other can be used for observing the method for the perturbation in Nucleotide and the aminoacid sequence, also is well-known in the art (referring to the for example USP 5 of promulgation on September 7th, 1999 like single-strand conformation polymorphism analysis; 382; 510 with January 17 nineteen ninety-five promulgation 5,952,170).

In addition, can check the methylation state of psca gene in the biological sample.The unusual demethylation on CpG island and/or excessively methylate often betides in immortalization and the transformant in gene 5 ' regulatory region, and can change the expression of several genes.For example; As if the promotor of pi-class glutathione S-transferase (in normal prostatic, express but in＞90% prostate cancer not expressed protein) excessively methylates can for good and all reticent this gene transcription; And be that the genome that the most often is detected in the prostate cancer changes (De Marzo et al., Am.J.Pathol.155 (6): 1985-1992 (1999)).In addition, this change be present at least 70% high level prostatic epithelium in the tumour (PIN) (Brooks et al, CancerEpidemiol.Biomarkers Prev., 1998,7:531-536).In another example; Induce the LAGE-1 tumour-specific gene in the lymphoblastoid (in normal prostatic, not express through deoxidation-U-18496; But in the prostate cancer of 25-50%, express) expression; The hint tumour express be demethylation cause (Lethe et al., Int.J.Cancer 76 (6): 903-908 (1998)).The test of multiple inspection gene methylation state is well-known in the art.For example, can in the Southern hybrid method, use the methylation state of the responsive Restriction Enzyme that methylates being estimated the CpG island, said enzyme can not cracking contains the sequence in methylated CpG site.In addition, the methylation state in MSP (methylation status of PTEN promoter) all the CpG sites that can describe fast to exist in the CpG island of given gene.Originally this method comprises uses sodium sulfite anhy 96 (can change all unmethylated cytosine(Cyt)s into uridylic) modifying DNA, then uses to be specific to the primer that methylates to non-methylate DNA and to increase.The method that relates to the intervention that methylates also can be referring to for example Current Protocols In Molecular Biology, and Unit 12, Frederick M.Ausubel et al.eds., 1995.

Gene amplification is the method that another kind can be used for estimating the PSCA state.The sequence that can provide based on this paper; It is suitable to label probe to use; Northern trace (the Thomas that directly transcribes through for example conventional Southern trace or quantification of mrna; 1980, Proc.Natl.Acad.Sci.USA, 77:5201-5205), dot blotting (DNA analysis) or in situ hybridization comes the gene amplification in the working sample.Perhaps, use ability identification specificity duplex, comprise the antibody of DNA duplex, RNA duplex and DNA-RNA heterozygosis duplex or DNA-protein duplex.Traget antibody carries out the test of duplex and surface bonding conversely, makes through the formation of surperficial duplex, can detect the existence with duplex bonded antibody.

Use for example Northern, dot blotting or RT-PCR analyzing and testing psca expression, can analyze whether there is cancer cells in biopsy or the peripheral blood easily.The existence of the PSCA mRNA that RT-PCR can increase provides the indication that has cancer.The RT-PCR test is well-known in the art.Recently, the RT-PCR of tumour cell detects to test and is estimated in the peripheral blood, with diagnosis and the processing that is used for the various human noumenal tumour.In the prostate cancer field, said test comprises RT-PCR test (Verkaik et al., 1997, the Urol.Res.25:373-384 that is used to detect the cell of expressing PSA and PSM; Ghossein et al., 1995, J.Clin.Oncol.13:1195-2000; Heston et al, 1995, Clin.Chem.41:1687-1688).

Another aspect of the present invention is to estimate individuality to being in the susceptibility of developing cancer.In one embodiment, prediction comprises PSCAmRNA or PSCA albumen in the test set tissue samples to the method for the susceptibility of cancer, and it exists expression to the cancer susceptible, and wherein PSCA mRNA expression levels is relevant with the degree of susceptibility.In specific embodiments, whether there is PSCA in inspection prostate gland or other tissue, exist PSCA to represent in the sample to prostate cancer susceptible (or appearance or have tumor of prostate).Similarly, the integrity that can estimate PSCA Nucleotide and aminoacid sequence in the biological sample is to identify the perturbation in these molecular structures, like insertion, disappearance, replacement etc.The susceptibility (or the appearance of tumour or existence) to cancer is represented in the existence of interior one or more perturbations of psca gene product in the sample.

The present invention also comprises the method for estimating tumor challenge property.In one embodiment; The method of estimating tumor challenge property comprises: measure expressed PSCA mRNA or the proteic level of PSCA of tumour cell; The level of being measured is compared with reference to PSCA mRNA that expresses in the sample or the proteic level of PSCA with the related normal tissue of taking from same individual or healthy tissues; Wherein for normal specimens, the PSCA mRNA or the PSCA protein expression level of tumor sample demonstrate aggressive level.In specific embodiments, estimate the aggressiveness of tumour through the level of measuring tumor cells expression PSCA, higher expression level representes that tumour is more aggressive.Another embodiment is to estimate the integrity of PSCA Nucleotide and aminoacid sequence in the biological sample to identify the perturbation in these molecular structures, like insertion, disappearance, replacement etc.The existence of one or more perturbations representes that tumour is more aggressive.

Another embodiment of the invention relates to the method for observing the individual development of malignant tumour in the whole time.In one embodiment; The method of observing the individual development of malignant tumour in the whole time comprises: measure expressed PSCA mRNA or the proteic level of PSCA of cell in the tumor sample; The level of being measured is compared with take from PSCA mRNA or the proteic level of PSCA that being equal to of same individual express in the tissue sample at different time, and wherein PSCA mRNA in the tumor sample or PSCA protein expression level provide the information of relevant cancer development in the whole time.In specific embodiments, estimate the development process of cancer through measuring the psca expression in the whole time in the tumour cell, the development of cancer is represented in the increase of wherein in the whole time, expressing.The integrity that also can estimate PSCA Nucleotide and aminoacid sequence in the biological sample is to identify the perturbation in these molecular structures, and like insertion, disappearance, replacement etc., wherein the development of cancer is represented in the existence of one or more perturbations.

Above-mentioned diagnostic method can with multiple prediction known in the art and diagnostic method in anyly unite use.For example; Another embodiment of the invention relates to psca gene and the expression (or the perturbation in psca gene and the psca gene product) of psca gene product and the conforming method between the malignant tumour correlation factor of observing, and said method can be used for diagnosis and prediction group tissue samples state.Can utilize the multiple factor relevant with malignant tumour; Malignant tumour Expression of Related Genes (the PSA for prostate cancer etc. for example for example; PSCA and PSM express) and the general cell observation (referring to 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): 918-24).For example; Because the existence of the specificity factor that a cover conforms to disease can provide vital information for diagnosis and prediction group tissue samples state; Therefore, it is useful observing the psca gene and the expression (or the perturbation in psca gene and the psca gene product) of psca gene product and the conforming method between another malignant tumour correlation factor.

In one embodiment; PSCA mRNA or proteinic the mistake are expressed in the essential test set tissue samples of the expression (or the perturbation in psca gene and the psca gene product) of observation psca gene and psca gene product and the conforming method between another malignant tumour correlation factor; PSA mRNA or proteinic cross to express in the test set tissue samples (or PSCA or PSM express), and observe PSCA mRNA or protein is crossed the consistence between the expression (or PSCA or PSM expression) with PSA mRNA or protein.In specific embodiments, detect the expression of PSCA and PSA mRNA in the prostata tissue, the consistence that wherein PSCA and PSA mRNA cross expression in the sample is represented the existence of prostate cancer, the appearance of prostate cancer susceptibility or tumor of prostate or state.

This paper has described the method that detects with quantitative PSCA mRNA or protein expression, and the nucleic acid of standard and protein detection and quantitative technique are well-known in the art.Detection comprises with the standard method of quantitative PSCA mRNA: use the in situ hybridization through mark PSCA ribose probe; Use the Northern trace and the correlation technique of PSCA polynucleotide probes; Use the RT-PCR of PSCA Auele Specific Primer to analyze; With other amplification type detection method, like branched DNA, SISBA, TMA etc.In specific embodiments, use half-quantitative RT-PCR to detect and express with quantitative PSCA mRNA.The primer of any number of PSCA of increasing all can be used for this purpose, includes but not limited to the specifically described multiple primer cover of this paper.In specific embodiments, can in the immunohistochemical methods test of biopsy, use polyclone or monoclonal antibody with the reaction of wild-type PSCA protein-specific.

IX.) identify and the interactional molecule of PSCA

Disclosed PSCA albumen of this paper and nucleotide sequence can identify and PSCA interacting proteins, small molecules and other reagent those skilled in the art, and via in the multiple method of accepting for this area any by PSCA activated approach.For example, can utilize a kind of so-called interaction trap system (being also referred to as " two-the heterozygosis test ").In said system, molecule and the transcription factor interaction that mediates the reporter gene expression, and rebuild said transcription factor, measure the expression of reporter gene thus.Other system identifies body internal protein-protein interaction through rebuilding the eukaryotic transcription activator.Referring to the for example USP 5,955,280 of promulgation on September 21st, 1999; 5,925,523 of promulgation on July 20th, 1999; 6,004,746 of the promulgation on December 21st, 5,846,722 and 1999 of promulgation on December 8th, 1998.Relevant algorithm based on genome predicted protein matter function also be known in the art (referring to for example Marcotte, et al., Nature 402:4 November 1999,83-86).

Perhaps can screen peptide library to identify and the interactional molecule of PSCA protein sequence.In said method, the library through screening is encoded at random or controlled amino acid is gathered identifies and PSCA bonded peptide.The peptide of library coding is expressed as the fusion rotein of phage coating protein, screening and the protein bound phage particle of PSCA then.

Therefore, need not to know earlier the information of any relevant expection part or acceptor molecule structure, also can identify the peptide that serves many purposes, like treatment, prediction or diagnostic reagent.Can be used for identifying issue on March 31st, 5,723,286 and 1998 that typical peptide library and screening method with the interactional molecule of PSCA protein sequence are disclosed in for example promulgation on March 3rd, 1998 5,733,731.

Perhaps, the clone of expression PSCA can be used for identifying the protein-protein interaction by the PSCA mediation.Can use the said interaction of immunoprecipitation technology for detection (referring to for example Hamilton B.J., etal.Biochem.Biophys.Res.Commun.1999,261:646-51).Use anti--psca antibody can be from the clone of expressing PSCA immunoprecipitation PSCA albumen.Perhaps, can in the clone of the fusions (above-mentioned carrier) that after transforming, can express PSCA and His-tag, use anti-His-tag antibody.Through such as Western trace, proteinic ³⁵S-methionine(Met) mark, protein microsequencing, silver dyes and the method for two-dimensional gel electrophoresis, can detect the protein bound situation of immunoprecipitation complex.

Related embodiment through said shaker test can identify and interactional small molecules of PSCA and part.For example; Can identify the small molecules that can hinder protein function, comprise can hinder PSCA mediation phosphorylation and dephosphorylation, with the molecule of the ability of the interaction (conducting or tumorigenic indication) of DNA or RNA molecule as Cycle Regulation, second messenger's signal.Similarly, identify the small molecules that can modulate PSCA-relevant ions passage, protein pump or cell communication function, and with they be used to treat suffer from the cancer of expressing PSCA the patient (referring to for example Hille; B.; IonicChannels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland; MA, 1992).In addition, combine PSCA and the ability that activates the report construct to identify the part of regulating the PSCA function based on it.Typical method is discussed at the for example USP 5,928,868 of promulgation on July 27th, 1999, and it comprises the method that forms the heterozygosis part, and wherein at least one part is a small molecules.In illustrative embodiment, use cell coexpression heterozygosis part/small molecules and the proteinic fusion rotein of cDNA library transcriptional activator that to express PSCA and the protein-bonded fusion rotein of DNA-through transforming.Cell also contains reporter gene, and in the expression of first and second fusion roteins nearside adjusting reporter gene each other, this incident only just can take place when the heterozygosis part combines the target site on two kinds of hybrid proteins.Select to express the cell of reporter gene, and the small molecules of identified unknown or unknown part.This method provides the method that can activate or suppress the modulator of PSCA of identifying.

Embodiment of the present invention comprise screening can with the method for the interactional molecule of PSCA aminoacid sequence shown in Figure 1; Said method comprises the steps: to make molecule colony to contact with the PSCA aminoacid sequence; Helping that molecule colony and PSCA aminoacid sequence are interacted; The existence of mensuration and the interactional molecule of PSCA aminoacid sequence will not separate with the interactional molecule of PSCA aminoacid sequence together with the interactional molecule of PSCA aminoacid sequence then.In specific embodiments, this method further comprises purifying, sign and evaluation and the interactional molecule of PSCA aminoacid sequence.Use the function of the molecule modulation PSCA enforcement of identifying.In preferred embodiments, the PSCA aminoacid sequence contacts with peptide library.

X.) Treatment and composition for

PSCA is accredited as generally in a limited cover tissue, expresses, but also in the cancer listed like Table I expressed protein developed the said treatment for cancer method of multiple treatment.

It should be noted that the targeting anti-tumor therapy is useful, promptly convenient healthy tissues, or even important normal organ tissue is when expressing the protein of target, this therapy also is useful.Vitals be earn a bare living necessary, like heart or colon.Unessential organ is to extract the individual organ that still can survive in back.The example of inessential organ is ovary, mammary gland and prostate gland.

For example; Herceptin

is the medicine of being made up of antibody through FDA approval; Said antibody capable be called HER2 differently, the protein of HER2/neu and erb-b-2 carries out immunoreation.This medicine is introduced to the market by Genentech, is a kind of at commercial very successful antineoplastic agent.2002 Herceptin

sales volume reach nearly 400,000,000 dollars.Herceptin can treat the positive metastatic breast cancer of HER2.Yet the expression of HER2 is not limited to this tumour.Multiple healthy tissues is also expressed this protein.Have HER2/neu in particularly known normal kidney and the heart, so these tissues are present in everyone acceptor of Herceptin.Latif, Z., et al., B.J.U.International (2002) 89:5-9 has also confirmed the existence of HER2/neu in the normal kidney.Shown in this piece document (this article has been estimated, and whether anti--HER2 antibody such as Herceptin preferably indicate renal cell carcinoma), benign renal is organized and is both produced protein and also produce mRNA.HER2/neu albumen overexpression consumingly significantly in the benign renal tissue.

Although HER2/neu expresses in the vital tissue such as heart and kidney, that Herceptin is still is very useful, through the FDA approval, at the medicine of commercial achieving success.Herceptin is to the effect of heart tissue, and promptly " cardiac toxic " only is the spinoff of treatment.When only with Herceptin treatment patient, have only the patient of very low per-cent significant cardiac toxic can occur.For cardiac toxic is minimized, for stricter access requirement being arranged with the HER2/neu treatment.Before treating, estimate factors such as cardiopathic susceptibilities.

Show normal expression although it should be noted that nephridial tissue especially, even maybe be higher than the expression of heart tissue, kidney does not have perceptible Herceptin spinoff.In addition, in the multiple array of the healthy tissues of expressing HER2, seldom any spinoff can appear.Have only heart tissue some perceptible spinoffs can occur.HER2/neu expresses tissue especially significantly, is not the basis of any spinoff like kidney.

In addition, found targeting epidermal growth factor receptor (EGFR); The favourable result of treatment of antitumor therapy of Erbitux (ImClone).EGFR is also at multiple normal tissue expression.Use after the anti-EGFR therapeutical agent, very limited spinoff is only arranged in the healthy tissues.The spinoff of accompanying with the EGFR treatment generally is in 100% patient who receives treatment, to observe serious fash.

Therefore, healthy tissues, or even the important healthy tissues protein expression that hits can not hinder this proteinic target medicament as this proteinic tumor treatment agent of some overexpressions.For example, the expression in vitals itself is not deleterious.In addition, be considered to nonessential organ, can picked-off and can not influence mortality ratio like prostate gland and ovary.Finally, because the immunity preferential treatment, the expression in the normal organ can not influence some vitals.The organ that receives the immunity preferential treatment is protected through blood-organ barrier, avoids the influence of blood, thereby makes immunotherapy can't arrive this organ.The example that receives the organ of immunity preferential treatment is brain and testis.

Therefore, the treat-ment of inhibition PSCA protein-active is useful to suffering from the cancer patients who expresses PSCA.These treat-ment generally are divided three classes.The function of first kind modulation PSCA because it relates to growth of tumour cell, causes the inhibition or the delay of growth of tumour cell or induces it to kill and wound.Second type comprises that multiple inhibition PSCA albumen combines counterpart or other protein bound or associating method with it.The 3rd type comprises that multiple inhibition psca gene is transcribed or the method for PSCA mRNA translation.

X.A.) anti--Theratope

The invention provides the cancer vaccine that contains PSCA-related protein or PSCA-associated nucleic acid.Because the expression of PSCA, cancer vaccine can prevent and/or treat the cancer of expressing PSCA, and very little or do not have an influence to the influence of non--target tissue.In vaccine, use the tumour antigen conduct produce cell-mediated HI resist-the cancer therapy is well-known in the art; And be used for the immunogenic prostate cancer vaccine of end user PSMA and rodent PAP (Hodge et al.; 1995, Int.J.Cancer63:231-237; Fong et al, 1997, J.Immunol.159:3113-3117).

Through utilizing the PSCA-related protein, or the nucleic acid molecule of coding PSCA with can express and present the PSCA immunogen recombinant vectors of (generally containing multiple t cell epitope or antibody), can easily implement this method.Those skilled in the art understand the vaccine system of multiple premunition reaction epi-position be known in the art (referring to for example Heryin et al., Ann Med 1999 Feb 31 (1): 66-78; Maruyama etal., Cancer Immunol Immunother 2000 Jun 49 (3): 123-32).Briefly; The method that describedly in Mammals, produces (for example cell-mediated and/or body fluid) immunne response comprises the steps: immune system is exposed to immunoreation epi-position (epi-position that exists in PSCA albumen as shown in Figure 1 or its analogue or the homologue), so that Mammals produces the immunne response (for example producing the antibody of this epi-position of specific recognition) that is specific to this epi-position.

Can and transmit complete PSCA albumen through the several different methods combination, its immunogenicity zone or its epi-position.Said vaccine composition for example can comprise lipopeptid (Vitiello for example; A.et al., J.Clin.Invest.95:341,1995); Be wrapped in the peptide combinations that gathers in (DL-lactide-co-glycolide) (" PLG ") microsphere (referring to for example Eldridge; Et al., Molec.Immunol.28:287-294,1991; Alonso etal., Vaccine 12:299-306,1994; Jones et al., Vaccine 13:675-681,1995), be contained in peptide combinations in the immunostimulating complex (ISCOMS) (referring to for example Takahashi et al, Nature344:873-875,1990; Hu et al., Clin Exp Immunol.113:235-243,1998), multiple antigenic peptide system (MAP) is (referring to 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), be mixed with the peptide of multivalence peptide; The peptide that is used for the trajectory transfer system, the peptide of typical crystalization, the viral carrier (Perkus, M.E.et al., In:Concepts in vaccine development, Kaufmann, S.H.E., ed., p.379,1996; Chakrabarti, S.et al., Nature 320:535,1986; Hu, S.L.et al., Nature320:537,1986; Kieny, M-P.et al., AIDS Biol Technology 4:790,1986; Top, F.H.et al., J.Infect Dis.124:148,1971; Chanda, P.K.et al., Virology 175:535,1990), the particle in virus or synthetic source (Kofier for example, N.et al., J.Immunol.Methods.192:25,1996; Eldridge, J.H.et al., Sem.Hematol.30:16,1993; Falo, L.D., Jr.etal., Nature Med.7:649,1995), adjuvant (Warren, H.S., Vogel, F.R., and Chedid, L.A.Annu.Rev.Immunol.4:369,1986; Gupta, R.K.et al., Vaccine 11:293,1993), liposome (Reddy, R.et al., J.Immunol.148:1585,1992; Rock, K.L., Immunol.Today 17:131,1996), or exposed or be adsorbed in particulate cDNA (Ulmer, J.B.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.Hemato.30:16,1993).Also can use toxin-targeted delivery technology, also be known as receptor-mediated target, like AvantImmunotherapeutics, Inc. (Needham, technology Massachusetts).

Patient as far as the PSCA-associated cancer; Vaccine composition of the present invention also can be used for the therapy of cancer with other; Unite use like surgical operation, chemotherapy, pharmacotherapy, radiotherapy etc., comprise and immunological adjuvant, unite use like IL-2, IL-12, GM-CSF etc.

Cell vaccine:

Use particular algorithm can measure the CTL epi-position, with identify in the PSCA albumen with corresponding HLA allelotrope bonded peptide (referring to for example Table IV; Eimer ^TMAnd Epimatrix ^TM, Brown University (URL brown.edu/Research/TB-HlV_Lab/epimatrix/epimatrix.html); And BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/).In preferred embodiments; The PSCA immunogen contains one or more aminoacid sequences that uses technical evaluation well-known in the art; Sequence shown in Table V-XVIII and XXII-LI or by 8 of HLAI class primitive/hyper-base unit specialization; 9,10 or 11 amino acid whose peptides (like Table IV (A), Table IV (D) or Table IV (E)) and/or contain at least 9 amino acid whose peptides (like Table IV (B) or Table IV (C)) of HLAII class primitive/hyper-base unit.Those skilled in the art should understand that the HLAI class combines the essential closed end of ditch, make the peptide that has only certain size range be fit to get in the ditch and are combined, and usually, the length of I class HLA epi-position is 8,9,10 or 11 amino acid.On the contrary, the HLAII class combines the essential open end of ditch, and therefore, about 9 or more a plurality of amino acid whose peptide can be combined by the HLAII quasi-molecule.Because combine the difference of ditch between HLAI class and the II class, HLAI class primitive is that length is specific, promptly the 2nd of I class primitive the is second amino acid of amino to the carboxyl direction of peptide.The amino acid position of II class primitive only is relative to each other, rather than for whole peptide, the amino acid that promptly can combine other at the amino and/or the C-terminal of the sequence that carries primitive.The length of HLAII class epi-position often is 9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 or 25 amino acid, or be longer than 25 amino acid.

The multiple method that in Mammals, produces immunne response is (for example as the first step that produces hybridoma) known in the art.The method that in Mammals, produces immunne response comprises mammiferous immunity system is exposed to immunogenicity epi-position on the protein (like PSCA albumen) to produce immunne response.Typical embodiment is made up of the method that in the host, produces anti-PSCA immunne response, and at least a PSCA B cell through making host and q.s or cytotoxic T-cell epitope or its analogue contact; At least after the certain interval of time, make host and PSCA B cell or cytotoxic T-cell epitope or its analogue once more-contact and can implement this method.Specific embodiments is made up of the method for the immunne response that produces anti-PSCA-related protein or artificial multi-epitope peptide; Said method comprises: give the PSCA immunogen (like PSCA albumen or its peptide fragment, PSCA fusion rotein or analogue etc.) that comprises in people or the another kind of administration vaccine product.Usually, said vaccine product also contains suitable adjuvant (referring to for example USP 6,146,635) or general auxiliary epi-position, like PADRE ^TMPeptide (EpimmuneInc., San Diego, CA; Referring to for example Alexander et al., J.Immunol.2000 164 (3); 164 (3): 1625-1633; Alexander et al., Immunity 1,994 1 (9): 751-761 and Alexander et al., Immunol.Res.1998 18 (2): 79-92).Another kind of selectable method comprises through giving in individual muscle or the skin volume uses dna molecular; In individuality, produce the immunogenic immunne response of anti-PSCA; Said dna molecular contains the immunogenic dna sequence dna of coding PSCA, and said dna sequence dna can be operated with the adjusting sequence that the control dna sequence dna is expressed and link to each other; Wherein dna molecular is by cellular uptake, and dna sequence dna is expressed in cell, thereby produces anti-immunogenic immunne response (referring to for example USP 5,962,428).Choose wantonly and also use gene vaccine promotor, like the negatively charged ion lipid; Saponin(e; Lectin; Estrogen compound; The hydroxylation low alkyl group; DMSO 99.8MIN.; And urea.In addition, in order to produce, can use the antiidiotypic antibody of simulation PSCA to the replying of target antigen.

Nucleic acid vaccine:

Vaccine composition of the present invention comprises the vaccine of nucleic acid-mediation.Can use code book invention protein DNA or RNA to the patient.Can use the genetic immunization method to produce preventative or therapeutic body fluid and cellullar immunologic response to the cancer cell of expressing PSCA.Can make muscle or skin cells picked-up construct, and express the PSCA albumen/immunogen of coding containing coding PSCA-related protein/immunogenic DNA muscle or the skin extremely individual with the construct direct injection of suitably regulating sequence.Perhaps, vaccine contains the PSCA-related protein.The immunogenic expression of PSCA-related protein causes producing preventative or therapeutic body fluid and the cellular immunity to the cell that carries PSCA albumen.Can use multiple prevention known in the art and therapeutic genes immunological technique (relevant commentary can referring to disclosed information of internet address genweb.com and reference).Transmission based on nucleic acid is described in for example Wolff et.al., Science 247:1465 (1990) and USP 5,580,859,5,589,466,5,804,566,5,739,118,5,736,524,5,679,647 and WO98/04720.Example based on the transmission of DNA technology comprises " exposed DNA ", promotes that the transmission of (bupivicaine, polymkeric substance, peptide-mediation) transmission, cation lipid mixture and particle-mediation (" particle gun ") or pressure-mediation is (referring to for example USP 5; 922,687).

In order to treat or preventative immunity, can express protein of the present invention via virus or bacteria carrier.The multiple viral gene delivery system that can be used for the present invention's practice includes but not limited to that vaccinia virus, fowlpox virus (fowlpox), canary pox virus (canarypox), adenovirus, influenza virus, poliovirus, gland-associated virus, slow virus and sindbis virus (sindbis virus) are (referring to for example Restifo; 1996, Curr.Opin.Immunol.8:658-663; Tsang et al J.Nat.Cancer Inst.87:982-990 (1995)).Also can import patient's (for example intramuscular or intracutaneous) through the naked DNA of the PSCA-related protein of will encoding utilizes non--viral system induction Anti-tumor to reply.

With the carrier of vaccinia virus as the nucleotide sequence of for example expressing code book invention peptide.Through importing the host, vaccinia virus recombinant marking protein immunogenic peptide takes this to cause host immune response.The vaccinia virus vector and the method that are used for immunization scheme are described in for example USP 4,722,848.Another kind of carrier is BCG (Bacille Calmette Guerin).The BCG carrier is described in Stover et al., Nature 351:456-460 (1991).Description according to this paper; Multiple other carrier that can be used for therapeutic administration or immunization peptide of the present invention, for example anthrax toxin carrier of gland and gland-accompanying virus carriers, retroviral vector, salmonella typhi carrier, detoxification etc. is conspicuous to those skilled in the art.

Therefore, can use gene delivery system to transmit PSCA-associated nucleic acid molecule.In one embodiment, utilized total length people PSCA cDNA.In another embodiment, utilized the PSCA nucleic acid molecule of coding specific cytotoxic T lymphocyte (CTL) and/or antibody epitope.

(Ex vivo) vaccine of ex vivo:

Can utilize the strategy of multiple ex vivo to produce immunne response.A kind of method comprises uses antigen presenting cell (APC), presents PSCA antigen like dendritic cell (DC) for patient's immune system.Dendritic cell expresses the MHCI class and II quasi-molecule, B7 are total to stimulator and IL-12, is the antigen presenting cell of eggcase therefore.In prostate cancer, be used to the I clinical trial phase to stimulate immunity system (Tjoa et al., 1996, the Prostate 28:65-69 of patients with prostate cancer with self dendritic cell of Prostato-specific membrane antigen (PSMA) peptide pulse; Murphy et al., 1996, Prostate 29:371-380).Therefore, divide in the subenvironment, can use dendritic cell that the PSCA peptide is and pass T cell in MHCI class or mhc class ii.In one embodiment, with can with MHCI class and/or II quasi-molecule bonded PSCA peptide pulse self dendritic cell.In another embodiment, with complete PSCA albumen pulse dendritic cell.Another embodiment comprises uses multiple tool carrier known in the art; Like adenovirus (Arthur et al., 1997, Cancer Gene Ther.4:17-25), retrovirus (Henderson et al.; 1996; Cancer Res.56:3763-3770), slow virus, gland-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res.57:2865-2869) or derive from RNA transfection (the Ashley et al. of tumour; 1997, J.Exp.Med.186:1177-1182) psca gene in the dendritic cell is crossed expression and transform.Also can transform the cell of expressing PSCA so that it expresses immunomodulator, like GM-CSF and as immunizing agent.

X.B.) PSCA is used as the target based on the therapy of antibody

PSCA is the target of attractive therapeutic strategy based on antibody.Multiple targeted cells known in the art outer with cell in the antibody strategy (referring to the use of killing and wounding of mediating of for example complement and ADCC and endosome) of molecule.Because for corresponding normal cell; The cancer cell of multiple pedigree is expressed PSCA; Prepare general and use PSCA-immunoreation compsn; Said composition shows fabulous susceptibility, does not show to combine the toxicity that causes, non--specificity and/or non--target effect by immunoreation compsn and non--target organ and tissue.With the antibody conduct of PSCA structural domain specific reaction and the conjugate of toxin or therapeutical agent, or, can be used for the cancer that systemic treatment is expressed PSCA as the exposed antibody that can suppress cell proliferation or function.

Can psca antibody be imported the patient so that antibody combines with PSCA and modulation function, as with the interaction that combines counterpart, mediate the destruction of tumour cell and/or the growth of inhibition tumour cell subsequently.The mechanism of said antibody performance therapeutic action comprises that physiological function, inhibition part combination or signal transduction pathway, the differentiation of modulation tumour cell, the change tumor angiogenesis factor of the lysis of complement-mediation, the cell-mediated cytotoxicity that depends on antibody, modulation PSCA distribute and/or apoptosis.Examples include for non - Jieshi lymphoma Rituxan

, for the transfer of breast cancer Herceptin and for colorectal cancer Erbitux

.

Those skilled in the art understand can use antibodies specific target and binding immunoassay originality molecule, the immunogenicity zone of PSCA sequence for example shown in Figure 1.In addition, those skilled in the art understand that the coupling of antibody and cytotoxic agent is routine techniques (referring to for example Slevers et al.Blood 93:113678-3684 (June 1,1999)).For example, when through making cytotoxicity and/or therapeutical agent and being specific to the antibody coupling of the expressed molecule of cell (like PSCA) and when they directly were passed to cell, cytotoxic agent will be to said cell performance its known biological action (being cytotoxicity).

Using antibody-cytotoxic agent conjugate is known in the art with the multiple compsn and the method for cell killing.In the context of cancer; Typical method uses the conjugate of biologic effective dose must for the animal that suffers from tumour; Said conjugate contains selected cytotoxic agent and/or the therapeutical agent that is connected with target agent (like anti--psca antibody); Said target agent combines with the mark (like PSCA) of expression, is easy to combine or be positioned cell surface.Typical embodiment is the method that cytotoxic agent and/or therapeutical agent is passed to the cell of expressing PSCA; Said method comprises the antibody coupling that makes cytotoxic agent combine the PSCA epi-position with immunologic opsonin, and with cellular exposure in antibody-cytotoxic agent conjugate.Another illustrative embodiment is the method that the individuality of suffering from metastatic cancer is suspected in treatment; Said method comprises the step to the non-enteron aisle drug administration of said individuality compsn, said compsn contain the treatment significant quantity with cytotoxic agent and/or therapeutical agent link coupled antibody.

Can use the immunotherapy for cancer of anti--psca antibody according to the multiple method for cancer that successfully is used to treat other type, said cancer includes but not limited to: and colorectal carcinoma (Arlen et al., 1998, Crit.Rev.Immunol.18:133-138); Multiple myeloma (Ozaki et al., 1997, Blood90:3179-3186, Tsunenari et al.; 1997, Blood 90:2437-2444), cancer of the stomach (Kasprzyk etal., 1992; Cancer Res.52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother.Emphasis Tumor Immunol.19:93-101); White blood disease (Zhong et al., 1996, Leuk.Res.20:581-589); Colorectal carcinoma (Moun et al., 1994, Cancer Res.54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403) and mammary cancer (Shepard et al., 1991, J.Clin.Immunol.11:117-127).Some treat-ment comprise makes exposed antibody and toxin or ri coupling, like Y ⁹¹Or I ¹³¹Respectively with anti-CD 20 antibodies coupling (Zevalin for example ^TM, IDEC Pharmaceuficals Corp.or B exxar ^TM, CoulterPharmaceuficals), and other treat-ment comprises common administration of antibodies and other therapeutical agent, as using Herceptin simultaneously ^TM(trastuzuMAb) with paclitaxel (Genentech, Inc.).Antibody can with the therapeutical agent coupling.For example, in order to treat kidney, can when using psca antibody, use radiotherapy, chemotherapy or hormone to break away from therapy.In addition, antibody can be with toxin conjugated, and said toxin such as calicheamycin are (like Mylotarg ^TM, Wyeth-Ayerst, Madison, NJ; A kind of and Anti-tumor microbiotic calicheamycin link coupled recombinant humanized IgG4 κ antibody) or maytansinoid (like the tumour-activation prodrug based on taxane, TAP, platform; ImmunoGen, Cambridge, MA; Also can be referring to for example USP 5,416,064) or Ao Ruisitading E (Nat Biotechnol.2003 Jul; 21 (7): 778-84. (SeattleGenetics)).

Although the psca antibody therapy can be used for the cancer in all stages, antibody therapy is particularly suitable for late period or metastatic cancer.To accept one take turns or take turns more chemotherapy the patient carry out antibody therapy of the present invention.Perhaps, the patient who did not accept chemotherapy is carried out antibody therapy of the present invention and chemotherapy or radiotherapy scheme.In addition, antibody therapy can make the chemotherapy dosage that accompanies reduce, and is all the more so for toxic patient that can not the well tolerated chemotherapeutic agents.(Cancer Res.53:4637-4642 such as Fan; 1993); The use of uniting of multiple antibody and chemotherapeutics has been described in (Cancer Res.51:4575-4580,1991) such as Prewett etc. (International J.of Onco.9:217-224,1996) and Hancock.

Although the psca antibody therapy can be used for the cancer in all stages, antibody therapy is particularly suitable for late period or metastatic cancer.To accept one take turns or take turns more chemotherapy the patient carry out antibody therapy of the present invention.Perhaps, the patient who did not accept chemotherapy is carried out antibody therapy of the present invention and chemotherapy or radiotherapy scheme.In addition, antibody therapy can make the chemotherapy dosage that accompanies reduce, and is all the more so for toxic patient that can not the well tolerated chemotherapeutic agents.

Preferred use the immunohistochemical methods evaluation of tumor tissues, quantitatively PSCA video picture or other can represent reliably that the technology of existence and the level of psca expression estimates the existence and the level of psca expression among the cancer patients.Preferably tumor biopsy or surgical samples are carried out immunohistochemical analysis for this reason.The method of tumor tissues being carried out immunohistochemical analysis is well-known in the art.

Anti--PSCA monoclonal antibody of treatment prostate cancer and other cancer comprises and can cause strong anti-tumor immune response or directly have Cytotoxic monoclonal antibody.In this; Anti--PSCA monoclonal antibody (MAb) can cause the tumour cell cracking through cell-mediated cytotoxicity complement-mediation or that depend on antibody (ADCC) mechanism; These two kinds of mechanism all need the complete Fc part of immunoglobulin molecules, with complement protein on effector cell Fc acceptor site interact.In addition, the anti--PSCA MAb that tumor growth is directly brought into play biological action can be used for treating the cancer of expressing PSCA.Directly the mechanism of action of cell toxicant MAb comprises: cell growth inhibiting, modulation cytodifferentiation, modulation tumor angiogenesis factor distribute and cell death inducing.Use the in vitro tests of any evaluation necrocytosis, as the lysis of ADCC, ADMMC, complement-mediation wait estimate specific anti--mechanism of PSCAMAb performance Anti-tumor effect, said test generally is known in the art.

In some patients, use mouse or other non--human monoclonal antibodies, or the chimeric MAb of people/mouse can induce anti--non-human antibody's moderate to intensive immunne response.This can cause antibody from circulation, to be eliminated and to render a service reduction.In the majority of severe case, said immunne response can cause the extensive formation of immunocomplex, and said mixture can potentially cause renal failure.Therefore, the preferred monoclonal antibody that is used for treat-ment of the present invention is those total mans or humanization, and combines with target PSCA antigen-specific with high-affinity, but in the patient, shows the monoclonal antibody of low antigenicity or no antigen.

Treat-ment expectation of the present invention is used anti--PSCA MAb and associating separately or is mixed and use different MAb.Said MAb mixture owing to the MAb that contains the different epi-positions of target, utilize different effect thing mechanism or direct union cell toxicant MAb and rely on the functional MAb of immunoeffectors and have some advantages.Said MAb combination can show synergistic therapeutic action.In addition, anti--PSCA MAb can with include but not limited to that multiple chemotherapeutics, male sex hormone-blocker, immunomodulator are (like IL-2, GM-CSF), other form of therapy of surgical operation or radiotherapy uses together.Anti--PSCA the MAb of " exposing " or not coupling form be can use, therapeutical agent and said MAb coupling perhaps made.

Can the approach that antibody is passed to tumour cell be used anti--PSCA MAb preparation via any.Route of administration includes but not limited in the intravenously, intraperitoneal, intramuscular, tumour, intracutaneous etc.Treatment generally comprises via acceptable route of administration, as intravenous injection (IV) repetitive administration anti--PSCA MAb goods, dosage range generally is about 0.1 .2 .3 .4 .5 .6 .7 .8 .9,1,2,3,4,5,6,7,8,9,10,15,20 or the 25mg/kg body weight.Generally speaking, 10-1000mg MAb is effectively and can be by tolerance preferably weekly.

Based on using Herceptin ^TMThe clinical experience of the mammary cancer that the MAb treatment is shifted, acceptable dosage is that original intravenously loading dose is about the 4mg/kg weight in patients, then, intravenous dosages weekly is about 2mg/kg and resists-PSCA MAb goods.Preferably with 90 minutes or the longer time infusion use original loads dosage.If original doses can be by tolerance preferably, with 30 minutes or infusion administration period maintenance dose for more time.Those skilled in the art understand that multiple factor can influence the optimal dosage scheme of concrete case.Said factor comprises that binding affinity and half life, the psca expression level among the patient, the circulation of for example used Ab or MAb flow out the antigenic degree of PSCA, required stable state AC level, therapeutic frequency and unite chemotherapeutics or the influence of other medicament and the healthy state of particular patient of use with treat-ment of the present invention.

The level of PSCA in the given sample of optional evaluate patient (the for example level of the cell of cycle P SCA antigen and/or expression PSCA) is to assist definite the most effectively dosage etc.Said evaluation also can be used for the monitoring in the therapeutic process; And can be used for other parameter (like urinary cytology in the bladder cancer therapy and/or ImmunoCyt level; Or similarly, the Serum PSA level in the prostate cancer therapy) evaluation combine and estimate whether success of treatment.

In anti--cancer therapy, anti--idiotype is anti--and psca antibody also can be used as vaccine to cause the immunne response to the cell of expressing the PSCA-related protein.Particularly, the method that produces anti--idiotype antibody is well-known in the art; Can easily change this method with the anti--idiotype that produces the epi-position on the simulation PSCA-related protein anti--psca antibody is (referring to for example Wagner et al., 1997, Hybridoma16:33-40; Foon et al., 1995, J.Clin.Invest.96:334-342; Herlyn et al., 1996, Cancer Immunol.Immunother.43:65-76).Said resisting-idiotype antibody can be used for the cancer vaccine strategy.

The purpose of this invention is to provide psca antibody, it can suppress or delay to express the growth of the tumour cell of PSCA.Thereby another object of the present invention provides and suppresses vasculogenesis and other biological function minimizing Mammals; The method of tumor growth in the preferred human body; Said method has been used psca antibody, particularly when using psca antibody, also unites and has used radiation and chemotherapy or the two.

In one embodiment, use psca antibody and chemotherapeutics or radiotherapy or its combined therapy tumour, when comprising people's tumour, can produce synergy when uniting.In other words, when with chemotherapeutics or radiotherapy or the use of its combinatorial association, psca antibody can be than the enhancing to some extent of expection to the restraining effect of tumor growth.For example, the desired result that combination therapy is only treated with psca antibody the restraining effect ratio of tumor growth, or use the Overlay of psca antibody and chemotherapeutics or radiotherapy stronger, this point can demonstrate synergy.Preferably illustrate synergy, wherein all can not cause the alleviation of cancer with the combined therapy expection of exposed psca antibody treatment or use psca antibody and chemotherapeutics or radiotherapy through the alleviation of cancer.

The method of using psca antibody and chemotherapeutics or radiotherapy or both combinations to suppress growth of tumour cell comprises: before beginning chemotherapy or the radiotherapy, during or afterwards and arbitrary combination (promptly before beginning chemotherapy and/or the radiotherapy and during; Before and afterwards; During this time and afterwards; Or before, during and afterwards) use psca antibody.For example, generally 1 to 60 day before beginning radiotherapy and/or chemotherapy preferred 3 to 40 days, was more preferably used psca antibody in 5 to 12 days.Yet, different according to regimen and patient's specific demand, it is that aim is implemented said method that Ying Yineng provides efficacious therapy and the final patient of prolongation life.

Administration of chemotherapeutic agents comprises through non-enteron aisle and the administration of enteron aisle approach whole body in many ways.In one embodiment, psca antibody and chemotherapeutics are treated as independently molecule administration.In another embodiment, psca antibody through for example coupling combine with chemotherapeutics (referring to be entitled as " through end user in the body anti--people's clinical trial of psca antibody treatment and diagnosis human cancer " embodiment and the trifle that is entitled as " PSCA is as the target based on the therapy of antibody ").The special example of chemotherapeutics or chemotherapy comprises: cis-platinum, dicarbazine (dacarbazine; DTIC), gengshengmeisu (dactinomycin), mechlorethamine (mustargen), streptozotocin (streptozocin), endoxan (cyclophosphamide), carmustine (carmustine) (BCNU), lomustine (lomustine, CCNU), adriamycin (adriamycin), daunorubicin, the third kappa nitrile (procarbazine), MTC, cytosine arabinoside (cytarabine), VP, methotrexate (methotrexate), 5 FU 5 fluorouracil (5-fluorouracil), vincaleucoblastine, vincristin, bleomycin (bleomycin), taxol (paclitaxel), Japanese yew terpene (taxotere), rIL-2 (aldesleukin), asparaginase (asparaginase), busulfan (busulfan), carboplatin (carboplatin), carat upright shore (cladribine), dicarbazine, floxuridine (floxuridine), fludarabine (fludarabine), hydroxyl urea (hydroxyurea), suitable Floex acid amides (ifosfamide), interferon alpha, leuprorelin (leuprolide), megestrol (megestrol), melphalan (melphalan), purinethol (mercaptopurine), Plicamycin (plicamycin), mitotane (mitotane), pegaspargase (pegaspargase), penta system rhzomorph (pentostatin), pipobroman, tamoxifen (tamoxifen), teniposide (teniposide), testolactone, thioguanine (thioguanine), plug for send (thiotepa), uracil mustard (uracil mustard), dewater vincaleucoblastine (vinorelbine), Amboclorin (chlorambucil), taxol and combination thereof.

The radioactive source of uniting use with psca antibody can place to be waited to treat in patient's the external or body.When radioactive source is positioned at the patient when external, therapy is outside ray radiotherapy (EBRT).When radioactive source was positioned at patient's body, treatment was called as short therapy (brachytherapy) (BT).

Use for this purpose and the standard equipment of making like AECL Theratron and Varian Clinac, is used radiotherapy according to well-known standard technique.The dosage of radiotherapy depends on a plurality of factors, and this point is well-known in the art.Said factor comprises that the organ of being treated, the healthy organ that in the radiotherapy path, possibly inadvertently affect adversely, patient are to the tolerance degree of radiotherapy and the health area of needs treatment.Dosage is generally 1 to 100Gy, more particularly is 2 to 80Gy.Some dosage of having reported comprise 35Gy (to spinal cord), 15Gy (to kidney), 20Gy (to liver) and 65-80Gy (to prostate gland).Yet what should stress is that the present invention is not limited to any specific dosage.The doctor is according to confirming dosage to the specific factor under the stable condition in treatment.

External radiation source and the distance that gets between patient's the site can be any distances, and said distance is being represented and killed and wounded target cell and making spinoff obtain acceptable balance between minimizing.Generally speaking, external radiation source and to get into distance between patient's the site be 70 to 100cm.

Generally, radioactive source lacks therapy through being placed in patient's body.Usually radioactive source is placed and tissue to be treated at a distance of the place of about 0-3cm.Known technology comprises in the tissue, the short therapy in the chamber and surface.Can be for good and all or implant radioactive source provisionally.Some typically have been used for the permanent radioactive atom of implanting and have comprised iodine-125 and radon.Some typically have been used for the interim radioactive atom of implanting and have comprised radium, caesium-137 and iridium-192.Some other radioactive atoms that have been used for short therapy comprise americium-241 and gold-198.The radiation dose of short therapy can be identical with the dosage of said external ray radiotherapy.Except the factor of above-mentioned definite outside ray radiotherapy dosage, when confirming the dosage of short therapy, the also characteristic of the used radioactive atom of considered.

X.C.) PSCA is as the target of cellullar immunologic response

Other embodiment of the present invention is vaccine and the method for preparing vaccine, and said vaccine contains one or more HLA-binding peptides as herein described of immunogenicity significant quantity.In addition, vaccine of the present invention comprises the compsn of one or more peptides that require.Peptide can separately exist in the vaccine.Perhaps, peptide can be used as the homopolymer that contains the identical peptide of multiple copied and exists, or the heteropolymer of conduct and multiple peptide exists.The advantage of polymkeric substance is enhancing immunity to react, and when using different peptide epitopes to form polymkeric substance, can also induce the antibody and/or the CTL that produce with the different antigenic determinants of pathogenic organisms or the tumour of immunne response institute target-related peptides reaction.Compsn can be antigenic natural zone, can prepare said compsn through for example reorganization or chemosynthesis.

Can be well-known in the art, comprise for example Thyroprotein, BSA, Toxoid,tetanus, polyamino acid, influenza virus, hepatitis B virus core protein etc. as gathering L-Methionin and L-glutamic acid like human serum albumin with the carrier that vaccine of the present invention uses.Vaccine can contain (can accept) thinner that physiology can tolerate, and like water or salt solution, is preferably PBS.Vaccine generally also comprises adjuvant.Adjuvant like incomplete Freund's adjuvant, phosphagel phosphaljel, white lake or alum is the example of Adjuvanting material well-known in the art.In addition, as disclosed herein, through making peptide of the present invention and lipid, like three palmitoyl (tripalmitoyl)-S-glyceryl cysteinyl seryl-Serine (P ₃CSS) coupling can cause ctl response.In addition, found can ctl response be strengthened 10 to 100 times (referring to for example Davila and Celis, J.Immunol.165:539-547 (2000)) such as the adjuvant of the oligonucleotide that contains synthetic cytosine(Cyt)-thiophosphoric acidization-guanine (CpG).

Through via injection, aerosol, oral, through skin, in mucous membrane, pleura, in the sheath or other suitable approach carry out immunization with peptide combinations of the present invention, host's immunity system is specific to required antigenic CTL and/or HTL reacts to vaccine in a large number through producing.As a result, the host is at least to the expression that produced afterwards or cross the antigenic cell of expression PSCA and produced partial immunity power, or when antigen is tumour-related antigen, can obtain the benefit that some are treated at least.

In some embodiments, need combination use I class peptide compsn with can induce or promote to the neutralizing antibody of target antigen with or the component of helper cell reaction.The preferred embodiment of said compsn contains I class of the present invention and II class epi-position.The selectable embodiment of another of said compsn contains the HTL epi-position of I class of the present invention and/or II class epi-position and cross reaction, like PADRE ^TM(Epimmune, San Diego, CA) molecule (being described in for example USP 5,736,142).

Vaccine of the present invention also can comprise antigen-be delivery cell (APC), like dendritic cell (DC) as the carrier of presenting peptide of the present invention.Mobilization with collect dendritic cell after, can be at external generation vaccine composition, thus at the external load that carries out dendritic cell.For example, use for example according to minigene transfection dendritic cell of the present invention, or with peptide pulse dendritic cell.Then can dendritic cell be applied to the patient to cause immunoreation in vivo.The vaccine composition that no matter is based on DNA or peptide also can be united vivo medicine-feeding with the dendritic cell mobilization, thereby carries out the load of dendritic cell in vivo.

Preferably, when selecting to be included in the epi-position array of the multi-epitope synthetics that is used for vaccine, or select to be included in the vaccine and/or during by the discrete epi-position of nucleic acid such as minigene coding, adopt following principle.Each of the preferred following principle of balance is to select.The a plurality of epi-positions that are included in given vaccine composition can be, but are not necessarily to obtain the contiguous sequence in the natural antigen of epi-position.

1.) select epi-position, said epi-position can be simulated after administration through observing with tumour and remove relevant immunne response.As far as the HLAI class, this comprises 3-4 epi-position from least one taa (TAA).As far as HLA II class, adopt similar principle; Select 3-4 epi-position (referring to for example Rosenberg et al., Science 278:1447-1450) again by at least one TAA.Epi-position from a TAA can be united use with the epi-position from one or more other TAA, and to produce the vaccine of target tumor, said tumour has the different expression patterns of the TAA of frequent expression.

2.) select epi-position, said epi-position has confirms the necessary binding affinity relevant with immunogenicity: as far as HLAI class, IC ₅₀Be 500nM or lower, be generally 200nM or lower; As far as II class, IC ₅₀Be 1000nM or lower.

3.) select enough peptides that carries hyper-base unit or enough peptide arrays that carries the allele-specific primitive to give population coverage rate widely.For example, preferably has at least 80% population coverage rate.MonteCarlo analyzes, and a kind of statistical estimation known in the art can be used to assess scope or the redundancy that population covers.

4.) when from cancer associated antigens, selecting epi-position,, select analogue normally useful because the patient can produce the tolerance to natural epi-position.

5. relevant especially epi-position refers to " nested epi-position ".Nested epi-position appears in the overlapping at least two epi-positions of given peptide sequence.Nested peptide sequence can comprise B cell, HLAI class and/or HLAII class epi-position.When nested epi-position was provided, a common purpose was the epi-position that each sequence provides maximum number.Therefore, be for fear of providing on the one hand than the aminoterminal of aminoterminal epi-position in the peptide sequence and the long peptide of carboxyl terminal of carboxyl terminal epi-position.When a multi-epitope sequence is provided, when for example containing the sequence of nested epi-position, the screening sequence with confirm that it does not have pathology or other deleterious biological nature normally very important.

6.) if preparation multi-epitope protein or preparation during minigene, purpose is to produce the minimum peptide that comprises the target epi-position.If the principle that this principle adopts during with the peptide selecting to have nested epi-position is inequality also to be similar.Yet for the artificial multi-epitope peptide, minimized purpose and the needs of between the proteinic epi-position of multi-epitope, integrating the arbitrary interval sequence balance each other.For example, can introduce at interval amino-acid residue avoiding connecting epi-position (epi-position by immune system recognition is not present in the target antigen, and is only produced by manual work epi-position arranged side by side), or the cracking of supplementary table interdigit, present thereby strengthen epi-position.Usually should avoid connecting epi-position, because the recipient possibly produce immunne response to the non-natural epi-position.Special concern be connection epi-position as " determinant epi-position ".The determinant epi-position possibly cause intensive to be replied, thereby the immunne response to other epi-position is lowered or inhibition.

7.) when the sequence of a plurality of variants of same target protein exists, also can select the potential peptide epitopes based on their conservative property.For example, the principle of conservative property may be defined as, and the whole 9-aggressiveness core of the complete sequence of HLAI class binding peptide or II class binding peptide is conservative in being estimated the sequence of the antigenic designated ratio of specific protein.

X.C.1. minigene vaccine (minigene vaccine)

Can use the multiple different method that allows to transmit simultaneously a plurality of epi-positions.The nucleic acid of peptide of the present invention of encoding is useful especially embodiment of the present invention.The guide of listing according to last part, the preferably epi-position in being included in minigene.Preferably using the mode of the nucleic acid of code book invention peptide has used coding to contain the minigene construct of the peptide of one or more epi-positions of the present invention.

The application of multi-epitope minigene such as hereinafter and 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 is said.For example, can transform the epi-position that carries hyper-base unit and/or primitive of the said plasmid-encoded PSCA of deriving from, PADRE to the Polyepitope DNA plasmid ^TMGeneral helper T cell epitope or derive from a plurality of HTL epi-positions (referring to for example Table V-XVIII and XXII-LI) of PSCA, and endoplasmic reticulum encoding transport signals sequence.Vaccine also can comprise the epi-position that derives from other TAA.

The immunogenicity of multi-epitope minigene can be confirmed in transgenic mice, with the reaction of assessment CTL inductive to the position of being taken temperature.In addition, the immunogenicity of the epi-position of dna encoding can be associated to the vitro reactions with the target cell of DNA plasmid transfection with specific CTL system in the body.Therefore, these experiments can show that minigene is used for: 1.) produce that CTL replys and 2.) cell of inductive CTL recognition expression coding epi-position.

For example, for producing dna sequence dna (minigene) that coding selects epi-position in people's cell, to express, the aminoacid sequence of epi-position can be reversed translation.People's codon use table can be used for instructing each amino acid whose codon to select.The dna sequence dna of these coding epi-positions can directly connect, and makes when by translation, can produce the successive peptide sequence.For optimized expression and/or immunogenicity, can in the minigene design, add additional element.The aminoacid sequence instance of can reverse translation and being included in the minigene sequence comprises: HLAI class epi-position, HLAII class epi-position, antibody epitope, ubiquitination signal sequence and/or endoplasmic reticulum target signal.In addition, the HLA of CTL and HTL epi-position presents can be through comprising synthetic (for example Polyalanine) or natural improving with CTL or the adjacent flanking sequence of HTL epi-position, and these bigger peptides that contain epi-position also within the scope of the invention.

Can the minigene sequence be converted into DNA through the oligonucleotide of assembling coding minigene normal chain and minus strand.Can be under suitable condition with well-known technology synthesize, phosphorylation, purifying and the overlapping oligonucleotide of annealing (a long 30-100 base).The end of oligonucleotide can be used for example T4 dna ligase connection, and the minigene of this synthetic coding epitope polypeptide can be cloned into a required expression vector.

Preferably standard adjustment sequence well known by persons skilled in the art is included in the carrier to guarantee the expression in the target cell.Need some carrier elements: have the downstream cloning site promotor of minigene insertion altogether; The polyadenylic acid signal that effective termination is transcribed; The intestinal bacteria replication orgin; And intestinal bacteria selective marker (like penbritin or kalamycin resistance).Can adopt multiple promotor for this reason, human cytomegalic inclusion disease virus (hCMV) promotor for example, other suitable promoter sequence can be referring to for example USP 5,580,859 and 5,589,466.

Possibly need other carrier modification to optimize the expression and the immunogenicity of minigene.In some cases, the efficient gene expression needs intron, one or more synthetic or zones of transcribing that natural intron can mix minigene.For increasing the expression of minigene, also can consider to comprise the mRNA critical sequences and in mammalian cell, duplicate required sequence.

In case selected expression vector, minigene be cloned into the polylinker zone in promotor downstream.This plasmid is converted into suitable coli strain, prepares DNA with standard technique.Confirm other all elements of comprising in direction and the dna sequence dna and the carrier of minigene with restriction map and dna sequence analysis.The bacterial cell that contains correct plasmid can save as master cell bank and working cardial cell storehouse.

In addition, as if immunostimulatory sequence (ISS or CpG) works in the immunogenicity of dna vaccination.Improve immunogenicity if desired, these sequences can be included in the carrier, are positioned at the outside of minigene encoding sequence.

In some embodiments, can adopt the two-cistron expression vector that allows to produce simultaneously minigene coding epi-position and second kind of protein (being included) to improve or to reduce immunogenicity.Coexpression can improve the protein of immunne response valuably or the instance of polypeptide comprises: cytokine is (like IL-2; IL-12; GM-CSF), cytokine induction molecule (like LeIF), costimulatory molecules, or the conjugated protein (PADRE of the pan-DR for HTL replys ^TM, Epimmune, San Diego, CA).Auxiliary (HTL) epi-position can combine with target signal in the cell, and separates expression with the CTL epi-position of expressing; This allows to guide the HTL epi-position to get into and CTL epi-position different cells compartment.In case of necessity, this helps the HTL epi-position more effectively to get into the HLAII classpath, therefore improves HTL and induces.Induce on the contrary with HTL or CTL, reducing immunne response through coexpression immunosuppression molecule (for example TGF-β) specificity is useful in some diseases.

The plasmid that can prepare therapeutic dose through the purifying then of for example in intestinal bacteria, fermenting.According to well-known technology, the aliquots containig in working cardial cell storehouse is used to inoculate growth medium, grow in shaking bottle or bio-reactor saturated.DNA can be used the bioseparation technology purifying of standard, for example QIAGEN Inc. (Valencia, the solid phase anionite-exchange resin that California) provides.If desired, can from open loop and linear forms, isolate super coiled DNA with gel electrophoresis or other method.

Can be used for injection with the DNA that several formulations prepares purifying.The most simply be that refrigerated DNA is rebuild in sterile phosphate buffered saline (PBS).This method that is known as " naked DNA " is used to intramuscular (IM) injection at present in clinical trial.For making the immunotherapy maximum effect of minigene dna vaccination, need the method for a selectable preparation plasmid DNA purification, several different methods has been described, also can use new technology.Cation lipid, glycolipid and fusion liposome also can be used in the prescription (referring to for example WO93/24640; Mannino & Gould-Fogerite, BioTechniques6 (7): 682 (1988); USP 5,279,833; WO91/06309; And Felgner, et al, Proc.Nat ' lAcad.Sci.USA 84:7413 (1987)).In addition, be generically and collectively referred to as protectiveness, activated, non-cohesion compound (PINC) peptide and compound can compound for example stability, intramuscular disperse or arrive the variable of certain organs or cell type with influence with the DNA of purifying.

Target cell sensitization can be as the expression of the CTL epi-position of minigene coding and the functional selection that the HLAI class is presented.For example, DNA is introduced the suitable mammal cell line that is used as the target of standard CT L chromium-release test.Used transfection method will depend on final prescription.Electroporation can be used for " naked " DNA, and cation lipid allows the mediation in-vitro transfection.The plasmid of expressing green fluorescent protein (GFP) can be by cotransfection with cell sorting (FACS) the enrichment cells transfected with fluorescence-activation.Then with these cells with press against-51 ( ⁵¹Cr) mark, and be used as the target cell that epitope specificity CTL is; Through ⁵¹Generation and HLA that Cr discharges the CTL epi-position of detected lysis hint minigene coding present.Can use and assess the active similarly mode of HTL and assess the expression of HTL epi-position.

Immunogenicity is the second method of functional assays minigene DNA prescription in the body.Express the suitable proteic transgenic mice of people HLA with the immunity of DNA product.Dosage and approach depend on prescription (for example, the DNA among the PBS uses IM, injects with intraperitoneal (i.p.) with lipid compound DNA).Back 21 days of immunity is collected splenocyte and in the presence of the peptide of each determined epi-position of coding, is stimulated a week again.Then, for CTL effector cell, load there is the warp of peptide with standard technique ⁵¹The lysis of the target cell of Cr mark is analyzed.Use the cracking of the target cell of the HLA sensitization that has the corresponding peptide epitopes of epi-position of encoding to show that dna vaccination is used for inducing in the body function of CTL with minigene.In transgenic mice, confirm the immunogenicity of HLT epi-position with similar fashion.

Alternatively, nucleic acid can be used the administration of described trajectory transfer mode, and for example USP 5,204, and 253.Adopt this technology, use the particle that only contains DNA.In further optional embodiment, DNA can be sticked on the particle, for example goldc grains.

Minigene also can be used other bacterium well known in the prior art or viral systems communicate, and for example, the expression construct of code book invention epi-position can be mixed virus vector, for example vaccinia virus.

The combination of X.C.2.CTL peptide and auxiliary peptide

The vaccine composition that contains ctl peptide of the present invention can be modified, and for example analogizes the attribute that needs to provide, the serum half life, the population fraction of coverage of expansion or the immunogenicity of raising that for example improve.

For example, the active ability of inducing peptide CTL can through with peptide with contain at least one sequence that can induce the epi-position that t helper cell replys and be connected and improve.Although ctl peptide can directly link to each other with the auxiliary peptide of T, normally CTL epi-position/HTL epi-position conjugate interconnects through spacer molecule.Spacer molecule is formed like uncharged basically amino acid or amino acid analogue under physiological condition usually by less relatively neutral molecule.Spacer molecule is selected from for example Ala usually, the neutral spacer molecule of Gly or other nonpolar amino acid or neutral pole acidic amino acid.Being understood that optional spacer molecule needn't be made up of identical residue, can be allos or homologous oligomer therefore.When occurring, spacer molecule has 1 or 2 residue usually at least, and more commonly 3 to 6 residues are 10 or more residue sometimes.The ctl peptide epi-position can directly be connected with the auxiliary peptide epitopes of T or connect through the aminoterminal or the carboxyl terminal of spacer molecule at ctl peptide.The aminoterminal of the auxiliary peptide of immunogenic peptide or T all can be by acidylate.

The htl peptide epi-position also can be modified changes its biological nature.For example, they can be modified to and comprise D-amino acid and improve them to resistance towards proteases, have therefore prolonged their serum half life.Or they can with other molecule, for example couplings such as lipid, protein, carbohydrate are to improve their biological activity.For example, the auxiliary peptide of T can be connected with the amino or the carboxyl terminal of one or more palmitinic acid chains.

The combination of X.C.3.CTL peptide and T cell initiator

In some embodiments, perhaps need at least a ability inducing B lymphocyte or the lymphocytic composition of T be included in the pharmaceutical composition of the present invention.Confirmed that lipid is a kind of reagent that can cause CTL in vivo.For example, palmitic acid residues can be connected to the ε of lysine residue-with the alpha-amino group group, then via for example one or more connects for example Gly of residue, Gly-Gly-, Ser, Ser-Ser etc. are connected on the immunogenic peptide.The fat peptide can mix liposome directly in microne or particle, or in like the adjuvant of incomplete Freund's adjuvant emulsification to carry out administration.In preferred embodiments, a kind of significantly effectively immunogenic composition contain with the ε of Lys-with alpha-amino group group bonded palmitinic acid, it is through joint, for example Ser-Ser combines with the aminoterminal of immunogenic peptide.

Cause the example that CTL replys as another lipid, intestinal bacteria lipoprotein is for example with the covalently bound three palmitoyl-S-glyceryl cysteinyl seryl-Serine (P of peptide that is fit to ₃CSS) can be used for causing the CTL (referring to for example Dereset al, Nature 342:561,1989) of virus-specific.Peptide of the present invention can with P ₃The CSS coupling for example, gives individual lipopeptid and causes the immunne response to target antigen with specificity.In addition, also can use and P owing to inducing of neutralizing antibody ₃CSS link coupled epi-position causes, thereby two such compsns can be united to use and more effectively caused body fluid and cell-mediated replying.

Vaccine composition embodiment of the present invention comprises to the PBMC in the blood samples of patients or therefrom isolating DC from the mixture of using the peptide that carries epi-position externally.Can use the medicine that helps to collect DC, for example, Progenipoietin ^TM(Pharmacia-Monsanto, St.Louis, MO) or GM-CSF/IL-4.After with peptide pulsed D C, and infusion is before the patient again, and DC is to remove unconjugated peptide in flushing.In this embodiment, vaccine comprises the DC of peptide pulse, exists and HLA molecule compound pulse peptide epitopes on its surface.

DC can use peptide mixt from external earth pulse, and some of them stimulate the CTL to PSCA to reply.Randomly, helper cell (HTL) peptide, the HLAII class peptide of for example natural or artificial lax restriction, includable so that CTL replys.Therefore vaccine of the present invention can be used to treat expression or crosses and express the cancer of PSCA.

X.D.) adoptive immunotherapy (adoptive immunotherapy)

Antigenicity PSCA related peptides also can be used for from cause CTL and/or HTL replys externally; CTL that obtains or HTL cell can be used for treating those not reactions of treatment to other conventionally form, or to therapeutic vaccine peptide or the responseless tumour patient of nucleic acid of the present invention.Through in tissue culture with the patient's or the compatible CTL of heredity or HTL precursor cell and antigen presenting cell (APC) source, be incubated jointly like dendritic cell and the immunogenic peptide that suits, induce next exogenic CTL or HTL to reply to specific antigen.After suitable soaking time (about 7-28 days usually); Wherein precursor cell is activated and expands to the effector cell; Cell is fed back in patient's body, and they will destroy (CTL) or promote destroy (HTL) their specific target cells (for example, tumour cell) there.The dendritic cell of transfection also can be used as antigen presenting cell.

X.E.) use vaccine for the purpose of treating or prevent

Medicine of the present invention and vaccine composition are generally used for treating and/or preventing expression or cross and express the cancer of PSCA.In therapeutic is used, to the antigenic amount that has effective B cell, CTL and/or HTL to reply and cure or at least partly contain or slow down symptom and/or complication, give patient's administration for peptides and/or nucleic acid composition with enough initiations.The amount of enough accomplishing these purposes is defined as " treatment effective dose ".The significant quantity of this application for example depends on, the particular composition of using, the mode of administration are treated stage and severity, patient's body weight and the general health situation and the doctor's that writes a prescription the judgement of disease.

For pharmaceutical composition, immunogenic peptide of the present invention or their DNA of encoding are given and are suffered from the individuality of the tumour of expressing PSCA usually.Peptide or their DNA of encoding can be separately or as the fusions administration of one or more peptide sequences.Can be separately with immunogenic peptide or suitably the time and other therapies, like surgical operation combination therapy patient.

Use for therapeutic, administration should begin from the diagnosis first of the relevant cancer of PSCA usually.Booster dose alleviates in fact and after for some time up to symptom at least then.Give with the embodiment (promptly including but not limited to like peptide mixt multi-epitope polypeptide, the embodiment of minigene or TAA-specific CTL or pulse dendritic cell) of patient's vaccine composition can be according to the stage of disease or patient's state of health adjustment.For example, in expressing the tumour patient of PSCA, the vaccine that contains the PSCA-specific CTL can more effectively be killed tumour cell than selectable embodiment in suffering from patient's body of high-stage disease more.

It generally is important that a certain amount of peptide epitopes through being enough to the mode of administration transmission that the effective stimulus cytotoxic T cell replys is provided; According to this embodiment of the present invention, the compsn that stimulates helper cell to reply can be provided also.

The immunizing dose of initial treatment property in the scope of unitary dose, is approximately 1,5 than low value usually, 50,500 or 1,000 μ g, and higher value is about 10,000; 20,000; 30,000; Or 50,000 μ g.People's dose value scope typically is about 500 μ g to the per 70 kilograms of weight in patients of about 50,000 μ g.The strengthened scheme of abideing by several weeks to the several months, about 1.0 μ g to 50, the peptide of the booster dose of 000 μ g is according to the administration through measuring that the CTL that from blood samples of patients, obtains and the definite patient of HTL specific activity reply with situation.Administration should continue up to clinical symptom at least or experiment detect show that tumour has been removed or dwindles and reach for some time after.According to methods known in the art adjustment dosage, route of administration and dosage timetable.

In some embodiments, peptide of the present invention and compsn use in serious morbid state, the promptly life-threatening or life-threatening situation of possibility.In this case, have the result of nontoxic relatively character of foreign matter and the peptide of minimum in the preferred composition of the present invention, the treatment doctor possibly need give the peptide combinations of excess in fact with respect to the dosage of regulation with feeling.

Vaccine composition of the present invention also can be used as preventive merely.The dosage of common initial epidemic prevention is unitary dose, is about 1,5 than low value, 50,500 or 1000 μ g, and high value is about 10,000; 20,000; 30,000 or 50,000 μ g.Common human dose value is that about 500 μ g are to the per 70 kilograms of weight in patients of about 50,000 μ g.After using vaccine first, about 4 thoughtful 6 months intervals to confirm give about 1.0 μ g to 50, the peptide of the booster dose of 000 μ g.Can assess the immunogenicity of vaccine than living through measuring the CTL and the HTL that obtain from patient's blood sample.

The pharmaceutical composition of therapeutic treatment can non-enteron aisle, in the surface local (topical), oral, nasal feeding, sheath or local (local) (as as emulsifiable paste or surface local ointment) administration.Preferably, pharmaceutical composition is by parenterai administration, for example, and intravenously, subcutaneous, intracutaneous or intramuscular administration.Therefore, the invention provides to contain to be dissolved or suspended in and to accept carrier, be preferably the parenterai administration compsn of the immunogenicity peptide solution in the aqueous carrier.

Can adopt multiple aqueous carrier, for example water, buffered water, 0.8% salt solution, 0.3% glycocoll, mucinase etc.These compositions can be used the well-known again sterilising technology sterilization of tradition, or become aseptic through filtering.But the aqueous solution pack that produces mixed freeze dried preparation before administration in order to using or freeze-drying with sterile solution.

It is acceptable near the required auxiliary substance of physiological condition that compsn can comprise medicine; Like pH-adjusting and buffer reagent, osmotic pressure regulator, wetting agent, sanitas etc., for example sodium acetate, Sodium.alpha.-hydroxypropionate, sodium-chlor, Repone K, calcium chloride, sorbitanic list laurate, trolamine oleate etc.

The concentration of peptide of the present invention in pharmaceutical prepn can be very different; Promptly from accounting for weight less than about 0.1%, be generally or be at least about 2% to nearly 20% to 50% or more, and will be according to the specific administration pattern of selecting; Main through liquid volume, viscosity etc. are selected.

The human unit dosage form of compsn is usually included in and contains people's unitary dose and can accept in the pharmaceutical composition of carrier, and in one embodiment, said carrier is an aqueous carrier; Be used for that this compsn is applied to human volume/consumption and carry out administration with well known by persons skilled in the art (referring to for example Remington ' s Pharmaceutical Sciences, 17 Edition, A.Gennaro; Editor, MackPublishing Co., Easton; Pennsylvania, 1985).For example, for one 70 kilograms patient, the peptide dosage of initial immunity can be about 1 to about 50,000 μ g, is generally 100-5,000 μ g.For example, for nucleic acid, can use in a plurality of sites expression vector to carry out initial immunity with the naked nucleic acid form of the amount IM of 0.5-5 milligram (or SC or ID) administration.Nucleic acid (0.1 to 1000 μ g) also can be used the particle gun administration.Behind the 3-4 soak in week, use the medicine of booster dose.Booster dose can be with 5 * 10 ⁷To 5 * 10 ⁹The recombinant fowlpox virus of pfu dosed administration.

For antibody, treatment generally includes anti-psca antibody preparation via multiple acceptable route of administration, and the repeat administration of for example intravenous injection (IV), dosage range are about 0.1 usually to about 10mg/kg body weight.Substantially, dosage is effectively for 10-500mg MAb and can be by better tolerance weekly.And, be about the initial IV load dosage of 4 milligrams of/kilogram weight in patients, then the anti-PSCA MAb preparation of about weekly 2 milligrams of/kilogram IV dosage has been represented acceptable dosage.Such as those skilled in the art knowledge, various factors can influence the optimal dosage of case-specific.These factors comprise the circulate frequency of effusive degree, required Css level, treatment and unite chemotherapy or the influence of other reagent and the state of health of particular patient of use with treat-ment of the present invention of expression degree, the PSCA antigen of PSCA in the immunogenicity, patient of binding affinity, the material of half life, the Ab of compsn for example.Unrestriced preferred people's unitary dose is for for example: 500 μ g-1mg, 1mg-50mg, 50mg-100mg, 100mg-200mg; 200mg-300mg, 400mg-500mg, 500mg-600mg, 600mg-700mg; 700mg-800mg, 800mg-900mg, 900mg-1g or 1mg-700mg.In some embodiments, dosage range is the 2-5mg/kg body weight, for example then uses the dosage weekly of 1-3mg/kg; 0.5mg, 1,2,3,4,5,6,7,8,9, the 10mg/kg body weight is then for example used dosage weekly in 2,3 or 4 week; 0.5-10mg/kg body weight is for example used dosage weekly in 2,3 or 4 week; Weekly 225,250,275,300,325,350,375,400mg/m ²Body surface area; 1-600mg/m weekly ²Body surface area; 225-400mg/m weekly ²Body surface area; Can carry out 2,3,4,5,6,7,8,9,10,11,12 or the dosage weekly in more weeks then.

In one embodiment, the human unit dosage form of polynucleotide comprises can provide suitable dosage range of any result of treatment or significant quantity.As is known to the person skilled in the art, result of treatment depends on a plurality of factors, comprises polynucleotide sequence, the molecular weight of polynucleotide and route of administration.Usually by doctor or other health care professional according to multiple parameter known in the art, wait like severity of symptom, patient's medical history and to select dosage.Usually, as far as the polynucleotide of about 20 bases, the dose value scope can be selected from the lower limit of for example independently selecting, as about 0.1,0.25,0.5,1,2,5,10,20; 30,40,50,60,70,80,90,100,200,300; 400 or 500mg/kg up to the big upper limit of independently selecting of ratio lower limit, as about 60,80,100,200,300,400,500,750,1000; 1500,2000,3000,4000,5000,6000,7000,8000,9000 or 10,000mg/kg.For example, dosage maybe for approximately following any one: 0.1 to 100mg/kg, 0.1 to 50mg/kg, 0.1 to 25mg/kg; 0.1 to 10mg/kg, 1 to 500mg/kg, 100 to 400mg/kg, 200 to 300mg/kg; 1 to 100mg/kg, 100 to 200mg/kg, 300 to 400mg/kg, 400 to 500mg/kg; 500 to 1000mg/kg, 500 to 5000mg/kg or 500 to 10,000mg/kg.Usually, and more directly nucleic acid is applied to sick tissue and compares, the parenteral route administration needs the polynucleotide of higher dosage, and the polynucleotide that increase length also are like this.

In one embodiment, the T cell of human unit dosage form contains suitable dosage range or the significant quantity that any result of treatment can be provided.Such as those of ordinary skills knowledge, result of treatment depends on a plurality of factors.Usually by doctor or other health care professional according to known multiple parameter, like severity of symptom, patient's medical history waits selects dosage.Dosage can be about 10 ⁴Each and every one cell is to about 10 ⁶Individual cell, about 10 ⁶Individual cell is to about 10 ⁸Individual cell, about 10 ⁸Individual to about 10 ¹¹Individual cell, or about 10 ⁸Individual to about 5 * 10 ¹⁰Individual cell also can be about 10 ⁶Individual cell/m ²To about 10 ¹⁰Individual cell/m ², or about 10 ⁶Cell/m ²To about 10 ⁸Cell/m ²

The nucleic acid of protein of the present invention and/or coded protein also can pass through the liposome administration, and said liposome can be used for: 1) make the specific tissue of protein target, like Lymphoid tissue; 2) selectivity target disease cell; Or 3) half life of increase peptide combinations.Liposome comprises emulsion, foam, microne, undissolved individual layer, liquid crystal, phosphatide dispersion agent, laminated layer etc.In these preparations; A part that can be used as liposome by the peptide that transmitted individually or with can and lymphocyte in the molecule of ubiquitous receptors bind; For example combine the antigenic monoclonal antibody of CD45, or curative or immunogenic composition is united and is impregnated in other.Therefore, the liposome of filling or modifying with peptide required for the present invention can be delivered to lymphocytic site, carries peptide combinations at the there liposome.The used liposome of the present invention forms lipid by the vesicle of standard and forms, and generally includes neutral and electronegative phosphatide and sterol, for example SUV.The selection of lipid is for example considered usually, the acid-labile property and the stability of liposome in liposome size, the blood flow.Several different methods can be used to prepare liposome, like Szoka et al, and Ann Rev.Biophys.Bioeng.9:467 (1980) and USP 4,235,871,4,501,728,4,837,028 and 5,019,369 is described.

Be the immune cell of target, the part that mixes liposome comprises that for example specificity is to antibody or its fragment of the cell surface determinant of required immune system cell.Modes such as containing the peptide lipid liquid suspension can intravenously, part, external application are with the dosed administration according to the stage adjustment of the peptide of administering mode, transmission and treatment disease.

For solids compsn, can adopt traditional non-toxic solid carrier, comprise N.F,USP MANNITOL, lactose, starch, Magnesium Stearate, soluble saccharin, talcum powder, Mierocrystalline cellulose, glucose, sucrose, magnesiumcarbonate of pharmacy grade for example etc.For oral administration, through making up the vehicle that adopts usually arbitrarily, the for example listed carrier in front; With the activeconstituents that is generally 10-95%; Be one or more peptides of the present invention, more preferably its concentration is 25%-75%, prepares the acceptable non-toxic composite of medicine.

For the aerosol administration, immunogenic peptide is preferably used with tensio-active agent and propelling agent with meticulous form of separating.The common weight percent of peptide is about 0.01%-20%, preferably approximately 1%-10%.Tensio-active agent must be nontoxic certainly, and preferably may be dissolved in the propelling agent.The representative of this type reagent is the lipid acid that contains 6 to 22 carbon atoms, for example, and ester or part ester that caproic acid, sad, LAURIC ACID 99 MIN, palmitinic acid, Triple Pressed Stearic Acid, linolic acid, linolenic acid, olesteric acid and oleic acid and multi-hydroxy fat alcohol or its cyclic anhydride form.Can adopt mixed ester, as mixing or natural glyceride.Tensio-active agent can constitute about 0.1%-20% of composition weight, preferably approximately 0.25-5%.The balance of compsn is used propelling agent usually.In case of necessity, carrier also can be included, as the Yelkin TTS that is used for transmitting in the nose.

XI.) embodiment of PSCA diagnosis and prediction

Disclosed like this paper; Diagnosis, prediction and curative analysis that PSCA polynucleotide, polypeptide, reactive cytotoxic T cell (CTL), reactive helper cell (HTL) and anti-peptide antibody are used to be widely known by the people; This analysis is used for confirming and the abnormal cell growth diseases associated; Like cancer, listed cancer (expressing) in the Table I particularly referring to tissue expression specificity's pattern described in the embodiment that for example is entitled as " expression analysis of PSCA in healthy tissues and the patient's sample " and crossing in certain cancers.

PSCA can class be pushed into prostate gland related antigen PSA, PSA be the former phenotypic marker that exists of the identification adopted for many years of medical practitioner and monitoring prostate cancer (referring to 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.CancerInst.91 (19): 1635-1640 (1999)).Similarly also can use multiple other diagnostic flag in the context; Comprise that p53 and K-ras are (referring to for example Tulchinsky etal.; Int J Mol Med 1999 Jul 4 (1): 99-102 and Minimoto et al., Cancer Detect Prev2000; 24 (1): 1-12).Therefore; The disclosing of PSCA polynucleotide and polypeptide (and being used for discerning PSCA polynucleotide probes and anti-psca antibody that these molecules exist) and their characteristic make those skilled in the art with method like these application class, for example utilize these molecules at the multiple diagnostic test that is used for definite cancer relative disease.

For example the diagnostic test of PSA polynucleotide, polypeptide, reaction-ive T cell and antibody is similar to utilize typical embodiments and those the established utilizations of diagnostic method of PSCA polynucleotide, polypeptide, reaction-ive T cell and antibody.For example; Be used as probe (for example in Northern analyzes as the PSA polynucleotide; Referring to for example Sharief et al., Biochem.Mol.Biol.Int.33 (3): 567-74 (1994)) and primer (for example in pcr analysis, referring to for example Okegawa et al; J.Urol.163 (4): 1189-1190 (2000)), to observe existing and/or level of PSAmRNA in the method for crossing expression or prostate cancer transfer at monitoring PSA.PSCA polynucleotide as herein described can detect the cancer metastasis of crossing expression or prostate gland and other this gene of expression of PSCA in the same way.Alternatively; As crossing in the method for expression at monitoring PSA albumen; The PSA polypeptide can be used to produce the specific antibody to PSA; Thereby be used for observing the transfer (referring to for example Alanen et al., Pathol.Res.Pract.192 (3): 233-7 (1996)) of proteic existence of PSA or level (referring to for example Stephan et al, Urology55 (4): 560-3 (2000)) or prostatic cell; PSCA as herein described can be used for producing antibody, is used for detecting that PSCA crosses expression or prostatic cell is expressed the transfer of the cancer cells of this gene with other.

Particularly; Because shift to relate to cancer cells and move to health different zones (for example lymphoglandula) from primitive organ (for example lung or prostate gland etc.), the analysis of existence of confirming to express in the biological sample cell of PSCA polynucleotide and/or polypeptide can be used to provide the evidence of transfer.For example; When the biological sample from the tissue that contains the psca expression cell unusually (lymphoglandula) comes to light when comprising the psca expression cell; For example among LAPC4 and the LAPC9 (separating the heterograft that shifts from lymphoglandula and bone respectively) during the visible psca expression, this discovery has indicated transfer.

Alternatively; For example; Express cell in the biological sample of PSCA comes to light and expresses PSCA or psca expression when strengthening (referring to the psca expression in listed cancer in the Table I for example and the patient's sample etc. as unconventionality expression PSCA or with different levels; Shown in accompanying drawing), PSCA polynucleotide and/or polypeptide can be used to provide the evidence of cancer.In such analysis; The technician can further hope through the second tissue limitations property mark (except that PSCA) in the detection of biological sample; PSA for example; The existence of PSCA etc. produces auxiliary transfer evidence (referring to for example Alanen et al., Pathol.Res.Pract.192 (3): 233-237 (1996)).

Use the existence of PSCA polypeptide in the section of immunohistochemical methods appraisement organization, the result shows that the state of some cells in this tissue changes to some extent.Know the aggressiveness of existence, the course of disease, progress and/or tumour that the ability of the expressed polypeptide of antibody detecting cancer cell can be used for diagnosing the illness with those skilled in the art know that.Said antibody also can detect with corresponding non--malignant tissue compares, the distribution that changes to some extent of the polypeptide in the cancer cells.

In view of the phenomenon that ubcellular protein positioning under the morbid state changes to some extent, PSCA polypeptide and immunogenic composition also are useful.Cell is by normally changing into the variation that morbid state causes morphocytology, and usually relevant with the variation of ubcellular protein positioning/distribution.For example, the cell membrane protein of in normal cell, expressing with the polarity mode changes when ill to some extent, causes protein to be distributed in whole cell surface with non--polar mode.

Through using the immunohistochemical methods method, illustrated the phenomenon that the ubcellular protein positioning changes to some extent under morbid state with MUC1 and the proteic expression of Her2.Normal epithelium cell is located on the nuclear of some gp, also have typical MUC 1 top and distribute, and malignant lesions often shows non-polar dyeing pattern (Diaz et al., The Breast Journal, 7; 40-45 (2001); Zhang et al., ClinicalCancer Research, 4; 2669-2676 (1998): Cao, et al., The Journal ofHistochemistry and Cytochemistry, 45:1547-1557 (1997)).In addition, the normal breast epithelial cell is that Her2 albumen is negative, perhaps only show the substrate outside and distribute, and malignant cell can be in whole cell surface expression protein (De Potter, et al., International Journal of Cancer, 44; 969-974 (1989): McCormick, et al., 117; 935-943 (2002)).Perhaps, protein distributes and can become the kytoplasm expression that comprises the disperse under the morbid state by only being positioned the surface.Can be observed such example (Diaz, et al., The Breast Journal, 7:40-45 (2001)) with MUC1.

Change through protein positioning/distribution in the detected cell of immunohistochemical methods method also can provide the valuable information relevant with the profitability of some therapeutic modalities.Last point can be illustrated by following situation: protein can be intracellular in healthy tissues, but in malignant cell, is positioned at cell surface; The cell surface location makes cell can successfully accept diagnosis and the regimen based on antibody.When PSCA this protein positioning takes place changed, PSCA albumen and relative immunne response were very useful.Therefore, confirm that the ability whether 24P4C12 the change of ubcellular protein positioning takes place makes PSCA albumen and relative immunne response become very useful.Use the PSCA compsn can make those skilled in the art make important diagnostic and treatment decision.

When this polypeptide having occurred in the tissue that can not produce PSCA under the normal circumstances, the immunohistochemical methods reagent that is specific to PSCA also can be used for detecting the transfer of the tumour of expressing PSCA.

Therefore, the PSCA polypeptide and by to its immunne response and the antibody that produces can be used for multiple free-revving engine well known by persons skilled in the art, as diagnosing, predict, prevent and/or treat purpose.

Be used in the method for monitoring PSA by those of skill in the art as PSA polynucleotide passage and polynucleotide variant, PSCA polynucleotide passage and polynucleotide variant can use similar mode to use.Particularly, probe or primer that the typical PSA polynucleotide that use in the method for monitoring PSA are made up of PSA cDNA sequence fragment.To this explanation for example, the used primer of pcr amplification PSA polynucleotide must comprise that the sequence of lacking than total length PSA sequence is to bring into play function in polymerase chain reaction,PCR.In the context of PCR reaction; Those of skill in the art usually preparation a plurality of different can be as the polynucleotide passage of primer with the different piece of amplification herbicide-tolerant polynucleotide or optimization amplified reaction (referring to for example Caetano-Anolles; G.Biotechniques 25 (3): 472-476,478-480 (1998); Robertsonet al., Methods Mol.Biol.98:121-154 (1998)).The example that another this fragment is used provides in the embodiment that is entitled as " expression analysis of PSCA in healthy tissues and the patient's sample ", and the PSCA polynucleotide passage shows the expression of PSCA in the cancer cells as probe.In addition; Different polynucleotide sequences usually also as the primer of corresponding mRNA in PCR and the Northern analysis and probe (referring to for example Sawai et al.; Fet al Diagn.Ther.1996 Nov-Dec 11 (6): 407-13 and CurrentProtocols In Molecular Biology, Volume 2, and Unit 2; Frederick M.Ausubel et aleds., 1995)).Under the condition of height stringency, it is useful can combining the polynucleotide passage of target polynucleotide sequence and variant (the PSCA polynucleotide that for example show among Fig. 1 or its variant).

And, containing and can be used to monitor in the method for PSA by the PSA polynucleotide of the epi-position of antibody or T cell recognition, said antibody or T cell can combine with this epitope specificity.PSCA polypeptide fragment and polypeptide analog or variant also can be utilized in a similar fashion.The practice of using polypeptide fragment or polypeptide variants to produce antibody (like anti-PSA antibody or T cell) is that this area is typical; Can use multiple systems; For example (referring to for example Current Protocols In MolecularBiology, Volume 2, Unit16 by the fusion rotein of practitioner's utilization; Frederick M.Ausubel et al.eds., 1995).In this context, the function of each epi-position is the structure that provides with antibody or t cell responses.Typically, for producing the immunne response to the target polypeptides different piece, those of skill in the art can produce multiple different polypeptides fragment (referring to for example USP 5,840,501 and USP 5,939,533).For example, the preferred polypeptide that contains the PSCA primitive of this paper discussion or carry the subsequence of primitive that utilizes, those skilled in the art can easily identify said polypeptide based on the primitive that this area can be used.In this case, can produce specificity to the antibody of target peptide sequence (the for example PSCA polypeptide shown in Fig. 1) or the epi-position of T cell as long as polypeptide fragment, variant or analogue contain, they are normally useful.

As shown here, PSCA polynucleotide and polypeptide (and being used for discerning PSCA polynucleotide probes and anti-psca antibody or T cell that these molecules exist) show specific character, and making them is useful in diagnosing the listed cancer of Table I for example.In order to assess the disease described herein for example existence or the beginning of prostate cancer, detect diagnostic test that the psca gene product exists and be used to identify the patient carrying out preventive measure or further monitoring, as utilizing the success realization of PSA institute.And; Under for example can not making situation such as clarifying a diagnosis of prostate gland origin transfer based on independent detection PSA; These materials have satisfied the needs (referring to for example Alanenet al, Pathol.Res.Pract.192 (3): 233-237 (1996)) of prior art to molecule with or additional character similar with PSA.Thereby, need be used to confirm the transfer in prostate gland source like the material (and PSCA polynucleotide probes and anti-psca antibody of being used for discerning these molecules existence) of PSCA polynucleotide and polypeptide.

At last; Except they application in diagnositc analysis; The disclosed PSCA polynucleotide of this paper also have some, and other is used, for example the application (face is entitled as the embodiment of " chromosome mapping of PSCA " as follows) in their chromosome abnormalties in the chromosomal region of identifying the psca gene mapping relevant with oncogenesis.And; Except they application in diagnositc analysis; PSCA related protein disclosed herein and polynucleotide also have other application, and the for example application aspect legal medical expert's evaluation of unknown sources tissue is (referring to for example Takahama K Forensic Sci Int 1996 Jun 28; 80 (1-2): 63-9).

In addition, PSCA related protein of the present invention or polynucleotide can be used for treating to express the pathologic conditions that PSCA is a characteristic excessively.For example, amino acid among Fig. 1 or nucleotide sequence, or their fragment can be used to produce the antigenic immunne response of PSCA.Can adopt the function of modulating this molecule, therefore curative benefit is provided with antibody or other molecule of PSCA reaction.

XII.) suppress the proteic function of PSCA

The present invention includes multiple inhibition PSCA combine counterpart to combine with it or with the method and composition of other protein bound and the method for inhibition PSCA function.

XII.A.) suppress PSCA with intrabody

In a method, will encode and the recombinant vectors of PSCA specificity bonded single-chain antibody is introduced the cell of expressing PSCA through gene transfer technique.Accordingly, therefore the anti-psca antibody of the strand of coding also suppresses its function at cell inner expression with the PSCA protein binding.The method of producing single-chain antibody in this cell is widely known by the people.This intrabody also is called as " endosome (intrabodies) ", is selectively targeted intracellular specific compartment, in the active place of concentrating of the inhibition of treatment control is provided.This technology successfully has been applied to (summary is seen Richardson and Marasco, 1995, TIBTECH vol.13) in the prior art.Endosome shown weakened other redundant cell surface receptor in fact expression (referring to for example Richardsonet al, 1995, Proc.Natl.Acad.Sci.USA 92:3137-3141; Beerli et al, 1994, J.Biol.Chem.289:23931-23936; Deshane et al, 1994, Gene Ther.1:332-337).

Single-chain antibody comprises heavy chain and the variable region of light chain that connects through the flexible joint polypeptide, and is expressed as single polypeptide.Randomly, the variable region of light chain fragment that is connected with constant region of light chain of single-chain antibody conduct is expressed.The transportation signal is introduced the recombinant polypeptide carrier of this single-chain antibody of coding in the cell that will know through biotechnology, so that the required intracellular region chamber of the accurate target of endosome.For example in the endosome of target endoplasmic reticulum, mix leading peptide and optional C end ER stick signal like KDEL amino acid primitive through biotechnology.Desiring in nucleus, to add nuclear localization signal through biotechnology in the active endosome of performance.In order endosome to be limited in the cytosol face of plasma membrane, the lipid moity is added into endosome.Endosome also can be used for bringing into play function at cytosol, and for example the cytosol endosome is used to isolate the factor in the cytosol, transports to their natural cell positions thereby suppress them.

In one embodiment, endosome is used to catch endonuclear PSCA, thereby suppresses it in endonuclear activity.In order to arrive required target, nuclear target signal is introduced into this PSCA endosome.Designing this PSCA endosome combines with specific PSCA structural domain.In another embodiment, be used to suppress PSCA with PSCA protein-specific bonded cytosol endosome and get into nucleus, in nucleus, bring into play biological activity (for example suppress PSCA and other factor and form transcription complex) thereby suppress it.

For specificity mediates this endosome in certain intracellular expression, transcribing of endosome placed under the regulation and control of suitable tumor-specific promoters and/or enhanser.For example, in order to make endosome expression specificity target prostate gland, can utilize PSA promotor and/or enhanser (publishing on July 6th, 5,919,652,1999) referring to for example USP.

XII.B.) suppress PSCA with recombinant protein

In another method, recombinant molecule combines and therefore suppresses the function of PSCA with PSCA.For example, these recombinant molecules prevent or suppress PSCA combine with it counterpart near/combine or with other protein bound.For example, this recombinant molecule can comprise the for example active part of PSCA specific antibody molecule.In a specific embodiment, PSCA combines the PSCA binding domains of counterpart to be transformed into dimer fusion protein, so this fusion rotein comprises 2 and human IgG, like human IgG ₁The PSCA ligand binding domains that partly connects of Fc.This IgG part can comprise for example CH ₂And CH ₃Structural domain and hinge area, but CH do not had ₁Structural domain.This dimer fusion protein suffers from the cancer patients relevant with psca expression with soluble form, takes this, and this dimer fusion protein combines with the PSCA specificity, and blocking-up PSCA combines the interaction of counterpart with it.With known antibody interconnection technique this dimer fusion protein is further made up with polymer protein.

XII.C.) suppress transcribing and translating of PSCA

The present invention also comprises and suppresses several different methods and the compsn that psca gene is transcribed.Similarly, the present invention also provides inhibition PSCA mRNA to translate into method of protein and compsn.

In a method, the method that the inhibition psca gene is transcribed comprises makes psca gene contact with the PSCA antisense polynucleotides.In another method, the method that suppresses PSCA mRNA translation comprises makes PSCAmRNA contact with antisense polynucleotides.In another method, the PSCA specific ribozyme is used to cracking PSCA mRNA, thereby suppresses translation.Thisly also can be imported into the regulatory region of psca gene, like PSCA promotor and/or enhancer element based on the method for antisense and ribozyme.The protein that similarly, can suppress the psca gene transcription factor is used to suppress PSCA mRNA and transcribes.Multiple polynucleotide and the compsn that is used for preceding method described in the above.The application in inhibition is transcribed and translated of antisense and ribozyme molecule is widely known by the people in the prior art.

Other factor of disturbing the inhibition PSCA of PSCA transcriptional activity to transcribe also can be used for treating the cancer of expressing PSCA.Similarly, disturb the factor of PSCA processing also to can be used for treating the cancer of expressing PSCA.The cancer treatment method that utilizes this factor also within the scope of the invention.

XII.D.) generality of therapeutic strategy is considered

Transgenosis and gene therapy technology can be used for the therapeutic polynucleotide molecule is transferred to (being antisense, ribozyme, the polynucleotide of coding endosome and other PSCA inhibition molecule) in the tumour cell that synthesizes PSCA.Known in the state of the art have a several genes treat-ment.Can with these gene therapy methods will encode PSCA antisense polynucleotides, ribozyme, can disturb the recombinant vectors of factor that PSCA transcribes etc. to be delivered in the target tumour cell.

Above-mentioned treat-ment can combine with other multiple surgical operation, chemotherapy or radiotherapy scheme.Use treat-ment of the present invention can make chemotherapy (or other treatment) reduce using dosage with and/or reduce therapeutic frequency, particularly those are favourable to the patient that chemotherapeutics toxicity can't better tolerate to all patients for this.

Can use the anti-tumor activity of the combination of multiple external and body inner analysis system evaluation particular composition (for example, antisense, nucleicacidase, endosome) or these compsns.The in vitro tests of assessment therapeutic activity comprises the cell growth test, and the soft agar test can be indicated the test of tumor proliferation active with other, can confirm that therapeutic composition suppresses PSCA and combines combination test of counterpart combination degree or the like with it.

In vivo, the effect of PSCA therapeutic composition can be assessed in suitable animal model.For example; Can adopt xenogeneic prostate cancer model, wherein the xenograft tissues of human prostata cancer explant or passage is introduced into the animal of non-responsiveness, for example nude mice or SCID mouse (Klein et al; 1997, Nature Mediane 3:402-408).For example, described the xenograft models of various human prostate cancer in PCT patented claim W098/16628 and the USP 6,107,540, said model can be recurred the growth of original tumour, the formation of the bone transfer characteristic in micrometastasis and stage terminal stage of a disease.Can forecasting power with measuring the test that tumour forms inhibition, tumour fails or shift.

Estimate the promoted in vivo tests of apoptosis and help to assess therapeutic composition.In one embodiment, can detect in the mouse that contains tumor xenogeneic graft with therapeutic composition treatment whether have the apoptosis focus, and with the untreated than in control mice that carries heterograft.The indication of combination treatment effect is provided by the degree of discovery apoptosis focus in the tumour of treatment mouse.

The therapeutic composition of in the practice of preceding method, using can be formulated into the pharmaceutical composition that contains the carrier that is suitable for required transmission method.The carrier that is fit to comprises can keep the anti-tumor activity of therapeutic composition when mixes with therapeutic composition, and the common arbitrary substance that does not react with patient's immune system.Example includes but not limited to any one of multiple standards pharmaceutical carrier, sterile phosphate buffered saline solution for example, bacteriostatic water etc. (generally referring to Remington ' s Pharmaceutical Sciences16tEdition, A.Osal., Ed., 1980).

The treatment preparation can be dissolved and through can therapeutic compsn being delivered to the administration of tumor sites arbitrarily.Potential effective route of administration includes, but are not limited to: in the intravenously, non-enteron aisle, intraperitoneal, intramuscular, tumour, in the intracutaneous, organ, ortho position etc.The preparation of preferred intravenous injection comprises the therapeutic composition that is kept in antiseptic and inhibiting bacteria function water, the aseptic not anticorrosion water, and/or comprises to be diluted in the SE that contains 0.9% aseptic sodium-chlor or the polyethylene bag and supply injection, the therapeutic composition of USP.Curative protein articles can freeze-drying, preserves as sterilized powder, preferably preserves in a vacuum, before injection, uses bacteriostatic water (containing sanitas, for example phenylcarbinol) or sterilized water to rebuild then again.

Dosage and dosage regimen with preceding method treatment cancer will change along with the change of method and target cancer, and the multiple other factors that depends in the prior art usually to be known.

XIII.) evaluation of PSCA modulator, sign and application

Identify and utilize the method for modulator

In one embodiment, screen the modulator that to induce or to suppress the particular expression distribution plan, suppress or induce particular approach to identify, the preferred modulator that produces relevant phenotype thus.In another embodiment, identify difference expression gene important under particular state, screened the modulator that changes independent genetic expression (no matter being to increase or reduce) to identify.In another embodiment, screen to identify the modulator of the biological function that changes the expressed product of difference expression gene.In addition, identified the importance of gene under the particular state, carried out examination to identify the reagent of combination and/or modulate gene product.

In addition, screening is to the gene of candidate agent reaction.Identifying modulator (expression patterns cause the normal expression pattern thereby those suppress cancer, or cause the modulator like the cancer gene of the genetic expression in the healthy tissues) afterwards, screens and identifies that those are to reagent react and by the synthetic gene of specificity.The expression and distribution figure of the cancer that relatively more normal tissue and with medicament were treated has disclosed those and can not in healthy tissues or cancerous tissue, express, but can be at the gene of the tissue expression of crossing through pharmaceutical treatment, and vice versa.Identify these medicament-specific sequences and use it for cancer gene or protein with method described herein.Especially, these sequences are used to mark or differentiate the cell that with medicament was treated with their encoded protein matter.In addition, produce, and be used for the cancerous tissue sample of new therapy target through treatment to the proteic antibody of medicament inductive.

Evaluation and shaker test that modulator is relevant:

The genetic expression correlation test

Protein of the present invention, nucleic acid and antibody can be used for shaker test.The protein of cancer-related protein, antibody, nucleic acid, modification and the cell that contains these sequences can be used for shaker test, and for example the evaluate candidate medicine is to the influence of the change of " gene expression profiles ", expression of polypeptides distribution plan or biological function.In one embodiment, expression and distribution figure preferably with high-throughout triage techniques combined utilization so that (like, Davis, GF waits J BiolScreen 7:69 (2002) with the situation of tracking monitor genetic expression behind candidate's pharmaceutical treatment; Zlokarnik, et al, Science 279:84-8 (1998); Heid, GenomeRes 6:986-94,1996).

Cancer protein, antibody, nucleic acid, modifying protein and comprise natural or the cancer protein modified or the cell of gene are used to shaker test.That is, the present invention includes the screening method for compositions, the phenotype or the cancer protein physiologic function of the present invention of said composition modulation cancer.Gene itself is carried out or passes through the influence of assessment of drug-candidate to " gene expression profiles " or biological function.In one embodiment, expression and distribution figure preferably with high-throughout triage techniques combined utilization so that with the situation of tracking monitor genetic expression behind candidate's pharmaceutical treatment, civilian Zlokamik sees before.

Carried out multiple test to gene of the present invention and protein.Test is carried out on each nucleic acid or protein level.That is to say, identify the specific gene that in cancer, raises, screening test-compound modulate gene is expressed the proteinic ability of cancer among the present invention that perhaps combines." modulation " in this case comprises the raising or the reduction of genetic expression.Preferred modulation voltage depends on that healthy tissues is to the initial change of the genetic expression of cancerous tissue in the cancer, and changing is 10% at least, and is preferred 50%, and more preferably 100-300% reaches 300-1000% or higher in certain embodiments.Therefore, compare,, often need 4 times minimizing if a gene demonstrates 4 times increase in cancerous tissue with healthy tissues; Likewise, compare with healthy tissues, 10 times minimizing often needs the increase of 10 times of target values of compound expression to be tested in cancerous tissue.The modulator that increases visible genetic expression type in the cancer also is useful, for example, in further analyzing, is used as the target of rise.

Come the amount of gene expression through the quantification of using nucleic probe and gene expression dose, perhaps, alternatively, the immunoassay monitoring gene product itself through use proteinic antibody of cancer and standard for example.The quantification that proteomics and stripping technique also can be expressed.

Identify the expression monitoring of the compound of the modification of gene expression

In one embodiment, monitor the genetic expression of a plurality of individualities simultaneously, i.e. expression and distribution figure.These distribution plans typically comprise the one or more genes among Fig. 1.In this embodiment, for example, fixedly the nucleic probe of cancer detects and quantizes the cancer sequence in the specific cells on biochip.Selectively, can use PCR.Therefore, can use a series of holes on the droplet plate, be dispersed with primer in the wherein required hole.Carry out PCR reaction then and each hole is analyzed.

Express monitoring and identify the compound that to modulate one or more cancer correlated serieses expression, like the polynucleotide sequence that shows among Fig. 1.Usually, before analyzing in the cell adding tried modulator.And, also provide screening to identify that medicament, said medicament can modulate cancer, modulation cancer protein of the present invention, perhaps disturb cancer albumen of the present invention to combine the combination of counterpart with antibody or other with cancer protein bound of the present invention.

In one embodiment, high-throughout screening method comprises provides the library that contains a large amount of potential treatment compounds (candidate compound).This " combinatorial chemistry library " tested to screen with evaluation with one or more subsequently and demonstrated the active library member of required characteristic (special compound kind or subclass).So compounds identified can be used as conventional " leading compound " as screening with compound or therapeutical agent.

In certain embodiments, potential can combine with cancer polypeptide maybe can modulate active modulator in the screening combinatorial library.By convention; Be tested and appraised and have some required character or activity, for example suppress active compound (being called as leading compound), the variant of the leading compound of preparation; And characteristic and the activity of assessing these variant compounds, can produce the new chemical entity that character capable of using is arranged.Usually, adopt high-throughput examination (HTS) method to carry out this analysis.

As stated, the genetic expression monitoring can be used to check candidate's modulator (for example protein, nucleic acid or small molecules) expediently.After adding candidate's medicament, make cell insulation for some time, the sample that contains target sequence that analyze is added into biochip.

If desired, with known technology preparation target sequence.For example, with lysing cell, electroporation carries out purifying and/or amplification like PCR in the time of suitably with processing samples such as known lysis buffers.For example, carry out the covalently bound in-vitro transcription of affinity tag and nucleic acid.Usually, nucleic acid is with vitamin H-FITC or PE, or with cy3 or cy5 mark.

Target sequence can be used for example fluorescence, chemiluminescent substance, and pharmaceutical chemicals, or radiated signal mark, thus the specificity bonded mode that detects target sequence and probe is provided.Mark also can be an enzyme, and for example SEAP or horseradish peroxidase when suitable substrate is provided, can produce detectable product.Alternatively, this mark is compound or the small molecules that is labeled, and enzyme inhibitors for example, this suppressor factor combine with enzyme but not by its catalysis or change.This mark also can be moity or compound, for example with Streptavidin specificity bonded epi-position mark or vitamin H, and as for the example of vitamin H, labelled streptavidin as stated, thus the signal surveyed to the bonded target sequence is provided.Unconjugatedly, labelled streptavidin removes before analyzing usually.

As is known to the person skilled in the art, these tests can be that direct cross experiment maybe can comprise " sandwich test ", comprise using multiple probe, like USP 5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246; With 5,681, run in 697.In this embodiment, run over the preparation target nucleic acid usually as before, under the condition that hybridization complex is formed, add and contain in the biochip of a large amount of nucleic probes then.

Use multiple hybridization conditions among the present invention, comprised height, moderate and the low stringency condition as above pointed out.Test is carried out under the stringency condition usually, makes to have only when target exists to form the label probe hybridization complex.Can control tight degree through the variable step parameter of adjustment thermodynamics, include but not limited to temperature, methane amide concentration, salt concn, chaotropic salt concentration pH, organic solvent concentration etc.These parameters also can be used for controlling non-specific binding, like USP 5,681, point out substantially in 697.Therefore, under higher tight degree condition, carrying out some step, to reduce non-specific binding be gratifying.

The reaction of this paper summary can be accomplished through number of ways.Reacted constituent can add with different orders simultaneously or in succession, and the embodiment preferred that runs over is arranged below.In addition, reaction can comprise multiple other reagent.Comprise salt, damping fluid, neutral protein matter (like BSA), stain remover etc., can be used for promoting best hybridization and detection, and/or reduce non-specific or the background interaction.Also can use the reagent that other improves test efficiency suitably, proteinase inhibitor for example, the ribozyme suppressor factor, antimicrobial reagent etc., this depends on the preparation method of sample and the purity of target.The analytical test data are confirmed heterogeneic expression level, and the expression level between different states changes the formation gene expression profiles.

The biological activity correlation test

The invention provides identify or screening modulation the present invention in the method for cancer related gene or protein active.These methods are included in to contain and add the test-compound that specifies like preamble in the proteinic cell of cancer of the present invention, and cell contains the proteinic recombinant nucleic acid of code book invention cancer.In another embodiment, in a large amount of cells, detect candidate's preparation storehouse.

On the one hand; Test physiological exist or do not exist or before or after assess under the situation about exposing, above-mentioned signal comprises for example hormone, antibody, peptide, antigen, cytokine, growth factor, effect potentiality, comprises the medicament of chemotherapy, radiotherapy, carcinogenic substance or other cell (being cell-cells contacting).In another example, evaluation is carried out in the different steps of cell cycle.Modulating gene of the present invention or proteinic compound is differentiated by this way.Compound with pharmaceutical active can strengthen or disturb cancer activity of proteins of the present invention.In case identified, assessed the key structure characteristic that similar structure is discerned compound.

In one embodiment, modulation (as suppressing) cancer cells splitted method is provided; This method comprise to the cancer modulator.In further embodiment, treatment cancer cell or the individual method of cancer stricken are provided; This method comprise to the cancer modulator.

In one embodiment, provide modulation to express the method for the cell state of gene of the present invention.The state that this paper uses is included as the parameter that this area is accepted, the for example growth of cell, propagation, survival, organ dysfunction, apoptosis, aging, location, enzymic activity, signal conduction etc.In one embodiment, the cancer suppressor factor is the antibody that preamble is discussed.In another embodiment, the cancer suppressor factor is an antisense molecule.As described herein, cell growth, propagation and the test of shifting are that those skilled in the art are known.

Identify the high-throughput examination of modulator

The test of identifying the appropriate modulation agent should be a high flux screening.Therefore, preferred test detects the enhancing or the inhibition of cancer genetic transcription, the inhibition of the inhibition of expression of polypeptides or enhancing and polypeptide active and enhancing.

In one embodiment, the modulator of assessing in the high-throughput screening method is a protein, is generally the fragment of natural protein or natural protein.Therefore, utilize and for example contain proteinic cell extract, or protein cell extract at random or direct digestion.In this way, the preparation protein library supplies method screening of the present invention.Particularly preferably be bacterium in this embodiment, fungi, virus, and mammalian proteins matter library, the preferred latter, preferred especially human protein.Kinds of protein under the valuable especially detection compound target target, for example substrate of enzyme, or part and acceptor.

Form with soft agar growth and colony and to identify and to characterize modulator

Normal cell need adhere on the solid substrate grows.When cell is transformed, they lose this phenotype, and break away from the matrix growth.For example, cell transformed can be at the suspension culture that stirs or the semi-solid state substratum of suspension, as growing on semi-solid state agar or the soft agar.When with tumor suppressor gene transfection cell transformed, can rebulid normal phenotype and need be adsorbed in the solid substrate growth once more.Soft agar growth or colony form the modulator that is used for identifying the cancer sequence in the test, and when in host cell, expressing, said modulator suppresses paracytic propagation and differentiation.Modulator reduces or eliminates host cell in the solid-state or semi-solid state substratum ability of suspension growth in the agar for example.

Soft agar growth or colony in the suspension test form technical description in Freshney, Culture ofAnimal Cells a Manual of Basic Technique (3rd ed., 1994).Also can be referring to Garkavtsev et al. (1996), the method part that document is the same.

Assessment contact inhibition and stand density limit identifies and characterizes modulator

Normal cell is grown in cell culture with tiling and organized mode usually and is touched other cell until them.When cell was in contact with one another, they were by contact inhibition and stop growing.Yet cell transformed does not receive contact inhibition, and the random focus of continued growth to high-density.Therefore, compare with corresponding normal cell, cell transformed can grow to the more density of high saturation.This can detect through being formed on the morphology of random monolayer cell or cell colony.Alternatively, under saturation density, ( ³H)-label index of thymidine is used to detect the limitations in density of growth.Similarly, the blue experiment of MTT or Alamar can disclose multiplication capacity and the modulator of the cell ability to its influence, and referring to Freshney (1994), document is the same.After with tumor suppressor gene transfection cell transformed, can produce normal phenotype, become and receive contact inhibition and be grown to lower density.

In this experiment, under the saturation density ³The H-thymidine labeling index is the preferred method that detects the stand density restriction.With cancer correlated series transfection transformed host cells, and under the condition of unrestricted substratum, grew 24 hours with saturation density.Confirm to use through mixing cpm ³The per-cent of the cell of H-thymidine mark.

The growth that does not rely on contact is used for identifying the cancer sequence, and said sequence causes unusual cell proliferation and conversion.Modulator can reduce or eliminate the growth that does not rely on contact, makes the cellular-restoring normal phenotype.

Assessment growth factor or serum dependency are to identify and to characterize modulator

Compare with its normal counterpart, transformant have lower serum dependency (referring to for example Temin, J.Natl.CancerInst.37:167-175 (1966); Eagle et al, J.Exp.Med 131:836-879 (1970)); Freshney, document is the same.This part is because the multiple growth factor that transformant discharges.The growth factor and the serum dependency level that can compare transformed host cell and control cells.For example, the growth factor and the serum dependency level of monitoring cell in the method for the compound of identifying and characterize modulation cancer correlated series of the present invention.

Identify and characterize modulator with the marker levels of tumour-specific

Compare with its normal counterpart, the amount that tumour cell discharges some factor (hereinafter referred to as " tumour-specific markers ") increases.For example; The human glioma has discharged the plasminogen activator (PA) that increases than normal brain activity cell (referring to for example Gullino; Angiogenesis, Tumor Vascularization, andPotential Interference with Tumor Growth; In Biological Responses in Cancer, pp.178-184 (Mihich (ed.) 1985)).Similarly, the tumor angiogenesis factor (TAF) of tumour cell release increases than its normal counterpart.(referring to for example Folkman, Angiogenesis and Cancer, Sem Cancer Biol. (1992)), and the endotheliocyte knurl discharges bFGF (Ensoli, B et al).

The technology that these factors of multiple detection discharge is by Freshney (1994), the same description of document.Also can be referring to Unkless et al, J.Biol.Chem.249:4295-4305 (1974); Strickland&Beers, J.Biol.Chem.251:5694-5702 (1976); Whur et al., Br.J.Cancer 42:305 312 (1980); Gullino, Angiogenesis, TumorVascularization, and Potential interferencewith Tumor Growth, in Biological Responses in Cancer, pp.178-184 (Mihich (ed.) 1985); Freshney, Anticancer Res.5:111-130 (1985).For example, monitoring tumour-specific markers level in the method for the compound of identifying and characterize modulation cancer correlated series of the present invention.

Invade Matrigel and identify and characterize modulator

The degree of invading Matrigel or extracellular matrix components can be as the test of the compound of identifying and characterize modulation cancer correlated series.Tumour cell shows grade of malignancy and cell is invaded the positive correlation between Matrigel or some other extracellular matrix components.In this test, tumour generation cell is usually as host cell.The expression of tumor suppressor gene will reduce the offensiveness of host cell in these host cells.Can adopt aforesaid Cancer Res.1999; 59:6010; Technology among the Freshney (1994).In brief, through adopting the strainer that is encapsulated by Matrigel or other extracellular matrix components to detect the attack level of host cell.Infiltrate through gel, or the end that penetrates into strainer is considered to have offensiveness, and cell count and distance through moving, or through using in advance ¹²⁵The I labeled cell is also counted radioactivity in the bottom of strainer far-end or dish and is carried out histological grade, and referring to for example aforesaid Freshney (1984), document is the same.

Modulator is identified and is characterized in the growth of assessment in-vivo tumour

In transgenic or immunosuppression organism, detect the influence of cancer correlated series cell growth.Prepare transgenic organism through the multiple method of accepting for this area.For example, preparation is like the mammiferous transgenic organism that knocks out of mouse, and wherein the cancer gene is destroyed or has inserted the cancer gene.Via homologous recombination, insert marker gene or other heterology gene in the genomic endogenous oncogene of mouse site, knock out transgenic mice with preparation.This mouse also can prepare through replace the endogenous oncogene with mutant cancer gene, perhaps through the transgenation of endogenous cancer is prepared, as through being exposed to carcinogenic substance.

For preparing genetically modified chimaeric animals,, in the nuclear of embryonic stem cell, import DNA construct like mouse.The cell that contains the genetic damage of new transformation is injected among the embryo of host mouse, and the embryo is implanted female receptor again.A part of bud among these embryos has the gomphosis mouse of sexual cell, and some in these cells derive from the clone of sudden change.Therefore, through raising this chimeric mouse, possibly obtain to comprise the mouse new lines (referring to for example Capecchi et al., Science 244:1288 (1989)) of the genetic damage of introducing.Gomphosis mouse can obtain according to following document: the USP 6,365,797 that on April 2nd, 2002 published; Be published in the USP 6,107,540 on August 22nd, 2000; Hoganet al, Manipulating the Mouse Embryo:A laboratory Manual, Cold SpringHarbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells:APractical Approach; Robertson; Ed., IRL Press, Washington; D.C., (1987).

Alternatively, can adopt panimmunity to suppress or the immunodeficient host animal.For example; Heredity athymic " naked " mouse is (referring to for example Giovanella et al; J.Natl.CancerInst.52:921 (1974)), SCID mouse, thymectomized mouse or through the mouse of irradiation (referring to for example Bradley et al, Br.J.Cancer 38:263 (1978); Selby et al, Br.J.Cancer 41:52 (1980)) all can be used as the host.Be injected into homogenic host's transplantable tumor cell (common about 10 ⁶Individual cell) in a high proportion of case, produce offensive tumour, the normal cell of similar origin then can not.In growing the host of offensive tumour, express the cell of cancer correlated series and injected by subcutaneous or coordination (orthotopically).Mouse is divided into group then, comprises control group and therapeutic test group (for example, treating with modulator).After the time of suitable length, in preferred 4-8 week, detect growth of tumor (for example, through the size of volume or its two maximums, or weight) and compare with contrast.The tumour of dwindling (adopting for example Student ' s T check) with significance,statistical is called as has growth-inhibiting.

Identify and characterize the in vitro tests of modulator

Evaluation has the test of the active compound of modulation and can carry out external.For example, at first make cancer polypeptide and potential modulator contact and be incubated one suitable period, for example, from 0.5 to 48 hour.In one embodiment, the level through measuring protein or mRNA is in external definite cancer polypeptide level.The antibody of use and cancer polypeptide or its fragment selective binding adopts for example Western trace, and the immunoassay of ELISA etc. detects protein level.The hybridization analysis that adopts the amplification method of for example PCR, LCR or for example be preferably Northern hybridization, RNAse protection, dot blotting detects mRNA.As described herein, with the detection reagent of direct or indirect mark, for example the antibody through fluorescence or radiolabeled nucleic acid, radioactivity or enzyme labelling waits protein or the mRNA level of detecting.

Alternatively, can adopt the sub-design report genic system of cancer bak promoter, this cancer bak promoter links to each other with can operating such as the reporter gene of luciferase, green fluorescent protein, CAT or P-gal.Usually with the transfection of reporter gene construct to cell.After handling with the potential modulator, according to standard technique well known by persons skilled in the art detect the transcribing of reporter gene, translation or active amount (Davis GF, literary composition sees before; Gonzalez, J.& Negulescu, P.Curr.Opin.Biotechnol.1998:9:624).

As stated, can carry out in-vitro screening to each gene and gene product.That is to say, identify the gene that the vital specific differences of particular state is expressed earlier, again the expression modulator of screening-gene or gene product itself.

In one embodiment, the expression modulator of screening specific gene.Usually, only estimate one or several expression of gene.In another embodiment, designing and screening is at first to find to combine the compound of differential expression protein.Estimate the ability of these compound modulation difference expression activities then.In addition, in case identify the primary candidate compound, can further screen variant with evaluation structure activity relationship better.

Identify and characterize the combination test of modulator

In combination test of the present invention, generally use purifying of the present invention or isolating gene product.For example, produce, carry out immunoassay to confirm proteinic amount and/or position to the proteinic antibody of the present invention.Perhaps, use contains the proteinic cell of cancer in test.

Therefore, method comprises mixes cancer protein of the present invention and candidate compound, like part, and measures compound and cancer combination of proteins of the present invention.Preferred embodiment has been utilized human cancer protein; Also can develop and use the animal model of human diseases.In addition, those skilled in the art understand and also can use other similar mammalian proteins matter.In addition, in some embodiments, variation or deutero-cancer protein have been used.

Generally speaking, cancer protein of the present invention or part non--combine to dispersivity with insoluble upholder.Upholder can be the upholder (droplet plate, array etc.) that for example has the zone of the sample separation accepted.Insoluble upholder can be processed by any composition of synthetics that can combine, and said upholder can easily separate with soluble material, otherwise is exactly can be compatible with whole screening method.The surface of said upholder can be a solid or poroid, and can be any shape easily.

The example of suitable insoluble upholder comprises droplet plate, array, film and pearl.They are generally by glass, plastics (like PS), polysaccharide, nylon, nitrocellulose or Teflon ^TMDeng processing.Droplet plate and array are convenient especially, because can use small quantity of reagent and sample to test in a large number simultaneously.For the not special restriction of synthetics and upholder bonded ad hoc fashion, if it can be compatible with entire method of the present invention with reagent, can keep the active of synthetics and be non-disperse.Preferred combining method comprises use antibody; Said antibody can spatially not seal ligand-binding site point or activation sequence when making protein combine, directly be incorporated into the synthetic or reagent of " viscosity " or ion upholder, chemically crosslinked surface protein etc. with upholder.After making protein or part/wedding agent and upholder combining, remove excessive not bond material through washing.Seal the sample region of acceptance through being incubated then with bovine serum albumin (BSA), casein or other nontoxic protein matter or other moity.

In case cancer protein of the present invention combines with upholder, adds in test test-compound.Perhaps, candidate's wedding agent is combined with upholder, add cancer protein of the present invention then.Wedding agent comprises non--natural wedding agent that specific antibody, screen chemical libraries identify, peptide analogs etc.

Making us interested especially is the test of identifying the medicament of people's cell low toxicity.Multiple test be can use for this reason, protein-protein bound test of proliferation test, cAMP test, external mark, immunoassay that electrophoretic mobility changes test, protein bound, function test (phosphorylation test etc.) etc. comprised.

Can measure test-compound (part, wedding agent, modulator etc.) and cancer combination of proteins of the present invention with several different methods.Can the mark test-compound; Through all or part of cancer protein of the present invention is combined with solid support; Interpolation washes excess reagent and measures whether there is directly mensuration combination of mark on the solid support through the candidate compound (like fluorescent mark) of mark.Can utilize multiple sealing and washing step in the time of suitably.

In certain embodiments, a kind of component of mark only, for example only mark protein of the present invention or part.Perhaps, with more than one component of different markers substance markers, as use I ¹²⁵Labelled protein is used the fluorine tagged compound.Contiguous reagent also is useful like cancellation or energy transfering reagent.

The competitive combination to identify and to characterize modulator

In one embodiment, combine test to measure the combination of " test-compound " through competition with " competitor ".Competitor is to combine moity with target molecule (like cancer protein of the present invention) bonded.Competitor comprises the compound like antibody, peptide, combination counterpart, part etc.In some cases, test-compound combines to have replaced test-compound with competition between the competitor.In one embodiment, test-compound is labeled.In protein, add test-compound, competitor, or the two, keep the sufficiently long time so that they combine.Helping under the temperature of optimum activity, be generally 4 to 40 ℃ and be incubated.Usually the optimization soaking time is to help the fast high-flux screening; General 0 to 1 hour just enough.Usually need remove or reagent that flush away is excessive.Add second kind of component then, whether mensuration exists through marker components combines to show.

In one embodiment, at first add competitor and add test-compound again.The displacement competitor is represented test-compound and cancer protein bound, therefore can combine and potential modulation cancer activity of proteins.In this embodiment, can any component of mark.Therefore, for example, if the mark competitor exists mark to represent to be replaced by test-compound in the back-test compounds washing soln.Perhaps, if the mark test-compound exists mark to represent displacement on the upholder.

In selectable embodiment, at first add test-compound, insulation and washing then add competitor.Lack competitor and combine expression: compare with competitor, test-compound is with higher affinity and cancer protein bound.Therefore, if the mark test-compound, the existence of upholder marked merges the competitor bonded and lacks therefore potential modulation cancer protein of the present invention of expression test-compound combination.

Therefore, the competition combining method comprises that differential screening is to identify the medicament that can modulate cancer protein active of the present invention.In this embodiment, this method is included in mixes cancer protein and competitor in first sample.Second sample contains test-compound, cancer protein and competitor.The competitor of measuring in two samples combines, between two samples bonded change or difference represent to exist can with cancer protein bound and its active medicament of potential modulation.That is, if for first sample, it is different that the competitor in second sample combines, this medicament can with the cancer protein bound.

Perhaps, the usage variance Screening and Identification go out can with natural cancer protein bound, but can not with the drug candidate through the cancer protein bound of modifying.For example, make the proteinic structural models of cancer, and use it for the rational drug design with synthetic can with the interactional medicament in this site, general not with the medicament of site through the protein bound of modifying.In addition, the ability that strengthens or reduce said protein active through screening of medicaments also can identify the drug candidate that can influence natural cancer protein active.

Positive control and negative control have been used in this test.Preferably at least three parts of tests, contrast with given the test agent and test to obtain the significant result of statistics.The soaking time of all samples is enough to make medicament and protein bound.After the insulation, from sample flush away non--specificity bonded material, measure bonded, normally through the amount of the medicament of mark.For example, when utilizing radio-labeling, can in scintillometer, count sample to measure the amount of binding compounds.

Can comprise multiple other reagent in the shaker test.It comprises the reagent like salt, neutral protein matter (like BSA), stain remover etc., and said reagent helps optimum protein matter-protein bound and/or reduces non--specificity or the background interaction.Also can use the reagent that can improve test efficiency, like proteinase inhibitor, ribozyme suppressor factor, anti--microbial reagent etc.Can provide required bonded order to add the mixture of component.

Use the polynucleotide downward modulation or suppress protein of the present invention

Of WO91/04753, through forming conjugate, the polynucleotide modulator of cancer imported contain in the cell of target nucleotide sequences with part-binding molecule.Suitable part-binding molecule includes but not limited to cell surface receptor, growth factor, other cytokine or other and cell surface receptor bonded part.The coupling of preferred ligand binding molecules can not disturbed the ability of ligand binding molecules its corresponding molecule of combination or acceptor in fact, or obstruction has justice or antisense oligonucleotide or its coupling form to get into cell.Perhaps, can be of WO90/10448, the polynucleotide modulator of cancer is imported contain in the cell of target nucleic acid sequence through for example forming polynucleotide-lipid complex.Should understand except treat-ment, can also in shaker test mentioned above, use antisense molecule or knock out and embed (knock in) model.

Inhibition and GEM 132

In certain embodiments; Through using the small nuclear rna (snRNA) of antisense polynucleotides or inhibition; Promptly be complementary to and preferred ability specific hybrid coding mRNA nucleotide sequence; Like cancer protein of the present invention, the activity of cancer-related protein can be reduced or suppressed fully to the nucleic acid of mRNA or its subsequence.The translation and/or the stability that combine to have reduced mRNA of antisense polynucleotides and mRNA.

In context of the present invention, antisense polynucleotides can contain natural nucleotide, or the synthesizing ribonucleotide that is formed by natural subunit or its close homologue.Antisense polynucleotides also can have in sugared moity that changes or sugar and connects key.For example thiophosphatephosphorothioate and other sulfur component known in the art.The present invention also comprises analogue, as long as they can be hybridized with Nucleotide of the present invention effectively.Referring to for example Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA.

Use recombination method can easily synthesize said antisense polynucleotides, perhaps also can be at external synthetic said polynucleotide.Said synthetic instrument can comprise AppliedBiosystems available from several manufacturers.Other oligonucleotide also is that those skilled in the art are well-known like the preparation of thiophosphatephosphorothioate and alkyl derivative.

The antisense molecule that this paper uses comprises antisense or MODN is arranged.For example, can use MODN to transcribe through combining to block with antisense strand.Antisense and the said target mrna (justice is arranged) that has MODN to contain to combine the cancer molecule or the single-chain nucleic acid sequence (RNA or DNA) of DNA (antisense) sequence.Antisense of the present invention or have MODN to contain generally to be at least about 12 Nucleotide, preferably to be about the fragment of 12 to 30 Nucleotide.The ability description that obtains antisense or MODN arranged based on the given proteinic cDNA sequence of coding is in for example Stein&Cohen (Cancer Res.48:2659, (1988)) and vander Krol et al. (BioTechniques 6:958 (1988)).

Ribozyme

Except antisense polynucleotides, also can use the ribozyme target and suppress transcribing of cancer-related nucleotide sequences.Ribozyme is can other RNA RNA molecule molecule of catalytic pyrolysis.Dissimilar ribozymes has been described; Comprise I group ribozyme, hammerhead ribozyme, hair clip ribozyme, RNase P and axehead ribozyme (commentary of the characteristic of relevant different ribozymes referring to for example Castanotto et al., Adv.in Pharmacology 25:289-317 (1994)).

The general feature description of hair clip ribozyme is in for example Hampel et al., Nucl.Acids Res.18:299-304 (1990); European Patent Publication No 0360257; USP 5,254,678.The preparation method is that those skilled in the art are well-known (referring to for example WO94/26877; Ojwang et al., Proc.Natl.Acad.Sci.USA 90:6340-6344 (1993); Yamada et al., Human Gene Therapy1:39-45 (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., Virology205:121-126 (1994)).

In phenotypic screen, use modulator

In one embodiment, the cancer cell that has an associated cancer expression and distribution figure to a group is used test-compound." using " of this paper or " contact " refer to and in cell, add modulator in a particular manner, make modulator through acting in picked-up and the cell or effect pair cell through cell surface plays a role.In some embodiments, the nucleic acid importing virus formulation body with coded protein reagent (being peptide) like adenovirus or retroviral construct body, and is added in the cell, thereby accomplishes the expression of peptide agent, referring to for example PCT US97/01019.Also can use adjustable gene therapy system.In case used modulator to cell, washed cell preferably makes cell under physiological condition, be incubated for some time in case of necessity.Collecting cell produces new gene expression profiles then.Therefore, for example, screening is modulated to cancerous tissue, as induces or suppress the medicament of cancerous phenotype.At least one gene, the change of preferred a plurality of gene expression profiles represent that this medicament works to the cancer activity.Similarly, changing biological function or signal transduction path is the active demonstration of modulator.Through limiting the characteristic of cancerous phenotype, design can change the screening of the new drug of phenotype.Use this method, needn't know the medicine target, also will not be presented on original gene/protein expression screening platform by the medicine target, also needn't change the level of target protein transcript.The modulator inhibit feature can be used as surrogate markers.

As stated, need screen, that is to say, identify the gene that the vital specific differences of particular state is expressed earlier, again the expression modulator of screening-gene or gene product itself to estimate gene or gene product.

Use modulator to influence peptide of the present invention

Use multiple test determination cancer polypeptide activity or cancerous phenotype.For example, measure the influence of modulator through detecting above-mentioned parameter to the cancer polypeptide function.Use the active physiology of influence to change of the influence of assessment test-compound to polypeptide of the present invention.When using intact cell or zoometry function as a result, can estimate multiple effect; For example, can estimate tumor growth, metastases, neovascularity generation, hormone release, known and the change (changing) of transcribing change (as through the Northern trace), cellular metabolism of unidentified genetic marker and the interior second messenger's (like cGNIP) of cell change like cell growth or pH as far as the cancer relevant with noumenal tumour.

The method of diagnostic characteristics property cancer correlated series

The expression of several genes sequence is relevant with cancer, correspondingly, has identified the disease based on sudden change or variation cancer gene.In a specific embodiments, the invention provides the method for identifying the cell that contains variation cancer gene, for example, confirm the existence of all or part of sequence of at least one endogenous cancer gene in the cell.This can accomplish through the sequencing technologies of utilization arbitrary number.The present invention includes and identify the genotypic method of individual cancers, for example, confirm the existence of all or part of sequence of at least a gene of the present invention in the individuality.This at least a tissue of individuality, for example carries out in the listed tissue of table 1 usually, can also comprise the assessment to multiple tissue or the different samples of same tissue.This method can comprise the sequence and the known cancer gene of sequenced genes; Be that wild type gene compares; Confirm the existence of family member, homology, sudden change or variation, can the sequence of all or part of sequence of this gene and known cancer gene be compared then and determine whether variant existence.This can carry out through the known homology program of any number, like BLAST, and Bestfit etc.As described herein, the existence of difference is relevant with morbid state or morbid state tendency between patient's cancer gene and the known cancer gene order.

In a preferred embodiment, confirm the number of cancer gene copy in the genome as probe with the cancer gene.Confirm its position on karyomit(e) with the cancer gene as probe.Particularly, when the chromosome abnormalty that in the locus of cancer gene, identifies like transposition etc., can be applied to provide diagnosis or prediction like the information of chromosomal localization.

XIV.) therepic use of RNAi and little RNA interfering (siRNA)

The invention still further relates to segmental the siRNA oligonucleotide, particularly double-stranded RNA that comprise PSCA coding region or 5 ' UTR district at least, or its complement, or any antisense oligonucleotide that is specific to the PSCA sequence.In one embodiment, use said oligonucleotide to illustrate the function of PSCA, or use the modulator of said oligonucleotide screening or evaluation PSCA function or expression.In another embodiment, use the siRNA transfection to reduce the genetic expression of PSCA, and then the antigenic multiplication capacity through the conversion cancer cells of endogenous expression is significantly reduced; Through using for example relevant cell viability metabolism readout to measure, demonstrate the survival rate of reduction with the cell of specificity PSCA siRNA processing with the multiplication capacity that reduces.Therefore, PSCA siRNA compsn contains the siRNA (double-stranded RNA) corresponding to PSCA proteic nucleic acid ORF sequence or its subsequence; The length of these subsequences is generally 5,6, and 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 the RNA Nucleotide of 35 adjacency, and contain with at least a portion of mRNA encoding sequence complementary with complementary sequence not.In preferred embodiments, the length of subsequence is 19-25 Nucleotide, and most preferably length is 21-23 Nucleotide.

It is novel method external and the interior silencer of body that RNA disturbs, and therefore, little double-stranded RNA (siRNA) is valuable therapeutical agent.At present, the active ability of the reticent specific gene of siRNA has been able to embody in the animal model of disease, and also can be used for the people.For example, confirmed and will have curative effect in solution fluid perfusion to the mouse body to the siRNA of particular target.

Formerly working of Song etc. shows: a kind of nucleic acid of pure natural; Be little RNA interfering (siRNA) even need not promptly to can be used as therapeutical agent (Song through further chemically modified; E.; Et al., " RNAinterference targeting Fas protects mice from fulminant hepatitis " Nat.Med.9 (3): 347-51 (2003)).This work provides to animal perfusion siRNA first can alleviate evidence in the body of disease.In this work, the author give injected in mice through design can reticent FAS albumen (a kind of necrocytosis acceptor, when its in Inflammatory response during by overactivity, but inducing hepatocyte and other necrocytosis) siRNA.Second day, use the antibody that is specific to Fas to animal.In subsequently several days, control mice is died from acute hepatic failure, and the mouse of handling through siRNA more than 80% does not suffer from serious disease, and is able to survival.About 80% to 90% liver cell of these mouse has been mixed exposed siRNA oligonucleotide.In addition, the RNA molecule can play a role and reach 10 days, and is just ineffective after 3 weeks.

When being used to treat man-hour, transmit siRNA through inducing the active effective system of long-term RNAi.Main warning to clinical application is should siRNA be passed to suitable cell.As if liver cell can accept exogenous rna especially.At present, because liver is the organ of nucleic acid molecule and the easy target of virus vector, therefore, the target that is positioned at liver is especially noticeable.Yet the target of other tissue and organ also is preferred.

With the compound preparation siRNA that can promote cross-cell membrane transhipment, improve using of siRNA in the therapeutic process with this.Another embodiment of the invention is the synthetic siRNA through chemically modified, and said siRNA can resist the effect of ribozyme, has serum stability, and with it together, the time length of RNAi effect prolongs.

Therefore, the siRNA technology is to suffer from the method for treating human malignancies like the individuality of the listed cancer of Table I through passing to the siRNA molecule of PSCA.Use the growth that siRNA can cause expressing the cancer cells of PSCA and weaken, and the Anti-tumor therapy is provided, reduced sickness rate relevant and/or mortality ratio with malignant tumour.

When in external or body, measuring, the effectiveness that this gene product is knocked down (knockdown) form is significant.Use siRNA through (as stated) to cells in culture, perhaps when using in vitro method to detect the PSCA protein expression that reduces, use siRNA for the aliquots containig of cancer patients's biopsy samples, can easily illustrate vitro efficacy.

XV.) test kit/goods

Use in order to be used for laboratory described herein, prediction, prevention, diagnosis and treatment, test kit also within the scope of the invention.Said test kit can comprise carrier, packing or container; Container can be separated into cell to hold one or more containers such as bottle, test tube etc.; Each container contains the used a kind of independent component of present method respectively, and with the label or the inset that use (application as described herein) to explain.For example, but but container can contain through mark note or can be by the probe of mark note.This probe can be antibody or the polynucleotide that are specific to protein of the present invention or gene or information respectively.When this method utilized nucleic acid hybridization to detect target nucleic acid, test kit also can comprise the container of the Nucleotide that contains amplification target nucleic acid sequence.Test kit can comprise the container that contains reporter molecule, as conjugated protein with reporter molecule such as enzyme, fluorescence or labelled with radioisotope bonded vitamin H, and for example avidin or Streptavidin; Said reporter molecule can use with for example nucleic acid or antibody.This test kit can comprise Fig. 1, Fig. 2 or all or part of aminoacid sequence or its analogue shown in Figure 3, the perhaps nucleic acid molecule of encode such amino acid sequences.

Test kit of the present invention generally includes above-mentioned container and one or more other relevant with it container, wherein contains the material that meets commercial and user's needs, comprises damping fluid, thinner, strainer, pin, syringe; Carrier, packing, container, bottle and/or test tube label are shown content and/or explanation that supply to use on it, and with the package insert of operation instruction.

Container can post or explain that with label compsn is used for the specific treatment or the application of non-treatment, like prediction, prevention, diagnosis or laboratory applications, also can explain in the body as described herein or the indication of in-vitro application.Indication and/or out of Memory also can be included in the appended or inset that labeled of test kit or on.Label can be affixed on the container or with it and be associated.When letter, numeral or other were formed the literal mold of label or be etched on the container, label placed on the container; When label was present in storage or the carrier of container, label was associated with described container, for example as the inset of packing.Label can explain that compsn is used for diagnosis, treatment, prevention or prediction disease, the tumour of shown in Table I, organizing.

Term " test kit " can be used as synonym with " goods ".

In another embodiment of the invention; A kind of goods that comprise like the compsn of aminoacid sequence, small molecules, nucleotide sequence and/or antibody are provided; For example, be used for diagnosing, predict, prevent and/or treat material like the tumour of the cited tissue of Table I.Goods generally include at least one container and at least one label.Suitable containers comprises for example bottle, bottle, syringe and test tube.Container can be made of a variety of materials, like glass, metal or plastics.This container can comprise aminoacid sequence, small molecules, nucleotide sequence, cell colony and/or antibody.In one embodiment, container comprises the polynucleotide that are used for detecting cell mRNA expression and distribution figure, contains the reagent that is useful on this purpose simultaneously.In another embodiment, container comprises and is used for estimating PSCA at the protein expression of cell with tissue, or the antibody of the laboratory that is used to be correlated with, prediction, diagnosis, prevention and therapeutic purpose, its binding fragment or binding proteins specific; Said container can post or have the explanation and/or the indication of said application, also can comprise the reagent and other compsn or instrument that are used for these purposes.In another embodiment, contain the material that is useful on trigger cell or HI and relevant explanation and/or indication in the container.In another embodiment, contain the material that is useful on adoptive immunotherapy in the container, like cytotoxic T cell (CTL) or helper cell (HTL), and relevant explanation and/or indication; Also can comprise the reagent and other compsn or instrument that are used for this purpose in the container.

Container also can optionally comprise the compsn that is used for treating effectively, diagnose, predicting or prevent certain disease, can contain sterile port (can be an intravenous solution bag or a bottle that has with the stopper of hypodermic needle perforation like container) simultaneously.Promoting agent in the compsn can be specificity to combine PSCA and the antibody of modulating its function.

Goods may further include second container, and it contains pharmaceutically acceptable damping fluid, for example PBS, Ringer's solution and/or glucose solution.It may further include other material that meets commercial and user's needs, comprises damping fluid, thinner, strainer, pin, syringe and/or the package insert of indicating and/or explaining with use.

Embodiment:

Some embodiment through following are described further and illustrate different aspect of the present invention, and all embodiment should not be understood that limitation of the scope of the invention.

Embodiment 1: the expression analysis of PSCA variant in healthy tissues and the patient's sample

In the past, PSCA (this paper is referred to as PSCAv.1) was accredited as the antigen of in prostate cancer, expressing.In surpassing the transfer of 80% primary prostate cancer and most of prostate gland, detect its expression.Confirmed that PSCA also expresses in bladder cancer, ovarian cancer and carcinoma of the pancreas; These cancers are listed in Table I.Through immunohistochemical analysis, confirmed that PSCA crosses expression in most of urothelial are divided a word with a hyphen at the end of a line cancer cells surface and 60% PaCa.The psca expression data be reported in patent publications (PCT/US98/04664, PCT/US/28883, PCT/US00/19967) and survey article (Saffran et al., Proc Natl AcadSci U S is Feb 27 A.2001; 98 (5): 2658-2663; Amara et al., Cancer Res.2001 Jun15; 61 (12): 4660-65; Reiter et al., Proc Natl Acad Sci USA.1998 Feb 17; 95 (4): 1735-40; Argani et al., Cancer Res.2001 Jun 1; 61 (11): 4320-24).

Normally with in cancer patients's sample studying the specific expressed of different PSCA variants.To PSCAv.1/v.2/v.4; The primer that PSCAv.3 and PSCAv.5 design differ from one another; PSCAv.1/v.2/v.4 causes producing the PCR product of 425bp, and PSCAv.3 causes producing the PCR product of 300bp, and PSCAv.5 causes producing big or small be the PCR product of 910bp (Fig. 1 I (a)).

Sample library by normal bladder, brain, heart, kidney, liver, lung, prostate gland, spleen, Skelettmuskel, testis, pancreas, colon, stomach and prostate cancer, bladder cancer, kidney, colorectal carcinoma, lung cancer, ovarian cancer, mammary cancer, metastasis of cancer and carcinoma of the pancreas prepares the first chain cDNA (Fig. 1 I (b)).PCR through using the Actin muscle primer carries out normalization method.Use the variant Auele Specific Primer, take turns amplification cycles with 30 and carry out half-quantitative PCR.

The result shows that PSCAv.5 mainly expresses in mammary cancer, metastasis of cancer and carcinoma of the pancreas, and the expression level in colorectal carcinoma and lung cancer is lower.In prostate cancer, bladder cancer, kidney, colorectal carcinoma, lung cancer, ovarian cancer, mammary cancer, metastasis of cancer and carcinoma of the pancreas, detect PSCAv.1/v.2/v.4 PCR product.In healthy tissues, only in prostate gland, stomach, detect PSCAv.1/v.2/v.4 PCR product, the expression level of this product in kidney and lung is lower, and in any healthy tissues, all detects less than PSCAv.5.The PCR that in any test sample, detects less than PSCAv.3 detects product.

Design the primer (Fig. 1 J (a)) that differs from one another to PSCAv.4 and PSCAv.5.PSCAv.4 causes producing the PCR product of 460bp, and PSCAv.5 causes producing the big or small PCR product of 945bp that is.

Cancer sample library and multiple-heterograft storehouse (prostate cancer, kidney and bladder cancer heterograft) preparation first chain cDNA (Fig. 1 J (b)) by normal bladder, brain, heart, kidney, liver, lung, prostate gland, spleen, Skelettmuskel, testis, pancreas, colon, stomach and prostate cancer, bladder cancer.PCR through using the Actin muscle primer carries out normalization method.Use the variant Auele Specific Primer, take turns amplification cycles with 30 and carry out half-quantitative PCR.

The result shows that PSCAv.4 expresses in prostate cancer, bladder cancer and multiple-heterograft storehouse, normal kidney and prostate gland.Only in normal prostatic and bladder cancer, detect PSCAv.5.

The expression of PSCA variant in healthy tissues is limited, and in cancer patients's sample, can detect expression, and this shows that the PSCA variant is treatment, prediction, laboratory, prevention and the diagnosis target of human cancer.

The splice variant of embodiment 2:PSCA

Term variant used herein comprises transcribes variant and SNP (SNP).Transcribe variant and be from the variant that produces through alternative transcription or alternative splicing from the ripe mRNA of same gene.But the alternative transcription thing is from identical gene at the transcript of difference transcriptional start.Splice variant is to derive from identical transcript but the different mRNA variant of montage.In eukaryote, when many-exon genes was transcribed by genomic dna, original RNA produced functional mRNA through montage, and it has only exon, is used for translating into aminoacid sequence.Correspondingly, a given gene can have zero or a plurality of alternative transcription thing, and each transcript has zero or a plurality of splice variant.Each is transcribed variant and all is made up of the exon of uniqueness, and compares with primary transcript, and different coding and/or non-coding (5 ' or 3 ' end) part can be arranged.Transcribing variant can encode and have identical, the similar or different protein of same or similar function or difference in functionality.Variant proteins can be simultaneously in identical tissue, simultaneously in different tissues or different time in identical tissue or different time in different tissues, express.Transcribe variant encoded protein matter similar or different ubcellulars or location, extracellular (for example, excretory is with respect to intracellular) can be arranged.

Can identify through the multiple method of accepting for this area and transcribe variant.For example, alternative transcription thing and splice variant can pass through full-length clone, or adopt total length transcript and est sequence to identify.At first, all human EST assemble cluster, show direct or indirect identity each other.Secondly, the EST in identical the clustering further is grouped into inferior bunch, and is assembled into consensus sequence.The original gene sequence is compared with consensus sequence or other full length sequence.Each consensus sequence all is the potential splice variant of this gene.It is known in the art that several kinds of conclusive evidence patterns are arranged, as identifying variant through Northern analysis, full-length clone or use probe library etc.Be not full-length clone even variant is accredited as, through adopting technology known in the art, this part of variant also is a research tool of great use to producing antigen or further cloning the total length splice variant.

In addition, the computer program of transcribing variant of identifying based on genome sequence known in the art.Comprise FgenesH (A.Salamov and V.Solovyev, " Abinitio gene finding in Drosophila genomic DNA, " Genome Research.2000 April based on the genomic variant qualification program of transcribing; 10 (4): 516-22); Grail (URL compbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (URL genes.mit.edu/GENSCAN.html).The generality discussion of relevant splice variant qualification program can be referring to for example: Southan, C., A genomic perspective on humanproteases, FEBS Lett.2001 Jun 8; 498 (2-3): 214-8; De Souza, S.J., et al., Identification of human chromosome 22 transcribed sequences with ORFexpressed sequence tags, Proc.Natl Acad Sci U S is Nov 7 A.2000; 97 (23): 12690-3.

For further confirming to transcribe the parameter of variant; Can use multiple technologies known in the art; For example full-length clone, proteomics are confirmed, the affirmation of PCR-based and 5 ' RACE confirm etc., and (referring to for example: proteomics is confirmed: Brennan, S.O., et al.; Albumin banks peninsula:a newtermination variant characterized by electrospray mass spectrometry, BiochemBiophys Acta.1999 Aug 17; 1433 (1-2): 321-6; Ferranti P, et al., Differentialsplicing of pre-messenger RNA produces multiple forms of maturecaprine alpha (s1)-casein, Eur J Biochem.1997 Oct 1; 249 (1): 1-7; The affirmation of PCR-based: WellmannS; Et al.; Specific reverse transcription-PCR quantification of vascularendothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem.200 1Apr; 47 (4): 654-60; Jia, H.P., et al, Discovery of new humanbeta-defensins using a genomics-based approach, Gene.2001 Jan 24; 263 (1-2): 211-8; The affirmation of PCR-based and 5 ' RACE: Brigle; K.E.; Et al., Organization of themurine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta.1997 Aug 7; 1353 (2): 191-8).

Genome area in the cancer known in the art is modulated.When modulated, variable transcript or gene splicing variant are also modulated in particular cancers for the genome area of mapping when gene.What this paper disclosed is that PSCA has and the relevant particular expression distribution plan of cancer (referring to for example Table I).For example at one or more these tissues with in some other tissue, the alternative transcription thing of PSCA is also relevant with cancer with splice variant.Therefore, variant can be used as tumour mark of correlation/antigen.

Use total length psca gene and est sequence, identify four other transcribe variant, be referred to as PSCAv.2, v.3, v.4 and v.5.The exon border that has shown initial transcript PSCAv.1 in the Table VI.Fig. 1 has shown the sequence of PSCA and PSCA variant.

The SNP of embodiment 3:PSCA

SNP (SNP) is that the single base pair of nucleotide sequence specific position changes.Any given point in genome all has four kinds of possible nucleotide bases right: A/T, C/G, G/C and T/A.Allelotrope used herein is one of a series of optional forms of given gene, and its dna sequence dna is different, and can influence product (RNA and/or protein).

The SNP that on cDNA, takes place is called cSNP.This cSNP can change the proteinic amino acid of coded by said gene, therefore changes proteinic function.Some SNP can cause inherited disease; The individual reaction of the number change of other caused phenotype and influence to the environmental factors that comprises diet and medicine.Therefore; SNP and/or allelotrope make up many useful purposes that have of (being called as haplotype); As diagnose inherited disease, confirm that drug reaction and dosage, evaluation cause the gene of disease and analyze genetic affinity (P.Nowotny between individuality; J.M.Kwon and A.M.Goate, " SNP analysis to dissect humantraits, " Curr.Opin.Neurobiol.2001 Oct; 11 (5): 637-641; M.Pirmohamed and B.K.Park, " Genetic susceptibility to adverse drug reactions, " Trends Pharmacol.Sci.2001 Jun; 22 (6): 298-305; J.H.Riley, C.J.Allan, E.Lai and A.Roses, " The use ofsingle nucleotide polymorphisms in the isolation of common disease genes, " Pharmacogenomics.2000 Feb; 1 (1): 39-47; R.Judson, J.C.Stephens andA.Windemuth, " The predictive power of haplotypes in clinicalresponse, " Pharmacogenomics.2000feb; 1 (1): 15-26).

SNP can identify (P.Bean, " Thepromising.voyage of SNP target discovery, " Am.Clin.Lab.2001 Oct-Nov through the multiple method of accepting for this area; 20 (9): 18-20:K.M.Weiss, " In search of human variation, " Genome Res.1998 Jul; 8 (7): 691-697; M.M.She, " Enabling large-scale pharmacogenefic studies byhigh-throughput mutation detection and genotyping technologies, " Clin.Chem.2001 Feb; 47 (2): 164-172).For example, can identify SNP like restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) through being that basic method checks order to the dna fragmentation that shows polymorphum with the gel.Also can be through finding SNP from the sequence of different DNA samples to collecting from the direct order-checking of the DNA of Different Individual sample or through comparing.Rapid accumulation along with the sequence data of both privately and publicly owned's DB; Also can use the computer program comparative sequences to find SNP (Z.Gu; L.Hillier and P.Y.Kwok, " Single nucleotide polymorphism hunting incyberspace, " Hum.Mutat.1998; 12 (4): 221-225).SNP can be determined; And individual genotype or haplotype can be confirmed through serial of methods; Comprise direct order-checking and high-throughput microarray (P.Y.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-throughputSNP genotyping with the Masscode system, " Mol.Diagn.2000 Dec; 5 (4): 329-340).

Use aforesaid method, identify 13 SNP of PSCAv.2 transcript.Use variant 2 rather than variant 1 for example, because for variant 1, variant 2 has less uncertain base.Therefore, in PSCAv.2, identify and be positioned at 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), the SNP of 878 (g/a) and 978 (c/g).Shown in Figure 1B and Fig. 1 G, have and to select allelic transcript or protein to be named as variant PSCAv.6 extremely v.18.

V.6 the Nucleotide in changes and has changed initiator codon v.1, and therefore, translation just can begin until next ATG (in mRNA, being AUG), causes v.1 short 9 amino acid of albumen of protein ratio.V.7 and Nucleotide v.8 to change be reticent on protein level.

12 among these 13 SNP also are present in variant 4.Extremely v.30 12 the SNP variants relevant with PSCAv.4 are called as PSCAv.19.Shown in Fig. 1 H, the selectable amino acid of variant 19 to 27 codings.

Embodiment 4: in prokaryotic system, produce reorganization PSCA

For express recombinant PSCA in prokaryotic cell prokaryocyte and PSCA variant, total length or part PSCA and PSCA variant cDNA sequence clone are gone into any of multiple expression vector known in the art, one or more column regions down of PSCA variant are expressed: the full length sequence shown in Fig. 1, or PSCA, its variant or its analogue any 8,9,10,11; 12,13,14,15,16,17,18; 19,20,21,22,23,24; 25,26,27,28,29,30 or the amino acid of more a plurality of adjacency.

A. in-vitro transcription with the translation construct:

PCRII:For the PSCA of production in situ detection RNA has justice and antisense RNA probes, preparation coding PSCA cDNA total length or segmental pCRII construct (Invitrogen, Carlsbad CA).The pCRII carrier has Sp6 and T7 promotor at the flank of insertion sequence, can start transcribing of PSCA RNA, as the probe of RNA hybridization in situ experiment.PSCA was in the expression of rna level during these probes can be used for analysis of cells and organize.The expression psca gene cDNA amino acid coding region be used for external translating system through transcribing PSCARNA, like TnT ^TM(Madison WI) synthesizes PSCA albumen to Coupled Reticulolysate System for Promega, Corp..

B. bacterium construct:

The pGEX construct:In order in bacterium, to produce the reorganization PSCA albumen that merges with glutathione S-transferase (GST) albumen; All or part of PSCA cDNA albumen coded sequence is cloned into (the Amersham Pharmacia Biotech of pGEX family of GST fusion vector; Piscataway, NJ).These constructs allow N-terminal to merge GST and C-terminal has merged 6 Histidine epi-position (controllable expression of reorganization PSCA protein sequence of 6 * His).GST and 6 * His mark allow to come the recombination fusion protein of self-induction bacterium through suitable affinity matrix purifying, and allow anti-GST of utilization and anti-His antibody to discern fusion rotein.6 * His mark produces for 6 Histidine codons of clone's primer interpolation of ORFs (ORF) 3 ' end through for example.Can use protease cracking site, for example the PreScission in the pGEX-6P-1 ^TMRecognition site can allow the cracking from the PSCA GAP-associated protein GAP of GST mark to get off like this.Ampicillin resistance gene and pBR322 source allow in intestinal bacteria, to select and keep the pGEX plasmid.

The pMAL construct:In order in bacterium, to produce the reorganization PSCA albumen that has merged SANMALT-S-conjugated protein (MBP); Through being cloned into pMAL-c2X and pMAL-p2X carrier (New EnglandBiolabs; Beverly, MA) on, with all or part of PSCA cDNA albumen coded sequence and MBP gene fusion.These constructs allow aminoterminals to merge the reorganization PSCA protein sequence that MBP and carboxyl terminal merged 6 Histidine epi-position marks controllably to express.MBP and 6 * His mark allow to come the recombinant protein of self-induction bacterium through suitable affinity matrix purifying, and allow anti-MBP of utilization and anti-His antibody to discern fusion rotein.6 * His epi-position label produces through adding 6 Histidine codons for 3 ' clone primer.The Xa factor recognition site allows the cracking from PSCA of pMAL mark is got off.Thereby pMAL-c2X and pMAL-p2X carrier are optimised express recombinant protein in tenuigenin or cell pericentral siphon respectively.The cell periplasmic expression has strengthened protein folding through disulfide linkage.

The pET construct:In order in bacterial cell, to express PSCA, all or part of PSCA cDNA albumen coded sequence is cloned in the carrier of pET family (Novagen, Madison, WI).These carriers allow strict control ground express recombinant PSCA albumen in bacterium; Said protein can with improve deliquescent protein, for example NusA and Trx (Trx) and help purifying and the epi-position mark of detection recombinant protein for example 6 * His and S-Tag ^TMMerge, also can not merge with them.For example, adopt pETNusA emerging system 43.1 to produce construct, make the proteic zone of PSCA be expressed as aminoterminal fusion rotein with NusA.

C. yeast construct:

The pESC construct:In order in yeast kind yeast saccharomyces cerevisiae, to express PSCA to produce recombinant protein and to carry out functional study; All or part of PSCA cDNA albumen coded sequence is cloned in the carrier of pESC family, and each carrier comprises 4 selective marker HIS3, TRP1; LEU2; With one of URA3 (Stratagene, La Jolla, CA).These carriers allow in same yeast cell controllably to be expressed until 2 by same plasmid and comprise Flag ^TMOr the different genes or the cloned sequence of Myc epi-position mark.This system can be used for confirming the protein-protein interaction of PSCA.In addition, the expression in the yeast produces similar posttranslational modification, and for example glycosylation and phosphorylation can be found these modifications when in eukaryotic cell, expressing.

The pESP construct:In order in grain wine sugar yeast (Saccharomyces pombe), to express PSCA, all or part of PSCA cDNA albumen coded sequence is cloned in the carrier of pESP family.These carriers allow PSCA protein sequence that aminoterminals or carboxyl terminal merged the GST that helps purification of recombinant proteins matter high expression level controllably.Flag ^TMThe epi-position mark allows the anti-Flag of utilization ^TMAntibody detects recombinant protein.

Embodiment 5: in high eukaryotic system, produce reorganization PSCA

A. Mammals construct:

For express recombinant PSCA in eukaryotic cell, all or part of PSCA cDNA sequence or its variant are cloned into any of multiple expression vector known in the art.Expressed one or more following PSCA zone: PSCA in these constructs v.1, the amino acid/11 to 123 of PSCA variant or its analogue, 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 a plurality of in abutting connection with amino acid.

This construct can be by transfection to any one of multiple mammalian cell, in the 293T cell.Can detect by the 293T product of cell lysis of transfection with anti-PSCA polyclonal serum described herein.

The pcDNA4/HisMax construct:In order in mammalian cell, to express PSCA, with the ORF of PSCA, or its part be cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA).Adopt cytomegalovirus (CMV) promotor and SP16 translational enhancer to come the expression of kinesin matter.The N-terminal of recombinant protein merges has Xpress ^TMWith 6 Histidine (6 * His) epi-positions.The pcDNA4/HisMax carrier also contains the stability that Trobest (BGH) polyadenylic acid signal and transcription termination sequence improve mRNA, contains the SV40 starting point that in the clone of expressing large T antigen, is used for episomal replication and simple carrier rescue simultaneously.The Zeocin resistant gene allows to select expressing proteinic mammalian cell, and ampicillin resistance gene and ColE1 starting point allow in intestinal bacteria, to select and keep plasmid simultaneously.

The pcDNA3.1/MycHis construct:In order in mammalian cell, to express PSCA, with PSCAORF, or its a plurality of parts, with total Kozak translation initiation site, be cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA).Protein expression drives through cytomegalovirus (CMV) promotor.The C-terminal of recombinant protein merges has myc epi-position and 6 * His epi-position.The pcDNA3.1/MycHis carrier also comprises the stability that Trobest (BGH) polyadenylic acid signal and transcription termination sequence improve mRNA, contains the SV40 starting point that in the clone of expressing large T antigen, is used for episomal replication and simple carrier rescue simultaneously.Can use neomycin resistance gene, but because it allows to select expressing proteinic mammalian cell, ampicillin resistance gene and ColE1 starting point allow in intestinal bacteria, to select and keep plasmid simultaneously.

The pcDNA3.1/CT-GFP-TOPO construct:In order in mammalian cell, to express PSCA, and allow to adopt the fluoroscopic examination recombinant protein, with PSCA ORF, or its a plurality of parts, with total Kozak translation initiation site, be cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA).Protein expression drives through cytomegalovirus (CMV) promotor.The C-terminal of recombinant protein merges has green fluorescent protein (GFP), helps interior the detection and RESEARCH ON CELL-BIOLOGY of body of Noninvasive.The pcDNA3.1/CT-GFP-TOPO carrier also comprises the stability that Trobest (BGH) polyadenylic acid signal and transcription termination sequence improve mRNA, contains the SV40 starting point that in the clone of expressing large T antigen, is used for episomal replication and simple carrier rescue simultaneously.Neomycin resistance gene allows to select expressing proteinic mammalian cell, and ampicillin resistance gene and ColE1 starting point allow in intestinal bacteria, to select and keep plasmid simultaneously.N-terminal merges has other construct of GFP in pcDNA3.1/NT-GFP-TOPO, to process, and it has crossed over the PSCA albumen of total length.

PAPtag:With the ORF of PSCA, or its part be cloned into pAPtag-5 (GenHunterCorp.Nashville, TN).This construct has produced that C-terminal has merged SEAP and N-terminal has merged the PSCA albumen of IgG κ signal sequence.Also prepared the construct that SEAP with N-terminal IgG κ signal sequence and PSCA albumen N-terminal merge simultaneously.Make the reorganization PSCA albumen optimization that obtains, to by in the substratum of transfection mammalian cell, and can be used for identifying the protein with the PSCA protein-interacting with secretion, like part or acceptor.Protein expression drives through the CMV promotor, and recombinant protein also contains the myc that helps purifying and detection and the 6 * His epi-position that merges at C-terminal.Zeocin resistant gene on the carrier allows the mammalian cell of expressing recombinant protein is selected, and ampicillin resistance gene allows in intestinal bacteria, to select plasmid simultaneously.

PTag5:PSCA ORF or its part are cloned into pTag-5.This carrier and pAPtag are similar but do not merge SEAP.The PSCA albumen that this construct produces has IgG κ signal sequence at N-terminal, and C-terminal has myc and the 6 * His epi-position mark that helps to detect with affinity purification simultaneously.With the reorganization PSCA albumen optimization that obtains, being secreted into, and can be used as immunogen or part and be used to identify the protein with the PSCA protein-interacting, like part or acceptor by the substratum of transfection mammalian cell.Protein expression drives through the CMV promotor, and the Zeocin resistant gene on the carrier allows to select expressing proteinic mammalian cell, and ampicillin resistance gene allows in intestinal bacteria, to select plasmid simultaneously.

PsecFc:PSCA ORF or its part also are cloned into psecFc.(Invitrogen California) assembles the psecFc carrier through immunoglobulin G while 1 (IgG) Fc (hinge area, CH2, CH3 district) is cloned into pSecTag2.This construct produces IgG1 Fc fusion rotein at the proteic C-terminal of PSCA, and has merged IgG κ signal sequence at N-terminal.Also used the PSCA fusion rotein that utilizes mouse IgG1 Fc zone.The reorganization PSCA albumen optimization that obtains being secreted into by the substratum of transfection mammalian cell, and being can be used as immunogen or part and is used to identify the protein with the PSCA protein-interacting, like part or acceptor.Protein expression drives through the CMV promotor, and the hygromycin gene on the carrier allows the mammalian cell of expressing recombinant protein is selected, and ampicillin resistance gene allows in intestinal bacteria, to select plasmid simultaneously.

Fig. 8 has shown proteic expression of PSCA.psecFc and purifying in the 293T cell.

PSR α construct:In order to produce the mammal cell line of constitutive expression PSCA, PSCAORF or its part are cloned into pSR α construct.Through respectively the transfection of pSR α construct being packed to 293T-10A1 be, or, produce two preferendums and ecotropic retrovirus pSR α and helper plasmid (packaging sequence that comprises disappearance) cotransfection to 293 cell.Use the multiple mammal cell line of said retroviral infection, cause clone gene PSCA to be integrated into host cell system.Protein expression drives through LTR (LTR); The neomycin resistance gene that exists on the carrier allows to select expressing proteinic mammalian cell, and ampicillin resistance gene and ColE1 starting point allow in intestinal bacteria, to select and keep plasmid simultaneously.Therefore retroviral vector can be used for infecting and producing multiple use such as PC3, NIH 3T3, TsuPr1,293 or the clone of rat-1 cell.

Fig. 6 has shown that use PSCA.pSR α construct expresses PSCA in reorganization among mouse, rat and the human cell line.With carrying people PSCA cDNA and neomycin resistance gene or only carrying mouse, rat and the human cell line shown in the retroviral infection of neomycin resistance gene.Produce stable recombinant cell lines through the G418 medicament selection.Through with 1G8 anti--PSCA MAb (5ug/ml) carries out FACS and dyes and measure psca expression.Shown in the figure is the FACS distribution plan of each clone, only infects demonstrating the fluorescence transformation in the system at PSCA, indicates the psca expression of cell surface.These clones can be used as immunogen and the MAb screening reagent is used for the MAb exploitation, and can be used for function test.

Having prepared the carboxyl terminal that makes the PSCA sequence in addition merges like FLAG ^TMThe pSR α construct of the epi-position mark of mark, thus allow to use anti-Flag antibody to detect.For example, with FLAG ^TMSequence 5 ' gattac aag gat gac gac gat aag 3 ' (SEQ ID NO:19) adds the terminal clone's primer of ORF 3 '.Prepare pSR α construct in addition, come proteic aminoterminal of production total length PSCA and carboxyl terminal GFP and myc/6 * His fusion rotein.

Other virus vector:Prepared in addition and be used for the construct that virus-mediated PSCA transmits and expresses.In viral system such as adenovirus carrier and simplexvirus amplicon vector, obtained to cause the high virus titer of PSCA high level expression.Through pcr amplification PSCA encoding sequence or its fragment, and subclone is to AdEasy shuttle vectors (Stratagene).Recombinate and the virus packing is produced adenovirus carrier according to producer explanation.Alternatively, PSCA encoding sequence or its fragment cloning to HSV-1 carrier (Imgenex) are produced herpesvirus vector.Then these virus vector are used to infect various kinds of cell system like PC3, NIH 3T3,293 or the rat-1 cell.

The expression system of conditioned:In order to regulate and control the expression of PSCA in the mammalian cell, mammalian expression system that encoding sequence or its part of PSCA is cloned into conditioned such as T-Rex system (Invitrogen), GeneSwitch system (Invitrogen) and the Ecdysone system (Sratagene) that closely regulates.The effect that these systems allow reorganization PSCA to be depended on time and concentration is studied.Can these carriers be used to control various kinds of cell system then, like PC3, NIH 3T3,293 or the rat-1 cell in psca expression.

B. baculovirus expression system

In order in baculovirus expression system, to produce reorganization PSCA albumen, PSCA ORF or its part are cloned among the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), the N-terminal of said carrier provides His-mark.Specifically, pBlueBac-PSCA and helper plasmid pBac-N-Blue (Invitrogen) cotransfection are gone into SF9 (fall army worm) insect cell and produce recombinant baculovirus (seeing the Invitrogen operational manual for details).From cell conditioned medium liquid, collect baculovirus then and carry out purifying with the plaque test.

Then through produce reorganization PSCA albumen with the baculovirus infection HighFive insect cell (Invitrogen) of purifying.Can use anti-PSCA or anti--His traget antibody to detect reorganization PSCA albumen.Can purifying PSCA albumen, use it for and multiplely produce specific polyclone of PSCA and monoclonal antibody based on the analysis of cell or as immunogen.

C.PSCA is directly to the homologue expression vector

With the mouse of PSCA and monkey directly to homologue be cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA).Protein expression is by cytomegalovirus (CMV) promoters driven.Recombinant protein has myc epi-position and the 6 * His epi-position that merges with C-terminal.These carriers can be expressed PSCA directly to the cross reactivity of homologue with detection mono-clonal Anti-Human psca antibody.

In addition, mouse and the monkey with PSCA directly is cloned into pSR α construct to homologue.PSR α construct can produce can constitutive expression PSCA directly to the mammal cell line of homologue.Protein expression is by cytomegalovirus (CMV) promoters driven.Recombinant protein has myc epi-position and the 6 * His epi-position that merges with C-terminal.These carriers can express PSCA directly to homologue detecting the cross reactivity of mono-clonal Anti-Human pSCA antibody, and research PSCA is directly to the functionally active of homologue.Through respectively with the transfection of pSR α construct to 293T-10A1 packing system, or with pSR α and helper plasmid (packaging sequence that contains disappearance) cotransfection to 293 cell to produce pair preferendums and ecotropic retrovirus.Use the multiple mammal cell line of retroviral infection, cause cloned genes PSCA directly to be integrated into host cell system to homologue.

Fig. 7 has shown in transfection to the 293T cell, the expression of mouse and monkey PSCA.pcDNA3.1/MycHis.With mouse PSCA.pcDNA3.1/MycHis or monkey PSCA.pcDNA3.1/MycHis or pcDNA3.1/MycHis vehicle Control transfection 293T cell.After 40 hours, collecting cell also uses anti--PSCA monoclonal antibody to analyze through flow cytometry.

Embodiment 6: antigenicity distribution plan and secondary structure

The amino acid distribution plan of

PSCA variant

1,2,3,4 can be positioned at the ExPasy molecular biology server acquisition of ProtScale website, World Wide Web (.expasy.ch/cgi-bin/protscale.pl) through visit.

These distribution plans: wetting ability (Hopp T.P., Woods K.R., 1981.Proc.Natl.Acad.Sci.U.S.A.78:3824-3828); Parent/hydrophobicity (Kyte J., Doolittle R.F., 1982.J.Mol.Biol.157:105-132); Can reach residue per-cent (Janin J., 1979 Nature 277:491-492); Average flexible (Bhaskaran R., and Ponnuswamy P.K., 1988.Int.J.Pept.Protein Res.32:242-255); β-corner (Deleage, G., Roux be Protein Engineering 1:289-294 B.1987) and any other can be used to identify the antigenicity zone of every kind of PSCA variant proteins in this area like the distribution plan that obtains at the ProtScale website.Use above-mentioned each amino acid distribution plan that following ProtScale parameter generates the PSCA variant for analysis: 1) window size is 9; 2) compare window edge with window center and account for 100% weight; And 3) numerical standardization of amino acid distribution plan is between 0 and 1.

Use wetting ability, parent/hydrophobicity with can reach residue percent profile figure and confirm wetting ability (be wetting ability and can reach the residue percent value, the value on parent/hydrophobicity profile is less than 0.5) amino acid fragment greater than 0.5.Therefore these zones are exposed to water surrounding probably, appear at proteinic surface, can be used for immunity identification, as by antibody recognition.

Average flexibility and βZhuan Jiao distribution plan can confirm not receive the amino acid fragment (that is, the numerical value of β-corner distribution plan and average flexible distribution plan is greater than 0.5) of secondary structure such as βZhe Die and the folding constraint of α.Therefore these zones also are exposed to proteinic surface probably, can be used for immunity identification, as by antibody recognition.

The antigenicity sequence of the PSCA variant proteins that shows through for example above-mentioned distribution plan can be used for preparing or the peptide or the immunogen of the nucleic acid of encoded peptide, in order to produce the anti-psca antibody of treatment and diagnosis usefulness.This immunogen can be any 5,6,7,8,9,10,11,12 of a listed PSCA protein variant among Fig. 1; 13,14,15,16,17,18,19; 20,21,22,23,24,25,30; The amino acid of adjacency more than 35,40,45,50 or 50, or their nucleic acid of respective coding, deducibility goes out the amino acid distribution plan of said variant because this variant contain with shown in the identical sequence of variant.Especially, peptide based immunogens of the present invention can contain increasing with any integer but be at least 5 amino acid whose peptides zones among Fig. 1, comprising numerical value in hydropathy profile greater than 0.5 amino acid position; Increasing with any integer but be at least 5 amino acid whose peptides zones among Fig. 1, comprising numerical value in parent/hydrophobicity profile less than 0.5 amino acid position; Increasing with any integer but be at least 5 amino acid whose peptides zones among Fig. 1, comprising numerical value in can reaching residue percent profile figure greater than 0.5 amino acid position; Increasing with any integer but be at least 5 amino acid whose peptides zones among Fig. 1, comprising numerical value in average flexible distribution plan greater than 0.5 amino acid position; And increasing with any integer but be at least 5 amino acid whose peptides zones among Fig. 1, comprising numerical value in β-corner distribution plan greater than 0.5 amino acid position.Peptide based immunogens of the present invention also can comprise aforementioned any the nucleic acid of coding.

All immunogen, peptide or the nucleic acid of the present invention all can be included in people's the unit dosage form, or is included in the compsn that comprises the drug excipient compatible with Human physiology.

Can use HNN-grade neural network method (NPS:Network Protein Sequence Analysis TIBS 2000March Vol.25 through the visit of World Wide Web (.expasy.ch/tools/) ExPasy molecular biology server; No 3 [291]: 147-150 Combet C.; Blanchet C.; Geourjon C.andDel é age G.http: //pbil.ibcp.fr/cgi-bin/npsa_automat.pl? Page=npsa_nn.html), predict

PSCA protein variant

1,3 by the one-level aminoacid sequence; 4 and 6 secondary structure, the i.e. appearance of α spiral, extended chain and random coil and position.Analyze to show PSCA variant 1 by 30.89% α spiral, 21.95% extended chain and 47.15% random coil are formed.PSCA protein variant 3 is by 14.89% α spiral, and 8.51% extended chain and 76.60% random coil are formed.PSCA protein variant 4 is by 9.52% α spiral, and 8.99% extended chain and 81.48% random coil are formed.PSCA protein variant 6 is by 24.56% α spiral, and 21.93% extended chain and 53.51% random coil are formed.

Possibly exist the analysis of membrane spaning domain to adopt the multiple film prediction algorithm of striding to carry out to the PSCA misfolded proteins through the visit of World Wide Web (.expasy.ch/tools/) ExPasy molecular biology server.

Embodiment 7: produce the PSCA polyclonal antibody

Polyclonal antibody can generate in Mammals, for example through the agent of one or many injecting immune, and injection adjuvant when needing.Typically, immunizing agent and/or adjuvant are injected to Mammals through multiple subcutaneous or peritoneal injection.In addition; For immune with total length PSCA protein variant; Adopt computerized algorithm to design immunogen; According to amino acid sequence analysis, said immunogen comprises the antigenicity characteristic and by the characteristic of immune host's immune system recognition (being the embodiment of " antigenicity distribution plan and secondary structure " referring to title).Such zone has been predicted to be wetting ability, flexibility, β arranged-corner conformation and be exposed to protein surface.

For example; Comprise wetting ability, the flexibility of PSCA protein variant, the recombinant bacteria fusion rotein or the peptide of β-corner area is used as antigen, in NZw (New Zealand White rabbits), produce polyclonal antibody or as be entitled as the said generation monoclonal antibody of embodiment of " producing PSCA monoclonal antibody (MAb) ".For example, in PSCA variant 1, this zone includes, but are not limited to amino acid 28-56 and amino acid 66-94.For variant 3, this zone includes, but are not limited to amino acid 7-39 and amino acid 70-94.For variant 4, this zone includes, but are not limited to amino acid 6-18, amino acid 27-39, amino acid/11 03-133 and 177-189.For variant 6, this zone includes, but are not limited to amino acid/11 9-35 and amino acid 57-85.With immunizing agent and known immune Mammals is had immunogenic protein coupling is useful.The instance of this immunogenic protein includes, but are not limited to keyhole relative hemocyanin (KLH), serum albumin, bovine thyroglobulin and Trypsin inhibitor SBTI.In one embodiment, peptide and the KLH coupling of the amino acid/11 03-133 of coding PSCA variant 4, and be used for immunize rabbit.Selectively, immunizing agent can comprise all or part of PSCA variant proteins, its analogue or fusion rotein.For example, can make PSCA variant aminoacid sequence and any fusion rotein counterpart well-known in the art merge the fusion rotein of said counterpart such as glutathione s-transferase (GST) and HIS mark mutually with recombinant DNA technology.In one embodiment, the aminoacid sequence 18-98 of PSCA variant 1 and GST are merged in the pGEX expression vector with recombinant technology, expression, purifying also are used for immunize rabbit and mouse to produce polyclone and monoclonal antibody respectively.Adopt suitable affinity matrix this fusion rotein of purifying from the inductive bacterium.

Other utilizable recombinant bacteria fusion rotein comprises that the constant region of maltose binding protein, LacZ, Trx, NusA or Tegeline is (referring to the part that is entitled as " in prokaryotic system, producing PSCA "; And Current Protocols In Molecular Biology; Volume 2; Unit 16, Frederick M.Ausubul et al.eds., 1995; Linsley, P.S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J.Exp.Med.174,561-566).

Except the fusion rotein of bacterial origin, also adopted Mammals expressed protein antigen.These antigens are expressed (referring to the part that is entitled as " in the eukaryote system, producing reorganization PSCA ") by mammalian expression vector such as Tag5 and Fc-fusion vector, and keep posttranslational modification, like the glycosylation in natural protein, found etc.In one embodiment, the cDNA of PSCA variant 1 is deducted the terminal leading peptide of N-sequence, be cloned into Tag5 Mammals secretion vector then, and in the 293T cell, express with C-end GPI anchor.Through metal chelate chromatography by purification of recombinant proteins matter in the tissue culture supernatant of the 293T cell of stably express recombinant vectors.Tag5 PSCA albumen with purifying is used as immunogen then.

During immune operation, mixing or emulsification antigen are useful to the immunne response that improves host animal in adjuvant.The instance of immunological adjuvant includes, but are not limited to complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (single phosphoryl liposome A, synthetic trehalose two rod bacillus acid esters (trehalosedicorynomycolate).

In typical operation, at first use 200 μ g at the most, typically be 100-200 μ g and complete Freund's adjuvant (CFA) blended and KLH link coupled fusion rotein or peptide rabbit is carried out subcutaneous immunity.Then with 200 μ g at the most, typically be the immunogen that 100-200 μ g is in the incomplete Freund's adjuvant (IFA) and give rabbit per 2 all subcutaneous injections once.Each immunity back was approximately got blood examination in 7-10 days and is tested, with ELISA monitoring antiserum(antisera) titre.

In order to check the reactivity and the specificity of immune serum (like the rabbit anteserum that obtains with

PSCA variant

3 or 4 proteic GST-fusion protein immunizations); Each total length PSCA variant cDNA is cloned into pCDNA3.1 myc-his expression vector (Invitrogen is referring to the embodiment that is entitled as " in eukaryotic system, producing reorganization PSCA ").To the 293T cell, (SantaCruz Biotechnologies, Santa Cruz CA) detect cell lysate, confirm the atopy to the sex change misfolded proteins with the Western engram technology with anti--His antibody with anti--variant serum with the construct transfection.In addition, detect immune serum, confirm specific recognition natural protein through fluorescent microscopy, flow cytometry and immunoprecipitation to the cell of 293T and other recombinant expressed PSCA variant.Western trace, immunoprecipitation, fluorescent microscopy and the low cytometric analysis of cell that also can use endogenous expression PSCA is with detection reaction property and specificity.

Comprise affinity column independent or that be in the fusion counterpart in other uncorrelated fusion rotein consumes and the antibody that merges the counterpart serial response through flowing through; Take this purifying with PSCA variant fusion rotein, like the antiserum(antisera) of GST and the acquisition of MBP fusion protein immunization rabbit.For example, at first through flowing through and the covalently bound AffiGel matrix of GST albumen (BioRad, Hercules, Calif.) antiserum(antisera) that obtains by GST-PSCA variant 1 fusion rotein of column purification.Then through flow through by with the substrate composed pillar affinity purification of the covalently bound AffiGel of MBP-PSCA fusion rotein antiserum(antisera).Then, further come purified blood serum to separate the IgG component through G albumen affinity chromatography.The base for post matter of forming by urporotein immunogen or free peptide through flowing through come affinity purification derive from other by the serum of the rabbit of the antigen of His mark and peptide immunity with exhaust the serum that merges counterpart.

Embodiment 8: produce PSCA monoclonal antibody (MAb)

In one embodiment; The therapeutic monoclonal antibodies of PSCA and PSCA variant (" MAb ") comprise and specificity to each protein or specificity MAb to the epi-position reaction of common sequences between variant; Said MAb can combination, the biological function of internalization, destruction or modulation PSCA or PSCA variant; For example, those can destruction and part and the interactional MAb that combines counterpart.The immunogen that is used to produce this MAb comprises that those are through the design coding or comprise ectodomain or whole zones of PSCA protein sequences; Warp is inferred the zone of containing functional primitive, and infers the zone with antigenic PSCA protein variant by the Computer Analysis of aminoacid sequence.Immunogen comprises peptide and recombinant bacteria protein, like GST-PSCA fusion rotein (Fig. 8), and through the PSCApET of His mark carrier protein (Fig. 6), the protein purification through the His mark (Fig. 7) and people and mouse IgG FC fusion rotein that Mammals is expressed.In addition, but the cell of the high level expression PSCA variant 1 that use is transformed through the retrovirus transduction, and for example RAT1-PSCA, 293T-PSCA, 3T3-PSCA or 300.19-PSCA come immune mouse (Fig. 5).

In order to produce the monoclonal antibody of PSCA, at first use and proper adjuvant blended 5-50 μ g protein immunogen or 10 ⁶-10 ⁷(FP) immune mouse in the cell foot pad of individual expression PSCA.The example that is used for the proper adjuvant of FP immunity is used Titermax (Sigma) and the immune subsequently used alum gel Immuneasy (Qiagen) of initial FP injection.After the initial injection, twice immune mouse is condemned to death until them weekly.Obtain the B-cell to be used for fusion by lymphoglandula.

In the process of immune operation, get blood examination and test to monitor immunoreactive titre and specificity.In most of the cases; In case confirm to have obtained the reactivity and the specificity that suit through ELISA, Western trace, immunoprecipitation, fluorescent microscopy or flow cytometry; Just (BTX ECM2000) merges generation with hybridoma to adopt electric cytogamy.

In one embodiment, the invention provides the monoclonal antibody that name is called H1-1.10.Through identifying and confirming, this antibody capable reacts with cell surface or immobilized PSCA and combines.

Use XenoMouse technology

to produce MAb to PSCA; Mouse heavy chain and κ light chain gene seat inactivation wherein, most people's heavy chain and κ light chain immunoglobulin loci are inserted into.Through producing the XenoMice of people γ 1 based on cellular immunization, produced H1-1.10 with the B300.19/PSCA cell.Anti--PSCA MAb H1-1.10 combines the expressed endogenous PSCA of cell surface of prostate cancer heterograft cell.

The antibody that is called as H1-1.10 is sent to American type culture collection (ATCC) on May 4th, 2005 (via Federal Express), and P.O.Box 1549, Manassas, and VA 20108, and specified preserving number is respectively PTA-6697.

(Life Technologies, Gibco BRL) isolates from each hybridoma after the mRNA with Trizol reagent, measures the dna sequence dna of the anti--PSCA MAb H1-1.10 that encodes.Purifying and quantitatively total RNA.Use Gibco BRL Superscript Preamplification system, cause, produce the first chain cDNA by total RNA with few (dT) 12-18.The variable light chain primer of end user's immunoglobulin variable heavy chain primer and the human normal immunoglobulin first chain cDNA that increases.With PCR product cloning to pCRScript carrier (Stratagene, La Jolla).Measure several clones' sequence, confirm variable heavy chain and light chain district.Fig. 2 A and 2B and Fig. 3 A and 3B have listed the nucleic acid and the aminoacid sequence in variable heavy chain and light chain district.It is the sequence alignment of V-D-J sequence with planting that Fig. 4 A and 4B have listed PSCA H1-1.10 antibody.

Embodiment 9: screening and evaluation psca antibody

At first use F-MAT screening to use to be entitled as the antibody that method produces described in the embodiment of " producing PSCA monoclonal antibody (MAb) ", use then to comprise that ELISA, FACS, epi-position are organized surely and with the test combinations screening of the affinity of the PSCA of cell surface expression with identify said antibody.

A. through FACS screening PSCA people MAb

Carry out the screening of PSCA MAb hybridoma of former generation through FACS.Method is following: 50 μ l/ hole hybridoma supernatants (purified) or antibody purified (through serial dilution) are added 96-hole FACS culture plate and mix with the cell of expressing PSCA (endogenic or reorganization, 50,000 cells/well).Mixture is placed 4 ℃ of insulations 2 hours.Be incubated when finishing, cell, detects antibody with 100 μ l and (resists-hIgG-PE) be incubated 45 minutes in 4 ℃ with the washing of FACS damping fluid.When insulation finished, cell was used the formaldehyde fixed cell with the washing of FACS damping fluid, and used FACScan to analyze.Use CellQuest Pro software analysis data.Solid histogram is represented the data of negative control IgG1, and hollow histogram is represented the data (Fig. 9) of PSCA-positive cell.

To be transferred to the 24-well culture plate by the positive hybridoma that identifies in the former generation screening thing, collect supernatant to carry out the authenticity screening.The authenticity screening comprises to B300.19-PSCA/300.19-neo Rat1-PSCA/Rat1-neo, PC3-PSCA/neo; SW780 (bladder cancer cell lines); LAPC9AI (prostate cancer cell line), the facs analysis that HPAC (pancreatic cancer cell system) carries out and use Tag5-PSCA, GST-PSCA; GST-PSCA N-term, the ELISA that Med.C-Term and pET-PSCA carry out.

The relative affinity analysis of B.PSCAH1-1.10 people MAb

Detect the hybridoma supernatant to confirm the relative binding affinity of itself and cell surface PSCA.With FACS damping fluid (FB) serial dilution hybridoma supernatant, from μ g/ml to sub-ng/ml; Combine to use in the test LAPC9AI cell to estimate at FACS.High-affinity antibody provides high MFI value.Use CellQuest Pro software to obtain the MFI value of each point, be used to use Graphpad Prism software (Table VII and Table VIII): Sigmoidal Dose-Response (variable slope) equation calculates affinity.Table I X has listed the result that relative affinity is analyzed.

C. epi-position is divided into groups

Through estimating its binding pattern, psca antibody is divided into groups according to epi-position to the LAPC9AI cell.Briefly, make a spot of various antibody biotinylation; Then in 4 ℃, excessive (100 *) non--biotinylated antibody in the presence of, make every kind of biotinylated antibody be incubated 1 hour with LAPC9AI.Usually, if with identical epi-position bonded words, excessive antibodies can with biotinylated antibody competition.When insulation finished, washed cell was incubated 45 minutes in 4 ℃ with Streptavidin-PE.Wash after unconjugated Streptavidin-PE, cell is analyzed with FACS.MFI mensuration is used for data analysis (Table VII).Shown in Table X I, represented self competition (100% competition) by the high bright xanchromatic cell that is shown as, the MFI value in these cells is the background contrast of every kind of biotinylated antibody.There is not the cell of color to represent that two kinds of antibody compete (low MFI) mutually, the epi-positions combinations that high MFI (the high bright blueness that is shown as) two kinds of antibody of expression are different with two kinds.In the antibody, the antibody with identical combination pattern can combine identical epi-position, and 6 epi-position groups are arranged in the antibody to be detected.Table X I demonstrates PSCA H1-1.10 and combines with its unique epi-position.

Embodiment 10: identify and express psca antibody

A. with the cross reactivity of monkey PSCA and mouse PSCA

The ability of the PSCA reaction in screening and identification of M Ab and mouse and monkey source.When using mouse and monkey animal model, this characteristic can be used for understanding MAb and cell and structural PSCA results of interaction.Clone cynomolgus monkey (cynomolgous mokey) and mouse psca gene are expressed in retrovirus, then with its transient transfection to the 293T cell.Be incubated with the 293-T cell of expressing monkey or mouse PSCA detecting antibody.293T-neo is used as negative control.Use anti--hIgG-PE to detect antibody and detect institute's bonded antibody.Result shown in the Table X show PSCA MAb H1-1.10 not with monkey-PSCA or mouse-PSCA cross reaction.

Embodiment 11:PSCA antibody internalization

Use the internalization of PC3-PSCA cell research H1-1.10.Briefly, in 4 ℃ H1-1.10 and cell are incubated 90 minutes, so that antibody combines with cell surface.Then cell is divided into two groups, one group places 37 ℃ to continue insulation so that the antibody internalization, and one group places 4 ℃ with comparing (not internalization).After 37 ℃/4 ℃ insulations, use pickling to remove and cell surface bonded PSCA 4.121.The PSCA bonded antibody that can detect with internalization is changed in passing through subsequently.After the second detection antibody insulation, cell is analyzed with FACS, or under fluorescent microscope, observes.37 ℃ the insulation 2 hours after, about 30%H1-1.10 is by internalization (Figure 10).

Embodiment 12: antibody-mediated kills and wounds

When being incubated jointly with the link coupled SA, PSCA H1-1.10 MAb can mediate in the PC-3/PSCA cell and kill and wound.The PC-3 cell that to after transforming, express neomycin resistance gene (neo) or PSCA is laid on 96 hole tissue culture wares in triplicate.Cell attachment is spent the night; Remove substratum then, replace with fresh culture, anti--PSCA MAb H1-1.10 of concentration shown in wherein containing or H1-1.10 and 3 times excessive with (the Advanced Targeting Systems of SA shown in the sapotoxin albumen coupling; SanDiego, CA).With cell insulation 4 days, use MTT test (Promega Corp) to measure survival per-cent.Psca antibody H1-1.10 is specific because it does not mediate the cell of not expressing PSCA kill and wound that (Figure 11 one The A group).When being incubated with the SA in identification people Fc zone rather than with the regional SA of identification goat antibody Fc, the H1-1.10 mediated cell kills and wounds (Figure 11 one B group).These results show: use suitable anti--PSCA MAb can medicine or cytotoxic protein matter optionally be passed to the cell of expressing PSCA.

In addition, when being incubated jointly with the link coupled SA, PSCA H1-1.10 MAb can mediate in the LNCaP/PSCA cell and kill and wound.The LNCap cell that to after transforming, express neomycin resistance gene (neo) or PSCA is laid on 96 hole tissue culture wares in triplicate.Cell attachment is spent the night; Remove substratum then; Replacement is with fresh culture; Anti--PSCA MAbH1-1.10 of concentration shown in wherein containing or H1-1.10 and 3 times of excessive anti-people or with anti-people's SA of sapotoxin albumen coupling (Advanced Targeting Systems, San Diego, CA).With cell insulation 4 days, use MTT test (Promega Corp) to measure survival per-cent.Psca antibody H1-1.10 is specific, because it does not mediate kill and wound (Figure 12) of the LNCap cell of not expressing PSCA.

Embodiment 13: antibody-mediated IC

Estimate psca antibody to measure the Cytotoxic ability of its mediation immune dependent type.With RHB damping fluid (RPMI 1640, Gibco Life Technologies, 20mM HEPES) dilution H1-1.10 (0-50 μ g/ml).With the cell of RHB damping fluid washing expression B300.19-PSCA, with 10 ⁶The density resuspension of individual cell/ml.In typical test, (Cedarlane, Ontario Can) add flat tissue culture 96 orifice plates with 50 μ l cell suspending liquids together with 50 μ l psca antibodies, the diluted rabbit complement of 50 μ l serum.In 37 ℃, at 5%CO ₂In the incubator mixture is incubated 2 hours so that the lysis of complement-mediation.(Biosource Intl.Camarillo CA), in 37 ℃, continues insulation 4-5 hour in each hole, to add 50 μ l Alamar Blue.Use 96 hole photofluorometers, read the fluorescence in each hole with the exciting light of 530nm and the emission light of 590nm.The result shows: people Ig1 H1-1.10 can mediate the target cell cracking (Figure 13) that depends on complement.

ADCC (depending on the cytotoxicity of the cell of antibody) is combined with on the cell of antibody by immune-mediated molten born of the same parents' attack, the specific cell-surface antigens of said antibody target.Immunocyte through with Fc γ receptors bind with identification antibody the Fc part, said acceptor is positioned at the surface of white corpuscle, monocyte and the NK cell that can cause the molten born of the same parents' attack that causes necrocytosis.Usually, external make the Panc0203 cell with ⁵¹ Cr insulation 1 hour.With after the fresh culture washing, with different effector cells to the ratio of target cell (E: the T ratio), will be incubated through the cell of mark PMBC with 2.5 μ g/ml people PSCA MAb and fresh separated.After 37 ℃ of insulations 4 hours, slow eccentric visual cell, counting contains and is discharged by cell in the β telltale ⁵¹The supernatant of Cr.The result shows: when effector cell and the increase of target cell ratio, the cell killing that mediation depends on antibody increases to some extent.Through confirm IgG1 contrast MAb and under the situation that lacks antibody insulation target cell and effector cell can not cause cell killing, specificity that can confirmed test.

Embodiment 14: and generation F (Ab ') 2 fragments

F (Ab ') 2 fragments that produce MAb are used in the effect of research MAb molecule in external and the interior therapeutic property model, and said MAb molecule has kept its divalence antigen binding site, but lacks immunoeffectors Fc structural domain.Method is following: the MAb H1-1.10 that 20mg is dissolved in 20mM sodium-acetate buffer pH4.5 with the for some time shown in immobilized stomach en-(Pierce.Rockford IL) insulation.Remove complete MAb and Fc fragment through the albumin A chromatography through digesting.Observe the SDS-PAGE gel of Coomassie blue stain, wherein have, undigested complete, non-reduced MAb, through the material of digestion shown in the time non-reduced aliquots containig of taking out, and the F of final digestion (ab ') 2 products go back raw sample.Said reagent can be used for treating the animal that carries the tumour of expressing PSCA.Can intrinsic biological activity and the activity that is mediated by the mechanism that depends on immunity be made a distinction with the observed Anti-tumor activity of said antibody fragment.

Embodiment 15: use recombinant DNA method expressing human antibody

For in cells transfected recombinant expressed anti--PSCAMAb, will resist respectively-PSCA variable heavy chain and sequence of light chain be cloned into the upper reaches of people's heavy chain IgG1 and light chain Ig κ constant region.With complete anti--PSCA people's heavy chain and light chain box be cloned into the downstream of CMV promotor/enhanser in the cloning vector.The polyadenylation site is positioned at the downstream of MAb encoding sequence.With express recombinant anti--the construct transfection of PSCA MAb is to 293T, Cos and Chinese hamster ovary celI.Evaluation is by the H1-1.10 antibody of reorganization 293-T emiocytosis and combining of cell surface PSCA, and the same antibody with original hybridoma generation compares then.

Embodiment 16:HLAI class and II class binding analysis

According to disclosed method, use the HLA molecule of purifying to carry out HLAI class and II class combination test (for example, PCT publication number WO94/20127 and WO94/03205; Sidney et al., Current Protocols inImmunology 18.3.1 (1998); Sidney, et al, J.1mmunol.154:247 (1995); Sette, etal., Mol.Immunol.31:813 (1994)).In brief, of it, with MHC molecule (5-500nM) and the multiple unlabelled inhibitor peptides and the 1-10nM of purifying ¹²⁵The radiolabeled probe peptide of I is incubated jointly.After the insulation, MHC-peptide complex and free peptide are separated, confirm the binding peptide component through gel-filtration.Typically, in trial test, at each MHC goods of titration in the presence of radiolabeled peptide of fixed amount to confirm to combine the required HLA molecular conecentration of gross activity of 10-20%.The inhibition of all postorders all uses these HLA concentration to carry out with directly combining to test.

Because under these conditions, [mark]＜[HLA] and IC ₅₀>=[HLA] is so the IC that measures ₅₀Value is reasonably real K _DApproximation.Typically, inhibitor peptides can be detected when concentration is 120 μ g/ml-1.2ng/ml, and fully independently detects in the experiment at 2-4.In order to suppress the IC of contrast through using the positive according to the data that from the difference experiment, draw ₅₀Tried the IC of peptide (typically being unmarked version) divided by each through radiolabeled probe peptide ₅₀, calculate the relative combination numerical value of each peptide.For the purposes of DB and the comparison of inner experiment, edited relative associated value.Subsequently, these values are through the inhibiting IC with positive control ₅₀The nM value becomes again to IC divided by the relative combination of target peptide ₅₀The nM value.This data editing method is to being accurate and consistent at different fates or with the peptide of the purifying MHC mensuration of different shares relatively.

The binding analysis of general introduction can be used to analyze the peptide (seeing Table IV) that carries HLA hyper-base unit and/or HLA primitive above.

Embodiment 17: the structure of " minigene " Polyepitope DNA plasmid

Present embodiment is discussed the structure of minigene expression plasmid.The minigene plasmid certainly comprises the B cell of various configurations as herein described, CTL and/or HTL epi-position or epitope analogs.

The minigene expression plasmid generally includes a plurality of CTL and htl peptide epi-position.In this embodiment, use carry HLA-A2 ,-A3 ,-peptide epitopes of B7 hyper-base unit and carry HLA-A1 and-peptide epitopes and the epi-position and/or the DR3 epi-position that carry DR hyper-base unit of A24 primitive.Selection carries the peptide epitopes that derives from PSCA of HLAI class hyper-base unit or primitive, makes multiple hyper-base unit/primitive be provided to guarantee population coverage widely.Similarly, HLAII class epi-position is selected from PSCA so that population coverage widely to be provided, and the epi-position that promptly carries HLA DR-1-4-7 hyper-base unit all is selected with the epi-position that carries HLA DR-3 primitive and is included in this minigene construct.Then, selected CTL and HTL epi-position are mixed minigene and in expression vector, express.

This construct can also comprise the sequence that the HTL epi-position is guided to endoplasmic reticulum.For example; Ii albumen can with merge like described one or more HTL epi-positions in this area, wherein remove CLIP sequence and replacement in this Ii albumen with HLAII class epitope sequences, thereby HLAII class epi-position guided to endoplasmic reticulum; At this, epi-position combines with the HLAII quasi-molecule.

This embodiment illustrates the method that is used to make up the expression plasmid that carries minigene.Other expression vector that can be used for the minigene compsn by those skilled in the art knowledge and can getting.

The medium and small gene DNA plasmid of present embodiment comprises total Kozak sequence and total mouse κ-Ig light chain signal sequence, and what follow is CTL and/or the HTL epi-position of selecting according to the disclosed principle of this paper.This sequence encoding is by the opening code-reading frame that merges with Myc and His antibody epitope mark of pcDNA 3.1-Myc-His vector encoded.

Synthetic and through the overlapping oligonucleotide of HPLC purifying, it is long and have 15 Nucleotide overlapping that said overlapping oligonucleotide can for example on average be about 70 Nucleotide.Peptide epitopes that this oligonucleotide coding is selected and suitable joint Nucleotide, Kozak sequence and signal sequence.Final multi-epitope minigene warp three cover PCR reactions prolong the eclipsed oligonucleotide and assemble.Use Perkin/Elmer 9600 PCR appearance, and use following condition to carry out 30 altogether and take turns circulation: 95 ℃ 15 seconds, annealing temperature (being lower than each primer) 30 seconds to 5 ℃ of minimum calculating Tm, and 72 ℃ 1 minute.

For example, prepare minigene as follows: for PCR reaction at first, two kinds of oligonucleotide are respectively got 5 μ g through annealing and extending: use 8 oligonucleotide in an example; That is, four pairs of primers, oligonucleotide 1+2; 3+4,5+6, and 7+8 is incorporated in the reaction solution of 100 μ l; Comprise in this reaction solution the Pfu polymerase buffer (1 *=10mM KCL, 10mM (NH ₄) ₂SO ₄, 20mM Tris-muriate, pH8.75,2mM MgSO ₄, 0.1%TritonX-100,100 μ g/ml BSA), the various dNTP of 0.25mM, and the Pfu polysaccharase of 2.5U.This total length dipolymer product is through gel-purified, and two reaction systems that comprise 1+2 and 3+4 product and 5+6 and 7+8 product are through mixing, anneal and extending 10 circulations.Mix the half the of two reactions then, and before adding the flank primer, carry out 5 round-robin annealing and extend with the amplification full length product.This full length product is through gel-purified and be cloned into pCR-blunt (Invitrogen), through each clone of order-checking screening.

Embodiment 18: the degree of plasmid construction body and induction of immunity originality thereof

Through transduceing or transfection APC with the nucleic acid construct of expressing epi-position, the epi-position that definite then APC is carried out is presented, and at external confirmation plasmid construction body, the plasmid that for example makes up according to front embodiment can the immunogenic degree of inductive.Like this determined " antigenicity " also allows to use human APC.The density of this test through quantitative cell surface epi-position-HLAI type mixture confirms that in by the environment of T cell recognition APC presents the ability of this epi-position.Carry out quantitatively (referring to for example Sijts et al, J.Immunol.156:683-692,1996 from the amount that APC goes up the peptide of wash-out through direct mensuration; Demotz etal, Nature 342:682-684,1989); Or through measuring ill or through the cracking of target cell inductive or the lymphokine burst size of transfection; Cracking or the lymphokine that confirm equates level then discharges required peptide concentration and estimates that the number of peptide-HLAI class mixture is (referring to for example; Kageyama et al; J.Immunol.154:567-576,1995).

Perhaps, also confirm immunogenicity in the activity of in-vitro evaluation CTL and HTL subsequently through injection in the mouse body, described activity uses cytotoxicity and proliferation test to analyze respectively; For example; Alexander et al at Immunity 1:751-761, describes in detail in 1994.

For example, for the ability that the DNA minigene construct that confirms to comprise at least one HLA-A2 hyper-base unit peptide is induced CTL in vivo, the exposed cDNA that uses 100 μ g for example is to HLA-A2.1/K ^bTransgenic mice carries out the intramuscular immunity.By the method for the CTL level of cDNA immune induction, control animals still uses the actual peptide combinations that comprises multi-epitope to carry out immunity as relatively, said a plurality of epi-position reason minigenes codings and be synthesized and be single polypeptide.

Splenocyte twice with each compsn (peptide epitopes of encoding in minigene or the multi-epitope peptide) immune stimulatory animal exists then ⁵¹Measure peptide-special cytotoxic activity in the Cr release test.The result has shown the magnitude that the CTL to the A2-restricted epitope replys, and shows the interior immunogenicity of body of this minigene vaccine and polyepitope vaccines thus.

Therefore, find that this minigene can cause the immunne response of the first peptide epitopes of anti-HLA-A2 hyper-base, this effect with the multi-epitope peptide vaccine is identical.Also use other HLA-A3 and HLA-B7 transgene mouse model similarly to analyze, find also that whereby this minigene can cause the suitable immunne response that is directed against the epi-position that is provided to estimate by HLA-A3 and HLA-B7 primitive or the epi-position inductive CTL of hyper-base unit.

For the ability that the minigene of the II class epi-position that confirms to encode is induced HTL in vivo, use the for example DNA intramuscular immunity DR transgenic mice of 100 μ g, or for the epi-position of those and suitable mouse MHC molecule cross reaction, use I-A ^b-restricted mouse.By the method for dna immunization inductive HTL level, the control animal group uses still that the actual peptide combinations of emulsive carries out immunity in complete Freund's adjuvant as relatively.Be purified into the CD4+T cell in the splenocyte by immune animal, i.e. HTL, and stimulate with each compsn (peptide of encoding in the minigene).Use ³The H-thymidine mixes proliferation test measurement HTL and replys (referring to for example Alexander et al Immunity 1:751-761,1994).The result shows the magnitude that HTL replys, and proves this minigene immunogenicity in vivo thus.

Use initiation booster immunization scheme to confirm to can be used as the vaccine of uniting use with promotor like the DNA minigene of the said structure of preceding text embodiment.This promotor can be by recombinant protein (Bamett etal for example; Aids Res.and Human Retroviruses 14 Supplement 3:S299-S309; 1998) or vaccinia virus recombinant, for example, the vaccinia virus of minigene or DNA that can express the complete desired protein of coding is (referring to 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; With Robinson et al, Nature Med.5:526-34,1999) form.

For example, originally in transgenic mice, estimate the usefulness of the DNA minigene be used to cause the booster immunization scheme.In these embodiment, use the DNA minigene intramuscular immunity A2.1/K of 100 μ g coding immunogenic peptide ^bTransgenic mice, this immunogenic peptide comprise that at least one carries the peptide of HLA-A2 hyper-base unit.Behind the soak (3-9 week), use 10 ⁷The vaccinia virus recombinant intraperitoneal booster immunization mouse of the sequence that the expression of pfu/ mouse is identical with this DNA minigene encoding sequence.Control mice uses the 100 μ gDNA or the vaccinia virus recombinant that do not contain this minigene sequence to carry out immunity, or uses the DNA of coding minigene to carry out immunity, but does not carry out the vaccinia virus booster immunization.After the extra insulation in two weeks by a definite date, use the peptide-specific activity of ELISPOT test determination mouse boosting cell immediately.In addition, the A2-restricted peptides epi-position at external use minigene and vaccinia virus recombinant coding stimulates splenocyte, test peptides-specific activity in α, β and/or γ IFN ELISA then.

It is found that, compare that the minigene that is used to cause the booster immunization scheme can cause the more strong immune response to HLA-A2 hyper-base unit peptide with independent use DNA.Also can use HLA-A11 or HLA-B7 transgene mouse model to carry out such analysis, thereby estimate by HLA-A3 or HLA-B7 primitive or the epi-position inductive CTL of hyper-base unit.Causing the use of booster immunization scheme in the mankind is described among the embodiment that hereinafter is entitled as " use cause the booster immunization scheme induce CTL to reply ".

Embodiment 19: derive from how antigenic polyepitope vaccines compsn

PSCA peptide epitopes of the present invention is used for uniting use with the epi-position of other target taa, can be used for preventing or treats the vaccine composition of expressing PSCA and this type of other antigenic cancer with generation.For example; Vaccine composition can be used as single polypeptide and provides; The a plurality of epi-positions and the antigen relevant of PSCA have been mixed in this single polypeptide with tumour; This antigen target cancer that quilt is relevant with psca expression is usually expressed, and vaccine composition can also be as the compsn administration, and said composition is the mixture formula compsn that comprises one or more discrete epi-positions.Perhaps, this vaccine can be used as the minigene construct or as in the dendritic cell administration of the external epi-position of loaded peptide.

Embodiment 20: use peptide to estimate immunne response

Peptide of the present invention can be used for analyzing immunne response, to confirm existing to antibodies specific, CTL or the HTL of PSCA.Such analysis can use Ogg etc. at Science 279:2103-2106, and the method described in 1998 is carried out.In this embodiment, peptide according to the present invention is used as the reagent of diagnosis or prediction purpose, but not as immunogen.

In this embodiment, highly sensitive human leucocyte antigen tetramer mixture (" tetramer ") is used to cross-section analysis, and for example, analysis is in the various disease stage or comprises A through comprising ^*HLA-A after the PSCA peptide immunity of 0201 primitive ^*The PSCA HLA-A of 0201-positive individuals ^*The frequency of 0201-specific CTL.Synthetic existing describe (Musey et al, N.Engl.J.Med.337:1267,1997) of tetramer mixture.In brief, the HLA heavy chain (A among these embodiment through the prokaryotic expression system synthesizing and purifying ^*0201) and B2M.Stride film-kytoplasm tail and modify this heavy chain through lacking this in the terminal sequence that comprises BirA enzymatic living beings elementization site that increases of COOH.Through dilution folding again this heavy chain, B2M and peptide.Separate the refolding product of 45-kD through fast protein liquid chromatography, subsequently vitamin H (Sigma, St.Louis, Missouri), adenosine 5 ' triphosphoric acid and magnesium exists down by the BirA biotinylation.Add Streptavidin-phycoerythrin conjugate with 1: 4 mol ratio, and concentrate this tetramer product to 1mg/ml.Products obtained therefrom is called the tetramer-phycoerythrin.

In order to analyze patient's blood sample, with about 1,000,000 PBMC with 300g centrifugal 5 minutes, and resuspending was in the cold PBS of 50 μ l.Use the tetramer-phycoerythrin, anti--CD8-three looks and anti-cd 38 to carry out three-chromatographic analysis.Be incubated the PBMC and the tetramer and antibody 30 to 60 minutes on ice, use formaldehyde fixed after washing twice.Use Gates to hold＞99.98% control sample.Tetrameric contrast comprises A ^*0201-negative individuals and A ^*The positive non-ill donor of 0201-.Through cells were tested by flow cytometry through the painted cell per-cent of the tetramer.The result has shown the cell number that comprises epi-position-Restricted CTL in the PBMC sample, easily indicates the degree for PSCA epi-position immunne response thus, and therefore indicates the state that is exposed to PSCA or is exposed to the vaccine that can cause that prevention or therapeutic are replied.

Embodiment 21: use and cause booster immunization scheme induce immune response

Cause booster immunization scheme potential principle and be used for confirming the principle of dna vaccination in the usefulness of transgenic mice; It is similar that for example preceding text are entitled as the principle described in the embodiment of " degree of plasmid construction body and induction of immunity originality thereof ", and this scheme can also be used for vaccine administration in the mankind.The dosage regimen of these vaccines can comprise initial administration, and for example exposed DNA then uses the recombinant virus of coding vaccine or recombinant protein/polypeptide or the peptide mixt that is present in the adjuvant to carry out booster immunization.

For example; Can use the expression vector of exposed nucleic acid form; For example be entitled as the expression vector that makes up among the embodiment of " structure of " minigene " Polyepitope DNA plasmid " and carry out first immunisation, said carrier is put muscle (IM) (or subcutaneous (SC) or or intracutaneous (ID)) administration with the amount of 0.5-5mg in multidigit.Can also use particle gun to use this nucleic acid (0.1 to 1000 μ g).3-4 is after week in insulation, gives booster immunization dosage.This stiffeners can be with 5-10 ⁷To 5 * 10 ⁹The recombinant fowlpox virus of the dosed administration of pfu.Another kind of recombinant virus, for example MVA, canary pox virus, adenovirus or adeno associated virus also can be used as stiffeners, maybe can use the mixture of multi-epitope protein or peptide.For vaccine evaluation usefulness, before the immunity and use and intermittently behind the vaccine of initial vaccine and booster dose obtain patient's blood sample.Isolate PMBC through Ficoll-Hypaque density gradient centrifugation from fresh heparinized blood, aliquots containig in refrigerant, and refrigerated storage.The CTL of specimen and HTL are active.

Analytical results shows: the magnitude of replying that has produced the treatment that is enough to realize anti-PSCA or protective immunity.

Embodiment 22: complementary polynucleotide

Can be used for detecting, reducing or suppress the expression of natural PS CA with coding PSCA sequence complementary sequence (Fig. 1 or Fig. 3) or its any part.Use the oligonucleotide that contains 15 to 30 base pairs of having an appointment though described, less in fact (9-aggressiveness or 10-aggressiveness) or the sequence fragment of big (40-90) also can use identical method.Use the for example suitable oligonucleotide of encoding sequence design of OLIGO 4.06 softwares (Naional Biosciences) and PSCA.In order to suppress to transcribe,, and use it for and prevent that promotor from combining with encoding sequence by unique 5 ' sequences Design complementary oligonucleotide.In order to suppress translation, design prevents the transcript bonded complementary oligonucleotide of rrna and coding PSCA.

Embodiment 23: use PSCA-specific antibody purifying natural or reorganization PSCA

Through the basic purifying natural of immunoaffinity chromatography of use PSCA specific antibody or the PSCA of reorganization.Resist-psca antibody and activatory chromatographic resin through covalent coupling, for example CNBr-activatory SEPHAROSE (Amersham Pharmacia Biotech) makes up immune affinity column.After the coupling, according to manufacturer's explanation sealing and flushing resin.

Make the medium that comprises PSCA through immune affinity column, wash this post down in the condition (the high ionic strength buffers liquid when for example, having stain remover to exist) that allows preferentially adsorbed PSCA.Destroy antibody/PSCA bonded condition (chaotropic agent of the for example damping fluid of pH2 to pH3, or high density, for example urea or thiocyanate ion) this post of wash-out down, and collecting GCR.P.

Embodiment 24: identify and the interactional molecule of PSCA

Use ¹²¹I Bolton-Hunter reagent mark PSCA or its biological activity segment (referring to (1973) Biochem.J.133:529 such as for example Bolton).Candidate molecules in the hole that is arranged in porous plate in advance is incubated with the PSCA through mark, and arbitrary hole that contains underlined PSCA mixture is analyzed in flushing then.The data that PSCA obtained of different concns are used to calculate various numerical value, comprise PSCA number, affinity and with the association of candidate molecules.

Embodiment 25: body inner analysis PSCA promotes the effect of tumor growth

Produce and growth through evaluation expression or the tumour that lacks the cell of PSCA, interior evaluating PSCA albumen is to the influence of growth of tumour cell.For example, respectively inject 1 * 10 in that the both sides of the chest of SCID mouse are subcutaneous ⁶3T3 or comprise the prostate cancer cell line (like the PC3 cell) of tkNeo empty carrier or PSCA.At least can use two kinds of strategies: (1) is constitutive expression PSCA under the adjusting of promotor; Said promotor is as deriving from the constitutive promoter of viral genome or allos mammalian promoter; Said virus such as polyomavirus, fowlpox virus (UK2,211,504; Be disclosed on July 5th, 1989), adenovirus (for example adenovirus 2), bovine papilloma virus, avian sarcomata virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40); Allos mammalian promoter such as actin promoter or immunoglobulin promoter, condition be said promotor and host cell systems is compatible and (2) at derivable carrier system; For example control such as ecdysone, tsiklomitsin adjustable ground expression down, condition is that said promotor is compatible with host cell systems.Measure the gross tumor volume that the palp tumour of monitoring shows through calipers then; Then measure the cell of whether expressing PSCA as time passes and shown the attack signature that changed (for example enhanced shifts, vascularization, and the reactivity of chemotherapeutics is reduced) with faster rate growth and the tumour that whether generates by the cell of expressing PSCA.

In addition, can be to mouse homotopic transplantation 1 * 10 ⁵Thereby same cell measures whether PSCA is influential to prostatic local growth, and whether PSCA influence the transfer ability of cell, particularly is transferred to ability (Miki T et al, the Oncol Res.2001 of lymphoglandula and bone; 12:209; Fu X et al, Int JCancer.1991,49:938).Estimate the influence that PSCA forms and grows bone tumor through injection prostate tumor cells in the shin bone.

This analysis also helps to measure the inhibition effect of candidate therapeutic property synthetics to PSCA, said synthetics such as PSCA endosome, PSCA antisense molecule and ribozyme.

The in-vivo tumour of embodiment 26:PSCA mediated monoclonal antibody suppresses

PSCA is in the remarkable expression on tumor tissue cell surface, with and restricted expression in healthy tissues make PSCA become the outstanding target spot of Antybody therapy.Similarly, PSCA is based on the target spot of T cellular immunization treatment.Therefore, through using recombinant cell lines, like PC3-PSCA and 3T3-PSCA (referring to for example Kaighn; M.E.; Et al., Invest Urol, 1979.17 (1): 16-23) and the human prostate heteroplastic transplantation model; (Saffran et al PNAS 1999 10:1073-1078) estimates the anti--curative effect of PSCAMAb in human prostata cancer xenotransplantation mouse model and human pancreas cancer xenotransplantation mouse model like LAPC 9AD.

Research antibody is to tumor growth and the usefulness that shifts formation in for example mouse coordination prostate gland or carcinoma of the pancreas heteroplastic transplantation model.Like what discussed in the present embodiment, antibody can be link coupled not, or can with other medicines coupling mutually, this point is that those skilled in the art are accessible.Anti--PSCA MAb suppresses the formation of pancreas, prostate gland and tumor of bladder heterograft.Anti--PSCA MAb has also hindered coordination growth of tumor of having set up and the survival time that has prolonged tumor-bearing mice.The validity of anti--PSCAMAb in treatment part and the listed cancer of advanced prostate cancer, carcinoma of the pancreas and Table I that the result shows is (referring to for example Saffran; D.et al, PNAS 10:1073-1078 or www.URL.pnas.org/cgi/doi/10.1073/pnas.051624698).

Resist-PSCA mAb can cause hindering the coordination growth of tumor of having set up and suppress the transfer to the site, distant place, thus the survival time of significant prolongation tumor-bearing mice.These are studied and show that PSCA is the attractive target spot of immunotherapy, and have shown the treatment potentiality of anti--PSCA MAb treatment part or transfer prostate gland and carcinoma of the pancreas.This embodiment shows the not effective growth that suppresses to grow in the intravital human prostate tumor xenogeneic graft of SCID mouse of link coupled PSCA monoclonal anti physical efficiency; Correspondingly, the associating of said effective monoclonal antibody also is effective.

Use a plurality of PSCAMAb to suppress tumour

Material and method

The PSCA monoclonal antibody:

As the description that is entitled as among the embodiment of " produce PSCA monoclonal antibody (MAb) " produces the monoclonal antibody to PSCA.Characterize the ability of this antibodies PSCA with ELISA, Western trace, FACS and immuno-precipitation.By the epi-position on the anti--PSCA MAb epitope mapping data identification PSCA albumen of ELISA and Western assay determination.Carry out the immunohistochemical analysis of prostate cancer tissue and cell with these antibody.

Through G albumen or A Protein S epharose chromatography monoclonal antibody purification from ascites or hybridoma tissue culture supernatant, dialyse facing to PBS, filter-sterilized, and in-20 ℃ of storages.(Bio-Rad, Hercules CA) measure protein through the Bradford test.Preparation treatment is with monoclonal antibody or comprise the mixture of the mixture of each monoclonal antibody, and is used to treat and accepts mouse subcutaneous or coordination injection LAPC-9 AD and HPAC tumour allogeneic.

Clone and heterograft

Prostate cancer cell line PC3 and LNCaP clone and fibroblast NIH 3T3 (American type culture collection) are maintained at respectively among the RPMI and DMEM that is supplemented with L-glutaminate and 10%FBS.

Like Hubert, R.S.et al.Proc Natl Acad Sci USA, 1999.96 (25): p.14523 said, shift generation PC3-PSCA and 3T3-PSCA cell colony through reverse transcription virus gene.

Through subcutaneous (s.c.) trocar implant (Craft; N.et al Nat Med.1999, the LAPC-9 heterograft that 5:280) will express the wild-type androgen receptor and generate PSA (PSA) is imported in 6 to 8 week male ICR-severe combined immunodeficiency in age (SCID) mouse (Taconic Farms) bodies.Press the single-cell suspension liquid of said preparation such as Craft LAPC-9 tumour cell.

The xenotransplantation mouse model

At right rib injection of male SCID mouse and the extent of dilution blended 1 * 10 of Matrigel (Collaborative Research) with 1: 1 ⁶Individual cancer cell, thus subcutaneous (s.c.) tumour generated.The usefulness that tumour is formed for test antibody begins peritoneal injection antibody on the same day at the injection tumour cell.As contrast, give the human IgG or the PBS of injected in mice purifying; Or the irrelevant antigenic sublimed monoclonal antibody do not expressed of identification people cell.The tumour size is measured through calipers, and gross tumor volume is by length x width ²/ 2 calculate.The Subcutaneous tumor diameter is condemned to death greater than the mouse of 1.5cm.

Carry out the coordination injection after using ketamine/xylazine anesthesia.In order to carry out the research of prostate gland coordination, the otch that belly is passed in incision one is to expose prostate gland.LAPC or PC3 tumour cell (2 * 10 ⁶) be injected into prostatic utriculus with the mixture of Matrigel with the volume of 10 μ l.In order to monitor tumor growth, mouse is carried out palpation, gather the mouse blood sample weekly to measure the PSA level.For suitable treatment with mice group, the anti-PSCA of peritoneal injection or the contrast MAb.

Anti--PSCA MAb suppresses the growth of the xenotransplantation tumor of expression PSCA

Use HPAC and the model measurement of LAPC9 coordination anti--PSCA MAb is to the influence of tumour formation.Compare with the s.c. tumor model, need respectively tumour cell is injected directly into mice pancreatic or prostaticly causes the local tumor growth with bit model, the development shifted of site at a distance; The deterioration that mouse is healthy, and death subsequently (Saffran, D.et al; PNAS, the same).These characteristics make more can be represented the process of human diseases and allow us to follow the tracks of the curative effect of mAb to the terminal point of being correlated with clinically with bit model.

Correspondingly, tumor cell injection is gone into the mouse prostate gland, after 2 days, mouse is divided into two groups, use a) anti--PSCA Ab or b of 250-1000 μ g) control antibodies, inferior on every Wendesdays, continued for two to five weeks.

The main advantage of coordination cancer model is to study the ability that shifts development.Analyze lung section research lotus and have the formation of the transfer in the mouse of the coordination tumour of having set up through IHC; The antibody of antineoplastic specificity cell surface protein is used in this research; Like anti--CK19 (Lin et al., Cancer Detect Prev. (2001) 25:202) as far as prostate cancer.

Another advantage of xenotransplantation cancer model is to study neovascularization and blood vessel generation.Tumor growth depends in part on the generation of neovascularity.Although capillary vessel system and the blood network source that generating are from the host, the initial sum of neovascularity structure structure is regulated (Davidoffet al., Clin Cancer Res. (2001) 7:2870 by the xenotransplantation tumour; Solesvik et al., Eur J Cancer Clin Oncol. (1984) 20:1295).According to methods known in the art, as study the influence of antibody and small molecules through IHC analysis tumor tissues and microenvironment on every side thereof to neovascularization.

In 4 time-of-weeks, injection PSCA MAb or control antibodies are carried out administration with the mouse that lotus is had the coordination tumour of having set up.Two groups of mouse all allow to set up high tumor load, thereby guarantee that mouse lung shifts the high frequency that forms.Put to death mouse then, through whether having tumour cell in IHC analysis their bladder, liver, bone and the lung.These researchs have proved that in the xenotransplantation mouse model anti--psca antibody has antitumor widely usefulness to the initial sum development of prostate cancer.Anti--psca antibody suppresses the formation of tumour, and hinders the growth of tumor of having set up, thereby prolongs the survival time of the mouse of treating.In addition, anti--PSCA MAb has shown the noticeable restraining effect that local tumor of prostate is diffused to the site, distant place, even same onset when big tumor load exists.Therefore, anti--PSCA MAb is effective to the prolongation and the health of the relevant clinically terminal point (tumor growth) of majority, survival time.

PSCA MAb is to the effect of human prostata cancer growth in the mouse body

Used aforesaid method, with people's male sex hormone-independent form prostate tumor cells UGB-1 (2.0 * 10 ⁶Individual cell/mouse) subcutaneous injection to male SCID mouse.With mouse random packet (every group of 10 mouse), as shown in the figure at the 0th day, intraperitoneal (i.p.) is used H1-1.10, PBS or contrast human IgG l MAb begin treatment.Give the animal two treatments weekly, till the 16th research day, 5 dosage of administration altogether.As shown in the figure, every at a distance from 3 to 4 days, use calipers to measure with the monitoring growth of tumor.Gross tumor volume is calculated as width ²* length/2, wherein width is minimum size, and length is maximum size.The result shows: and the people is anti--and growth (p＜0.01 is confirmed through the Kruskal-Wallis check) that PSCA monoclonal antibody H1-1.10 can significantly suppress to be transplanted to the subcutaneous human prostata cancer heterograft of SCID mouse is (Figure 14).

In another experiment, PSCA MAb H1-1.10 is suppressed at the subcutaneous growth of the people's male sex hormone-independent form prostate cancer heterograft of setting up in the SCID mouse.With people's male sex hormone-independent form human prostate tumour cell UGB-1 (1.5 * 10 ⁶Individual cell/mouse) subcutaneous injection to male SCID mouse.When tumour reaches about 70mm ³The time, with mouse random packet (every group of 10 mouse), as shown in the figure at the 0th day, intraperitoneal (i.p.) is with H1-1.10 or vehicle Control begin treatment.Give the animal two treatments weekly, till the 21st research day, 7 dosage of administration altogether.As shown in the figure, every at a distance from 3 to 4 days, use calipers to measure with the monitoring growth of tumor.Gross tumor volume is calculated as width ²* length/2, wherein width is minimum size, and length is maximum size.The result shows: and the people is anti--and growth (p＜0.05 is confirmed through Student ' s t-check) that PSCA monoclonal antibody H1-1.10 can significantly suppress to be transplanted to the subcutaneous human prostata cancer heterograft of having set up of SCID mouse is (Figure 15).

In another experiment, the subcutaneous growth of people's male sex hormone-dependent form prostate cancer heterograft of SCID mouse is transplanted in PSCAMAb H1-1.10 inhibition.With people's male sex hormone-dependent form tumour cell LAPC9-AD (2.0 * 10 ⁶Individual cell/mouse) subcutaneous injection to male SCID mouse.With mouse random packet (every group of 10 mouse), as shown in the figure at the 0th day, intraperitoneal (i.p.) is with H1-1.10 or contrast begin treatment.Give the animal two treatments weekly, till the 18th research day, 6 dosage of administration altogether.As shown in the figure, every at a distance from 3 to 4 days, use calipers to measure with the monitoring growth of tumor.Gross tumor volume is calculated as width ²* length/2, wherein width is minimum size, and length is maximum size.The result shows: and the people is anti--and growth (p＜0.01 is confirmed through the Kruskal-Wallis check) that PSCA monoclonal antibody H1-1.10 can significantly suppress to be transplanted to the subcutaneous human prostata cancer heterograft of SCID mouse is (Figure 16).

PSCA MAb is to the effect of human pancreas cancer growth in the mouse body

In another experiment, with human pancreas's tumour cell HPAC (3.0 * 10 ⁶Individual cell/mouse) subcutaneous injection to male SCID mouse.With mouse random packet (every group of 10 mouse), as shown in the figure at the 0th day, intraperitoneal (i.p.) is with H1-1.10 or contrast human IgG1 begin treatment.Give the animal two treatments weekly, till the 18th research day, 5 dosage of administration altogether.As shown in the figure, every at a distance from 3 to 4 days, use calipers to measure with the monitoring growth of tumor.Gross tumor volume is calculated as width ²* length/2, wherein width is minimum size, and length is maximum size.The result shows: and the people is anti--and growth (p＜0.05 is confirmed through Student ' s t-check) that PSCA monoclonal antibody H1-1.10 can significantly suppress to be transplanted to the subcutaneous human pancreas cancer heterograft of SCID mouse is (Figure 17).

PSCA MAb is to the effect of human bladder cancer's growth in the mouse body

In another experiment, with human bladder cancer cell SW780 (2.0 * 10 ⁶Individual cell/mouse) subcutaneous injection to male SCID mouse.With mouse random packet (every group of 10 mouse), as shown in the figure at the 0th day, intraperitoneal (i.p.) is with H1-1.10, PBS or contrast human IgG1 MAb begin treatment.Give the animal two treatments weekly, till the 18th research day, 6 dosage of administration altogether.As shown in the figure, every at a distance from 3 to 4 days, use calipers to measure with the monitoring growth of tumor.Gross tumor volume is calculated as width ²* length/2, wherein width is minimum size, and length is maximum size.The result shows: and the people is anti--and growth (p＜0.05 is confirmed through the Dunnett check) that PSCA monoclonal antibody H1-1.10 can significantly suppress to be transplanted to the subcutaneous SW780 human bladder cancer heterograft of SCID mouse is (Figure 18).

These result of experiment show: PSCA MAb can be used for treatment and diagnostic purpose, is used to treat the cancer listed with processing list I.

Embodiment 27: anti--psca antibody is to the mankind's treatment and diagnostic use

Anti--PSCA monoclonal antibody can be used for human diagnosis, prevention, prediction and/or therapeutic purpose safely and effectively.Use anti--PSCA MAb that the Western trace that cancerous tissue and cancer heterograft carry out is shown in the tumour have big area to dye by force with immunohistochemical analysis, and in the healthy tissues significantly attenuating or detection less than.In cancer and in the metastatic disease, detect PSCA proof MAb and can be used as diagnosis and/or prediction indicator.Therefore anti--psca antibody can be used for diagnostic use, and for example whether the immunohistochemical analysis of kidney biopsy samples suffers from cancer to detect suspicious patient.

Measure like flow cytometry, anti--PSCA MAb combines cancer cells specifically.Therefore; Anti--psca antibody is used to diagnostic total body opacification; For example radioimmunoscintigraphy and radioimmunotherapy (referring to for example Potamianos S.et al., Anticancer Res 20 (2A): 925-948 (2000)) show the part of psca expression and the cancer of transfer with detection.The PSCA extracellular domain comes off or is released to extracellular environment; (Meerson as viewed with alkaline phosphatase diesterase B10; N.R.; Hepatology 27:563-568 (1998)), allow through anti--psca antibody diagnostic assays PSCA in suspicious patient's serum and/or urine sample.

Specificity combines anti--psca antibody of PSCA to be used to treat the cancer of expressing PSCA.Anti--psca antibody is used with non-coupling pattern or is used with the coupling pattern, wherein in the coupling pattern, and a kind of in antibody and multiple treatment known in the art or the imaging modality, for example prodrug, enzyme or ri combination.In preclinical study; At SCID mouse cancer heteroplastic transplantation model, for example tested among kidney model AGS-K3 and the AGS-K6 not link coupled and link coupled anti--psca antibody is to tumor prevention and growth inhibiting usefulness (referring to the embodiment that for example is entitled as " in-vivo tumour of PSCA mediated monoclonal antibody suppresses ").Link coupled and not link coupled anti--psca antibody is used as human clinical trial's form of therapy, can use separately or with other treatment combined utilization, in following embodiment, be described.

Embodiment 28: in vivo through the end user anti--mankind of psca antibody treatment and diagnosis human cancer Clinical trial

Antibody used in the present invention can be discerned the epi-position on the PSCA, for example is used to treat some listed tumour in the Table I.Based on multiple factor, comprise the expression level of PSCA, be at present preferred indication like tumour listed in the Table I.Combine with each indication, successfully carried out three kinds of clinical methods.

I.) assisting therapy: in assisting therapy, use anti--psca antibody combined chemotherapy or antitumor drug and/or radiotherapy in the treatment patient.Through in standard one line and second line treatment, adding anti--psca antibody, according to the basic cancer target of standard scheme treatment, the for example listed cancer of Table I.The validity of conceptual design is through dwindling tumor mass and the ability of the routine dose of the chemotherapeutics that debases the standard is estimated.The dosage of the reduction of these dosage through reducing chemotherapeutics-xicity related allows treatment extra and/or that prolong.In several kinds of auxiliary clinical trials, anti--psca antibody and chemotherapy or antitumor drug Zorubicin (advanced prostate cancer), cis-platinum (late period head and neck cancer and lung cancer), taxol (mammary cancer) and Dx (before clinical) are united use.

II.) monotherapy:, give patient's antibody and do not give chemotherapy or antitumor drug about the anti--application of psca antibody in the tumour monotherapy.In one embodiment, monotherapy is used for extensive metastatic disease patient's cancer in latter stage clinically.Patient's the state of an illness demonstrates stability.Evidence to refractory cancers patient's effect.

III.) developer: through with radionuclide (for example, iodine or yttrium (I ¹³¹, Y ⁹⁰)) link to each other with anti--psca antibody, this immune labeled antibody can be used as diagnosis and/or developer.At this on, traget antibody is positioned the metastatic lesion of the cell of solid tumor and expression PSCA.About the application of anti--psca antibody as developer, said antibody is used as the assistant agent of surgical intervention solid tumor, follows up a case by regular visits to measure the residual and/or recurrence of tumour after comprising before the operation screening and operation.In one embodiment, ( ¹¹¹In)-and psca antibody is as the developer of human I clinical trial phase, and wherein patient's tumour is expressed PSCA (analogy referring to for example Divgi et al, J.Natl.Cancer Inst 83:97-104 (1991)).Anteposition (anterior) and position, back (posterior) γ camera with standard are followed up a case by regular visits to the patient.The result shows, has identified primary lesion and MET.

The dosage of administration and approach

As understood by one of ordinary skill in the art, the consideration of dosage can be through confirming with the comparison of clinical analogous products.Therefore, can be 5 to 400mg/m ²Dosage range in use anti--psca antibody, said dosage is relevant with safety research than low dosage.Is the parameter that those skilled in the art confirm similar dosage with respect to known antibodies for the avidity of anti--psca antibody of the avidity of its target spot.In addition, compare with chimeric antibody, as fully human antibodies anti--psca antibody is removed slowlyer; Correspondingly, the dosage of anti--psca antibody that is used for these complete peopleization of patient can be lower, probably 50 to 300mg/m ²Scope in, this moment still effective.With mg/m ²Expression dosage, and the dosage that is different from conventional mg/kg is represented mode, this is based on the balancing method of surface-area, also is the balancing method of dosage easily that can be used for comprising the patient of all bodily forms sizes from the baby to the grownup.

The method for releasing of three kinds of uniquenesses helps to transmit anti--psca antibody.Conventional intravenously transmission is a kind of standard transmission technology that is used for many tumours.But for Intraabdominal tumour, for example oophoroma, cholangioma, other conduit knurl etc., the intraperitoneal administration is proved to be effectively, because can assemble the antibody of high dosage and cleaning antibody is reduced to minimum in tumor site.In similar methods, some solid tumor has the vascular system of suitable regional perfusion.Regional perfusion allows tumor locus that the antibody of high dosage is arranged and makes the short-term removing of antibody reduce to minimum.

Clinical development plan (CDP)

General introduction: CDP follow and the treat-ment that developed anti--psca antibody together with assisting therapy, single therapy and as developer.In repeated doses, test has proved that at first security has confirmed validity subsequently.Test has been compared standard chemotherapy and standard care and has been added anti--psca antibody for open mark.Like what understood, can be used for selecting patient's a standard is the expression level through PSCA in its tumour of biopsy mensuration.

For any treatment of inculcating based on protein or antibody, security consideration relates generally to (i) cytokine release syndrome, that is, and and ypotension, heating, chatter, shiver with cold; (ii) to the generation (that is, the patient who accepts Antybody therapy produces people's antibody, or HAHA replys) of the immunogenic response of material; And, (iii) for the Normocellular toxicity of expressing PSCA.Standard testing and tracing study are used to monitor above-mentioned various security consideration.Find that it is safe that anti--psca antibody is used for the mankind.

Embodiment 29: the human clinical trial: the end user is anti--and the single therapy of psca antibody

Anti--psca antibody is safe for above-mentioned assisting therapy, II phase human clinical trial has confirmed the usefulness and the optimal dose of single therapy.Such test is accomplished, and has carried out identical security and interpretation of result, and different with above-mentioned assisting therapy is that the patient does not accept chemotherapeutics when accepting anti--psca antibody.

Embodiment 30: the human clinical trial: carry out diagnostic imaging with anti--psca antibody

Because above-mentioned assisting therapy is safe in above-mentioned safety standards, carry out once more about of the clinical trial of anti--psca antibody as diagnosing developing agent.The scheme that scheme of designing and this area are described is similar basically, Divgi et al for example, J.Natl.Cancer lnst.83:97-104 (1991).When finding this antibody as diagnostic medicine is safely and effectively.

Embodiment 31: the end user is anti--and the people of psca antibody and chemotherapy, radiotherapy and/or hormone disengaging therapy Class clinical trial adjuvant therapy

Start I phase human clinical trial with estimate about the people of treatment solid tumor anti--security of six kinds of intravenous dosages of psca antibody, the cancer of these solid tumors such as the listed tissue of Table I.In this research; Estimated the security of anti--psca antibody of single dose when being used for auxiliary antitumor or chemotherapeutics or hormone disengaging pharmacological agent; Wherein said medicine such as defined herein; For example, but be not limited to: cis-platinum, hycamtin, Dx, Zorubicin, taxol, Lupron, Zoladex, Eulexin, Casodex, Anandron.Test design comprises: according to following or similar scheme, transmit the anti--psca antibody of about six kinds of single doses, this antibody dosage is from about 25mg/m ²Rise to about 275mg/m ²

The 0th day the 7th day the 14th day the 21st day the 28th day the 35th day

MAb dosage 25 75 125 175 225 275

mg/m ² mg/m ² mg/m ² mg/m ² mg/m ² mg/m ²

Chemotherapy++++++

(standard dose)

After giving antibody and chemotherapeutics, the patient is followed up a case by regular visits to a week closely at every turn.Especially, the above-mentioned security consideration of evaluate patient: (i) cytokine release syndrome, that is, and ypotension, heating, chatter, shiver with cold; (ii) to the generation (that is, the patient of receiver's Antybody therapy agent produces people's antibody, or HAHA replys) of the immunogenic response of material; And (iii) for the Normocellular toxicity of expressing PSCA.Standard testing and tracing study are used to monitor above-mentioned each security consideration.The clinical effectiveness of evaluate patient also is particularly by the heavy reduction of knurl of MRI or other video picture proof.

Anti--psca antibody is proved to be safely and effectively, and the II phase tests and confirmed usefulness and optimized optimal dose.

Embodiment 32:RNA disturbs (RNAi)

RNAi disturbs (RNAi) technology to be applied to the multiple test cell line relevant with oncology.RNAi is by double-stranded RNA (dsRNA) activated PTGS mechanism.RNAi inducing specific mRNA degraded causes protein expression to change, and can change the function of gene subsequently.In mammalian cell, these dsRNA that are called as short RNA interfering (siRNA) have the degraded of correct composition with the RNAi approach, particularly some mRNA of the degraded of activation target.Referring to Elbashir S.M., et al., Duplexes of 21-nucleotide RNAs Mediate RNA interference in CulturedMammalian Cells, Nature 411 (6836): 494-8 (2001).Therefore, the RNAi technology successfully is used for reticent target gene in mammalian cell.

Cell proliferation is out of hand to be the sign of cancer cells; Therefore, estimating the effect of PSCA in cell survival/proliferation test is correlated with.Correspondingly, use the antigenic function of RNAi research PSCA.In order to produce the siRNA of PSCA, use can be predicted the algorithm of oligonucleotide, and said oligonucleotide shows vital molecular parameter (G: C content, melting temperature(Tm) etc.), when being imported into cell, can significantly reduce the proteic expression level of PSCA.According to this embodiment, employed PSCA siRNA compsn contains corresponding to the siRNA of the nucleic acid ORF sequence of PSCA albumen or its subsequence (two strands, short RNA interfering).Therefore, the general length of using in this way of siRNA subsequence is 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, and the RNA Nucleotide of adjacency more than 33,34,35 or 35.Complementary and the non--complementation of at least a portion of these siRNA sequences and mRNA encoding sequence.In preferred embodiments, the length of subsequence is 19-25 Nucleotide, and most preferably length is 21-23 Nucleotide.In preferred embodiments, these siRNA can knock down this protein in expressing the antigenic cell of PSCA, also have the functional effect that is described below.

In survival/propagation MTS test (mensuration cell metabolic activity), in a plurality of clones, detect selected siRNA (PSCA.b oligo).Colorimetric test (being MTS) based on tetrazole only can detect viable cell; Because viable cell has metabolic activity, therefore can reduce the amount that tetrazolium becomes colored first

compound; Yet dead cell but can not.In addition, use following method, this PSCA.b oligo can knock down this protein in expressing the antigenic cell of PSCA, also have the functional effect that is described below.

Mammals siRNA transfection: in siRNA transfection previous day, with 2 * 10 ³The density of individual cells/well (forms of 96 well culture plates) is laid on 80 μ l substratum (RPMI 1640+10%FBSw/o microbiotic) to survive/the MTS test with different clones.Oligo parallels with the PSCA specific siRNA, in each experiment, comprises that following sequence is as contrast: a) Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and the simulation transfectional cell of annealing buffer (no siRNA); B) luciferase-4 specific siRNA (the target sequence: 5 '-AAGGGACGAAGACGAACACUUC TT-3 ') (SEQ IDNO:20); And c) the Eg5 specific siRNA (the target sequence: 5 '-AACTGAAGACCTGAAGACAATAA-3 ') (SEQ ID NO:21).Use siRNA with 10nM and 1 μ g/mlLipofectamine, 2000 final concentrations.

Method is following: at first use OPTIMEM (serum-free transfection media, Invitrogen) dilution siRNA to 0.1 μ M (10 times concentrate) and in room temperature insulation 5-10 minute.To the transfection sum, Lipofectamine2000 is diluted to 10 μ g/ml (10 times concentrate) and is incubated 5-10 minute in room temperature (RT).An amount of diluted 10 times of spissated Lipofectamine 2000 are mixed with diluted 10 times of spissated siRNA with 1: 1 ratio, and in room temperature (RT) insulation 20-30 " (5 times of spissated transfection solution).5 times of spissated transfection solution of 20 μ l are added in each sample, and 37 ℃ of insulations performed an analysis in 96 hours again.

The MTS test: MTS test is based on tetrazotized zole compound [3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxylic methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole, inner salt; MTS (b)] and electron coupling reagent (phenazine methosulfate (ethosulfate); PES) measure viable count purpose colourimetry in propagation, cytotoxicity or the chemosensitivity test.Through in the culture hole, directly adding a small amount of SolutionReagent, be incubated 1-4 hour, the absorbance value that writes down 490nm with 96-well culture plate reader then is to make an experiment.The formazan

that absorbance value through 490nm is measured (formazan) in amount and the mitochondria activity and/or the culture of product the number of viable cell in direct ratio.

In order to study the function of PSCA in cell, the clone through transfection endogenous expression PSCA is with reticent PSCA.

Another embodiment of the invention is to analyze the method for PSCA relevant cell propagation, and what this method was measured is synthetic as the DNA of propagation mark.Use through the marker DNA precursor (promptly ³The H-thymidine), measure the amount that it mixes DNA.Through labelled precursor in DNA mix with culture in the fissional amount that occurs directly proportional.The method that another kind is used to measure cell proliferation is to clone to test.In these tests, the cell of restricted number is laid on the suitable matrix, count handle the back formed colony number of growing after for some time through siRNA.

When confirming PSCA cancer target, consider to replenish cell survival/proliferation assay with apoptosis and cell cycle distribution research.The biochemical marker of apoptosis process is the fragmentation of genomic dna, and fragmentation is the irreversible incident that causes necrocytosis.The method of fragmentation DNA is through immunoassay (be necrocytosis detect ELISA) immunodetection and histone compound dna fragmentation in the observation of cell, and this method is measured in the kytoplasm of apoptotic cell the enrichment with histone compound dna fragmentation (singly-and widow-nucleosome).This assay method need not labeled cell in advance, and can detect the dna degradation in external not proliferating cells (being firm isolating tumour cell).

The most important effector molecule that causes apoptotic cell death is a Caspase.Caspase is a proteolytic enzyme, and it can mediate the apoptosis of phase very early through activating at the multiple substrate of C-terminal site cracking of asparagicacid residue when being activated.All Caspases are synthesized and are proenzyme, activate to be included in the cracking of asparagicacid residue place.Specifically, as if Caspase 3 plays main effect in starting the apoptosis incident.Measure the test of Caspase 3 activated and detect apoptotic early stage incident.After the RNAi processing, the protease cracking of the product (being PARP) that the Western trace that has active Caspase 3 to exist detects or in apoptotic cell, finds has further been supported apoptotic activity inducement.Owing to cause apoptotic cell mechanism comparatively complicated, its advantage and limitation respectively arranged.Consider other standard/terminal point, like morphocytology, chromatin condensation, the growth of film water blister, it is apoptosis that the apoptosis body helps further sustenticular cell dead.Because the gene target of not all adjusting cell growth all is anti--apoptosis, mensuration is changed the dna content of cell thoroughly to obtain dna content distribution plan or cell cycle distribution figure.Because DNA drains in the kytoplasm, so the nuclear of apoptotic cell contains less DNA (Asia-G1 crowd).In addition, use DNA staining agent (being iodate third ingot) also can distinguish the different cell cycle phases of cell colony, this is because G0/G1, the DNA of the different amounts of existence among S and the G2/M.In these researchs, subgroup is by quantitative.

For psca gene, RNAi research helps understanding the effect of gene product to the cancer approach.This activated RNAi molecule can be used for test can be used as active Anti-tumor therapeutical agent with screening MAb.In addition, use siRNA as therapeutical agent to reduce several kinds of cancer types, comprise the malignancy of the listed cancer of Table I to the cancer patients.When PSCA worked in cell survival, cell proliferation, tumour generation or apoptosis, it can be used as diagnosis, predicts, prevents and/or treats the target of purpose.

Quoted the data content of a plurality of websites everywhere in the application, publication, patented claim and patent.(quoted the Uniform Resource Locator of website, or ULR, the address on the World Wide Web).Disclosed content is listed this paper in as a reference all in full in each reference.

The invention is not restricted to the scope of this paper disclosed embodiment, these embodiments are merely and illustrate all respects of the present invention separately, and the embodiment of any functional equivalent all within the scope of the invention.Remove described herein; The various variations of pattern of the present invention and method; For the those of skill in the art that describe with reference to preamble and instruct all is conspicuous; Desire equally to fall in the scope of the present invention, such variation or other embodiment can be implemented under the situation that does not depart from actual range of the present invention and spirit.

Table

Table I: the malignant tissue of expressing PSCA

Prostate gland

Pancreas

Bladder

Kidney

Colon

Lung

Ovary

Mammary gland

Table II: amino acid abbreviations

Table III: aminoacid replacement matrix

Revise according to GCG software 9.0 BLOSUM62 aminoacid replacement matrixes (piece substitution matrix (block substitution matrix)).Be worth highly more, might in relevant natural protein, find to replace (referring to www URL ikp.unibe.ch/manual/blosum62.html) more.

Table IV:

HLA I/II class primitive/hyper-base unit

Table IV (A): HLAI class hyper-base unit/primitive

The residue of runic is preferred residue, and the residue of italic takes second place: put in each one-level (primary) anchor position when a kind of peptide and have when going up the first one-level anchor of the table indicated primitive of institute or hyper-base, can think that this peptide carries primitive.

Table IV (B): HLAII class hyper-base unit

Table IV (C): HLAII class primitive

The residue of italic is represented the more preferably residue of (less preferred) or the residue of " tolerance ".

Table IV (D): HLAI class hyper-base unit

Table IV (E): HLAI class primitive

Table IV (G):

Primitive representes to limit the residue of superspecificity.These primitives insert based on the determined residue of common data, so that by the inner multiple allelotrope identification of said ultra type.Residue in the bracket is other residue, predicts that they also can be tolerated by the inner multiple allelotrope of said ultra type.

Table VI: transcript PSCA exon border v.1

Table VII: the MFI value that is used for each data point of affinity calculating

Table VIII: use Graphpad Prism software: single-point combines (hyperbolic line) by the affinity that combines saturation curve to calculate

Table I X: whole person PSCA MAb is based on the affinity of FACS

Affinity based on FACS

Table X: with the antibody of monkey PSCA and/or mouse PSCA cross reaction

Claims

1. contain the antibody of antigen binding site or contain the antibody fragment of antigen binding site; Wherein antigen binding site contains the variable light chain district that variable heavy chain district that aminoacid sequence is SEQ ID NO:13 and aminoacid sequence are SEQ ID NO:14, and wherein antigen binding site can specificity combine the PSCA albumen shown in the SEQ ID NO:2.

2. the antibody of claim 1 or antibody fragment, wherein antibody is monoclonal antibody.

3. the antibody of claim 1 or antibody fragment, wherein fragment is Fab, F (ab ') ₂, Fv or Sfv fragment.

4. the antibody of claim 1 or antibody fragment, wherein antibody with can survey affinity tag, toxin or therapeutical agent coupling.

5. the antibody of claim 4 or antibody fragment, therapeutical agent wherein is a chemotherapeutics.

6. the antibody of claim 4 or antibody fragment, wherein can survey affinity tag is ri, metal chelator, enzyme, fluorescent chemicals, bioluminescent compound or chemiluminescence compound.

7. the antibody of claim 6 or antibody fragment, wherein ri comprises ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, ¹⁸⁶Re, ²¹¹At, ¹²⁵I, ¹⁸⁸Re, ¹⁵³Sm, ²¹³Bi, ³²P or Lu.

8. the antibody of claim 4 or antibody fragment, wherein toxin comprises ricin, ricin A-chain, Zorubicin, daunorubicin, maytansinoid, taxol, ethidium bromide, MTC, VP, teniposide, vincristin, vincaleucoblastine, NST-757, dihydroxyl anthracin diketone, NSC-3053, diphtheria toxin, PE (PE) A, PE40, toxalbumin, abrin A chain, modeccin A chain, α-Zhou Qujunsu, sets toxalbumin in vain, silk splits lucky mycin, Restrictocin, phenomycin, enomycin, curcin, crotin, calicheamicin, Sapaonaria offcinalis suppressor factor, glucocorticosteroid, Ao Ruisitading, duomycin, yttrium, bismuth, Kang Bailaisitading, times ganmycin, many Luo Sitading, cc1065 or cis-platinum.

9. carrier contains the polynucleotide of the said antibody of coding claim 2.

10. pharmaceutical composition contains the claim 1 or the 2 said antibody or the fragments of human unit dosage form.

11. the pharmaceutical composition of claim 10, wherein antibody or fragment and cytotoxic agent or diagnostic reagent coupling.

12. the pharmaceutical composition of claim 11, wherein cytotoxic agent or diagnostic reagent are selected from and can survey affinity tag, toxin and therapeutical agent.

13. the pharmaceutical composition of claim 12, wherein can survey affinity tag is ri, metal chelator, enzyme, fluorescent chemicals, bioluminescent compound or chemiluminescence compound.

14. the pharmaceutical composition of claim 13, wherein ri comprises ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, ¹⁸⁶Re, ²¹¹At, ¹²⁵I, ¹⁸⁸Re, ¹⁵³Sm, ²¹³Bi, ³²P or Lu.

15. the pharmaceutical composition of claim 12, wherein toxin comprises ricin, ricin A-chain, Zorubicin, daunorubicin, maytansinoid, taxol, ethidium bromide, MTC, VP, teniposide, vincristin, vincaleucoblastine, NST-757, dihydroxyl anthracin diketone, NSC-3053, diphtheria toxin, PE (PE) A, PE40, toxalbumin, abrin A chain, modeccin A chain, α-Zhou Qujunsu, sets toxalbumin in vain, silk splits lucky mycin, Restrictocin, phenomycin, enomycin, curcin, crotin, calicheamicin, Sapaonaria offcinalis suppressor factor, glucocorticosteroid, Ao Ruisitading, duomycin, yttrium, bismuth, Kang Bailaisitading, times ganmycin, many Luo Sitading, cc1065 or cis-platinum.

16. the pharmaceutical composition of one of claim 10-15 has been adapted to make up with the chemotherapeutics administration or with radiotherapy.