CN1997751B - Antibodies and related molecules that bind to PSCA proteins - Google Patents

Antibodies and related molecules that bind to PSCA proteins Download PDF

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CN1997751B
CN1997751B CN200580017239.9A CN200580017239A CN1997751B CN 1997751 B CN1997751 B CN 1997751B CN 200580017239 A CN200580017239 A CN 200580017239A CN 1997751 B CN1997751 B CN 1997751B
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cancer
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J·古达斯
A·雅各波维茨
X·贾
R·K·莫里森
K·J·M·莫里森
H·邵
P·M·查利塔-埃德
A·B·莱塔诺
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    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

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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

Be incorporated into proteic antibody of PSCA and associated molecule
The cross reference of related application
Present patent application relates to the co 10/857,484 (being filed on May 28th, 2004) of the interim patent 60/475,064 of the requirement U.S. (being filed on May 30th, 2003) right of priority; The application relates to U.S. Provisional Patent Application 60/616,381 (being filed on October 5th, 2004), U.S. Provisional Patent Application 60/617,881 (being filed on October 12nd, 2004); U.S. Provisional Patent Application 60/621,310 (being filed on October 21st, 2004); U.S. Provisional Patent Application 60/633,077 (being filed on October 2nd, 2004); And the U.S. Provisional Patent Application of not specifying number of patent application as yet that is filed on April 14th, 2005.The present invention relates to PCT patent application PCT/US2004/017231 (being filed on May 28th, 2004).The content of this section listed each application is all included this paper in as a reference with it.
Ownership statement about the invention of government sponsored research project
Inapplicable.
Technical field
Invention described herein relate to be incorporated into the proteinic antibody that is called as PSCA and binding fragment thereof, and by its engineered molecule that obtains.The invention still further relates to the diagnosis, prediction, prevention and the Treatment and composition for that are used to treat the cancer of expressing PSCA.
Background of invention
Cancer is the second largest human cause of death that is only second to coronary heart disease.Have the millions of people to die from cancer every year in the world wide.Only in the U.S., like the report of American Cancer Society, cancer causes the people more than 500,000 dead every year, has every year 1200000 people to be suffered from cancer by new diagnosis simultaneously.Obviously reduce though die from cardiopathic number, the number of dying from cancer rises gradually.In the beginning of next century, estimate that cancer will become the No.1 cause of death.
In the world wide, it is main killers that several kinds of cancers are arranged.Especially lung cancer, prostate cancer, mammary cancer, colorectal carcinoma, carcinoma of the pancreas, ovarian cancer and bladder cancer, they are major causes of cancer mortality.These cancers and in fact all other cancers all have identical fatal characteristic.Almost not exceptionally, the metastatic carcinoma disease is fatal.In addition, even if for those for survival cancer patients down in the primary cancer, the universal experience demonstration, significant change has all taken place in their life.Many cancer patientss are owing to worrying that recurrence or treatment failure have produced the sense of intensive anxiety.Many cancer patientss are in poor health after treatment.In addition, many cancer patientss can experience recurrence.
In the world wide, prostate cancer is the fourth-largest popular cancer of the male sex.In North America and Northern Europe, it almost is a modal cancer among the male sex, is the second largest reason that causes male cancer dead simultaneously.Only in the U.S., there is every year the male sex more than 30,000 to die from this disease-be only second to lung cancer.Huge except these digital numerical value, also there is not effective treat-ment of metastatic prostate cancer.Surgery prostatectomy, radiotherapy, injection of hormone treatment, operation castrating and chemotherapy remain main treatment means.Unfortunately, these treat-ment are invalid for many people, and usually can bring unhappy result.
With regard to diagnosis, the tumor of prostate mark that shortage can accurately detect early stage limitation tumour still is a critical limitation in this medical diagnosis on disease and the treatment.Become a kind of effective tool although serum PSA (PSA) detects, thought that extensively it is in some importance lack of specific and versatility.
Promoted the evaluation of other prostatic cancer specific mark through producing the prostate cancer heterograft to reproduce disease different steps in the mouse.LAPC (Los Angeles prostate cancer) heterograft is the prostate cancer heterograft that in Reconstruction in Sever Combined Immunodeciency (SCID) mouse, has survival to go down to posterity; And demonstrate simulation from male sex hormone dependence changing into not dependency (Klein etc. of male sex hormone; 1997, Nat.Med.3:402).The prostate cancer markers of identifying recently comprises PCTA-1 (Su etc.; 1996; Proc.Natl.Acad.Sci.USA 93:7252), prostate specific membrane (PSM) antigen (Pinto etc.; Clin Cancer Res 1996-9-2 (9): 1445-51), STEAP (Hubert etc., Proc Natl Acad Sci U S A.1999-12-7; 96 (25): 14523-8) and PSCA (PSCA) (Reiter etc., 1998, Proc.Natl.Acad.Sci.USA 95:1735).
Although the mark such as PSA, PSM, PCTA and the PSCA that identified in the past have the effect that helps to diagnose and treat prostate cancer, other mark that also need identify prostate cancer and associated cancer is diagnosed and treatment with further promotion with the treatment target.
Renal cell carcinoma (RCC) accounts for into human malignant lesion's about 3%.In case reaching 2-3cm, the diameter of adenoma just possibly worsen.In the adult, two kinds of main malignant renal neoplasms are renal cell adenocarcinoma and renal plevis or transitional cell carcinoma of ureter.Estimation surpasses 29,000 examples at the sickness rate of U.S.'s renal cell adenocarcinoma, and has 11,600 patients of surpassing to die from this disease in 1998.Transitional cell carcinoma is comparatively rare, and its sickness rate in the U.S. is about annual 500 examples.
Recent decades, operation is the main treat-ment of renal cell adenocarcinoma always.Up to date, metastatic disease also is difficult to control with any constitutional treatment.Along with the development of nearest constitutional treatment especially immunotherapy, persistence reply may be in suitable patient the attack metastatic renal cell cancer.But, still need treat these patients' effective ways.
In all new cases of cancers of the U.S., bladder cancer accounts for 5% (the 5th kind of modal knurl) in the male sex, in the women, accounts for 3% (the 8th kind of modal knurl).This sickness rate slowly increases, and along with this numeral of increase of elderly population is also raising.In 1998, estimate at 54,500 cases, comprising 39,500 male sex and 15,000 women.In the U.S., this age adjustment sickness rate is per 100,000 male sex, 32 people, per 100,000 women, 8 people.3: 1 ratio of male/female may reduce owing to women's smoking in history.Estimating had 11,000 people to die from bladder cancer (7,800 male sex and 3,900 women) in 1998.The M & M of bladder cancer increases along with the age and sharply rises, and becomes serious day by day problem along with aging population.
Most of bladder cancer can recurrence in bladder.Bladder cancer is treated through the combination of transurethral resection,bladder (TUR) and intravesical chemotherapy or immunotherapy.Many focuses of bladder cancer and recurrence characteristic have been pointed out the limitation of TUR.Most of muscle wellability cancers can not only be cured through TUR.Radical cystectomy and diversion of urine are the most effectively to effect a radical cure this method for cancer, but can cause undeniable influence to uropoiesis and sexual function.Obviously treat-ment that also need be useful to bladder cancer patients.
Estimate that 130,200 routine colorectal carcinomas take place the U.S. in 2000 altogether, comprise 93, the 800 routine colorectal carcinomas and 36, the 400 routine rectum cancer.Colorectal carcinoma is the third-largest cancer in the masculinity and femininity.Its sickness rate significantly lowers (annual reduction by 2.1%) in the period of 1992-1996.Research shows that these reductions are because inspection and polypectomy increase, thereby have prevented that polyp from developing into the wellability cancer.Estimate to have in 2000 56,300 people dead (47,700 people die from colorectal carcinoma, and 8,600 people die from the rectum cancer), account for about 11% of american cancer total toll.
At present, surgical operation is the method for modal treatment colorectal carcinoma, often adopts this method for the cancer that does not also spread.For great majority degree of depth intestines wall perforation or be diffused into the cancer patients of lymphoglandula, before operation or after the operation, carry out chemotherapy usually, or chemotherapy adds radiotherapy.Colorectal carcinoma is also needed permanent colostomy (forming an opening to get rid of human body waste at belly) sometimes, and the rectum cancer also needs this operation once in a while simultaneously.The method that still needs effective diagnosis and treatment colorectal carcinoma.
Estimate newly-increased 164,100 routine lungs in 2000 and segmental bronchus cancer, account for 14% of the total cancer number of examining out of the U.S..The sickness rate of lung and segmental bronchus cancer disease significantly reduces in the male sex, from 1984 86.5/100,000 be reduced to 1996 70.0.In the nineties in 20th century, the increase impetus of sickness rate begins to slow down among the women.In 1996, the sickness rate among the women was 42.3/100,000.
Estimate that lung in 2000 and segmental bronchus cancer cause 156,900 people dead, account for 28% of total cancer mortality number.In the period of the 1992-1996, the mortality ratio of lung cancer significantly reduces (annual reduction by 1.7%) in the male sex, and this ratio significantly rises (every year 0.9%) in the women.Since 1987, compare mammary cancer, have every year many more women to die from lung cancer, and mammary cancer all is the major cause of women's cancer mortality in 40 years in the past.The lung cancer morbidity rate possibly reduce relevant with past 30 years smoking rate with the mortality ratio reduction is very big; Yet the reduction of women's smoking rate lags behind the male sex.What need concern is that although adult smoking rate slowly reduces, but young adult's smoking rate is raising.
The regimen of lung and segmental bronchus cancer is to be determined by the type of cancer and stage, comprising operation, radiation and chemotherapy.As far as many limitation cancers, select operative treatment usually.Because this disease spreads with discovery time usually, radiation and chemotherapy need be united use with operation usually.Can select chemotherapy itself or treat small cell lung cancer with combination with radiotherapeutic; When adopting this scheme, the symptom of Most patients can be alleviated, and wherein some is long lasting.Yet, still need efficacious therapy and the method for diagnosing lung and segmental bronchus cancer.
2000, estimating had 182,800 routine New Development mammary cancer cases in the American Women.In addition, about 1,400 routine mammary cancer case was newly diagnosed out in estimation in 2000 in the male sex.In the eighties in 20th century, among the women sickness rate of mammary cancer annual increase about 4%, and the nineties this sickness rate more steady, be about per 100,000 people, 110.6 examples.
Only in the U.S., estimating had 41,200 people (40,800 women, 400 male sex) to die from mammary cancer in 2000.Mammary cancer occupies second in women's cancer mortality.According to latest data, no matter be white man or Black people, this mortality ratio significantly reduces in the period of 1992-1996, and the reduction degree is maximum in young woman simultaneously.These reductions possibly be because early stage inspection and the improved result of treat-ment.
Consider medical environment and patient's situation, the treat-ment of mammary cancer can comprise lumpectomy's (local excision's tumour) and remove underarm lymphoglandula; Mastectomy (excision breast) is also removed underarm lymphoglandula; Radiotherapy; Chemotherapy; Or hormonotherapy.Usually can unite two or more methods of use.Big quantity research shows that for early stage disease, long-term surviving rate and the survival rate behind the modified radical mastectomy that lumpectomy adds after the radiotherapy are similar.The major progress of reconstruction technique can provide some options about breast reconstruction at the mastectomy postoperative.Recently, this reconstruction is carried out with mastectomy simultaneously.
An amount of NBT can prevent the DCIS recurrence around local excision's DCIS (DCIS) reached.Irradiation breast and/or tamoxifen treatment can reduce the chance that DCIS takes place the residue breast tissue.This is important, if will not develop into infiltrative breast carcinoma because DCIS does not treat.And these treat-ment have severe side effect or sequela.Therefore, need the effective ways of treatment mammary cancer.
Estimate 2000 in U.S.'s New Development 23,100 routine ovarian cancers.This account for all gynecological cancers 4% and be second largest gynecological cancer.In the period of the 1992-1996, the ovarian cancer sickness rate significantly reduces.The result of ovarian cancer is, estimating had 14,000 people dead in 2000.Compare other cancer of female genital system, ovarian cancer causes more people dead.
The method of treatment ovarian cancer has operation, radiation and chemotherapy.Operation generally includes the ovary of removing one or both sides, uterine tube (S&O) and uterus (uterectomy).In some very early stage tumours, have only relevant ovary to be excised, the young woman who especially bears child for those hope.In the disease, need remove interior all disease locations of abdomen late to strengthen chemotherapy effect.Effectively the method for treatment ovarian cancer is still important need.
Estimate 2000 in U.S.'s New Development 28,300 routine carcinoma of the pancreas.In 20 years, the sickness rate of male sex's carcinoma of the pancreas decreases in the past.It is almost constant that women's sickness rate still keeps, but also possibly begin to lower.Estimate to cause 28,200 people dead in U.S.'s carcinoma of the pancreas in 2000.In 20 years, mortality ratio has slight but significantly reduction (reduce by 0.9% every year approximately) among the male sex in the past, and this ratio slightly raises in the women.
Can treat carcinoma of the pancreas through operation, radiation and chemotherapy.In Most patients, these treat-ment can prolong survival time and/or relief of symptoms, but can not all cure.Press for the method for other treatment and diagnosing cancer.These methods comprise antibody, vaccine and small molecules as form of therapy (modality).In addition, also need these forms be used as cancer therapy and study diagnosis in all areas, detection, Study of Monitoring instrument, and state-of-art.
It has been recognized that the therepic use (G.Kohler and C.Milstein, Nature256:495-497 (1975)) of monoclonal antibody (MAbs).At present, monoclonal antibody grantedly be used for transplanting, the treatment of cancer, transmissible disease, cardiovascular disorder and inflammation.Different isoforms has different effector functions.Difference on these functions is corresponding to the different three-dimensional structures (P.M.Alzari etc., AnnualRev.Immunol., 6:555-580 (1988)) of various Tegeline isotypes.
Because it is very convenient with the most of heterogeneous human antigens of identification that mouse is used for immunity, is mouse source property usually to the MAbs with people's target of treating potentiality.Yet mouse MAbs itself has defective in the human treatment.Because the circulating half-life of MAbs in human body is shorter than people antibody, need to give more continually MAbs.More crucial is to repeat can cause human immune system through murine protein matter being identified as heterogeneous thing and producing replying of human anti-mouse antibody (HAMA) murine antibody administration of human immunity system.This HAMA replys and can cause anaphylaxis, and from immunity system, removes rapidly mouse source antibody, thereby makes and adopt failing to respond to any medical treatment of mouse source antibody.For fear of this influence, carried out in mouse, setting up the trial of human immune system.
Hope setting up in the trial in the early stage can be with the antigenic transgenic mice of the antibody response with human sequence (referring to Bruggemann etc.; But be subject to the amount that to stablize the DNA that keeps by the available cloning vector Proc.Nat ' 1.Acad.Sci.USA 86:6709-6713 (1989)).The macrospecies of adopting yeast artificial chromosome (YAC) cloning vector to open up people Ig seat is the road that fragment imports transgene mammal.Basically most people V, D and J regional gene are arranged with the same intervals of in the people's gene group, finding, and adopt YAC that human constant region is imported mouse.Known a kind of this type of transgenic mice strain is called XenoMouse (r) mouse, and it can be available from Abgenix, Inc. (Fremont CA).
Summary of the invention
The invention provides be incorporated into PSCA albumen and the segmental antibody of PSCA protein polypeptide and binding fragment thereof with they through engineered resulting molecule.The present invention includes polyclone and monoclonal antibody, mouse and other Mammals antibody, chimeric antibody, peopleization and total length people antibody and with the antibody of detectable label or therapeutical agent mark.In some embodiments, prerequisite be Fig. 3 nucleotide sequence not by the aminoacid sequence of whole coding and/or Fig. 2 not by whole preparation.In some embodiments, the whole aminoacid sequence of the whole nucleotide sequence of code pattern 3 and/or preparation Fig. 2, the arbitrary people's unit dosage that is separately among both.
The present invention also provides the method that detects PSCA polynucleotide and proteinic existence and state in the various biological samples, and the method for identifying the cell of expressing PSCA.One embodiment of the invention provide monitoring to contain or have been suspected to have some growth imbalance form such as the tissue of cancer or method of the psca gene product in the blood sample.
The present invention also provides the method for the cancer that various immunogenicities or therapeutic composition and treatment express PSCA (like the cancer of the tissue listed in the Table I), comprises to suppressing the therapy that PSCA transcribed, translates, processes or brought into play function, and cancer vaccine.On the one hand, the invention provides compsn and comprise method for compositions, express the cancer of PSCA with the treatment patient, wherein said compsn comprises and is fit to carrier and one or more inhibition PSCA manufacturing of people's unitary dose or the reagent of function that the people uses.Preferred said carrier is single people's carrier.In another aspect of this invention, said reagent is with PSCA albumen immunoreactive part to take place.The non-limitative example of this part includes but not limited to antibody (like strand, mono-clonal, polyclone, humanization, chimeric or people's antibody), its function equivalent (comprising natural generation or synthetic) and their combination.Said antibody can partly be puted together with diagnostic or therapeutic.On the other hand, said reagent is the small molecules of top definition.
Description of drawings
Fig. 1.CDNA and the aminoacid sequence of PSCA (be also referred to as PSCA v.1 " or " PSCA variant 1 ") are shown in Figure 1A.Initial methionine(Met) is represented with underscore.ORFs extends to amino acid No. 389 from No. 18 amino acid, comprising terminator codon.
The cDNA and the aminoacid sequence of PSCA variant 2 (being also referred to as " PSCA v.2 ") are shown among Figure 1B.The codon of initial methionine is represented with underscore.ORFs extends to amino acid No. 427 from No. 56 amino acid, comprising terminator codon.
The cDNA and the aminoacid sequence of PSCA variant 3 (being also referred to as " PSCA v.3 ") are shown among Fig. 1 C.The codon of initial methionine is represented with underscore.ORFs extends to amino acid No. 707 from No. 423 amino acid, comprising terminator codon.
The cDNA and the aminoacid sequence of PSCA variant 4 (being also referred to as " PSCA v.4 ") are shown among Fig. 1 D.The codon of initial methionine is represented with underscore.ORFs extends to amino acid No. 993 from No. 424 amino acid, comprising terminator codon.
The cDNA and the aminoacid sequence of PSCA variant 5 (being also referred to as " PSCA v.5 ") are shown among Fig. 1 E.The codon of initial methionine is represented with underscore.ORFs extends to amino acid No. 1479 from No. 910 amino acid, comprising terminator codon.
The cDNA and the aminoacid sequence of PSCA variant 6 (being also referred to as " PSCA v.6 ") are shown among Fig. 1 F.The codon of initial methionine is represented with underscore.ORFs extends to amino acid No. 427 from No. 83 amino acid, comprising terminator codon.
Fig. 1 G.PSCA v.2, PSCA is v.7 to v.18 SNP variant.V.7, PSCA contains 123 amino acid to protein v.18.V.7, v.18 variant PSCA is to have a Nucleotide to be different from PSCA variant v.2 extremely, and coding and v.2 identical protein.Though shown these SNP variants respectively, they also can above any combination and any variant of transcribing listed among Figure 1A-1F exist.
Fig. 1 H.PSCA v.4, PSCA is v.19 to v.30 SNP variant.V.19, PSCA contains 189 amino acid to protein v.30.V.19, v.30 variant PSCA is to have a Nucleotide to be different from PSCA variant v.4 extremely.PSCA v.9, v.10, v.11, v.24 and v.25 protein has only an amino acid to be different from PSCA v.1.V.23, v.28, v.29 with v.30 PSCA encodes and v.4 identical protein.Though shown these SNP variants respectively, they also can any combinations and v.3 and v.4 exist with any variant of transcribing.
Fig. 1 I.The expression of PSCA variant.(1I (a)) design primer distinguish PSCA v.1/v.2/v.4, PSCAv.3 and PSCA v.5.That above exon, indicate with small arrow among the figure is primer A and B, obtains the PCR product of PSCA 425bp v.1/v.2/v.4, the PCR product of PSCA 300bp v.3 and the PCR product of PSCA 910bp v.5 through these primers.The first chain cDNA of (1I (b)) preparation, it derives from: normal bladder, brain, the heart, kidney, liver, lung, prostate gland, spleen, Skelettmuskel, testis, pancreas, colon, stomach, prostate cancer mixture, bladder cancer, kidney, colorectal carcinoma, lung cancer, ovarian cancer, mammary cancer, metastatic carcinoma and carcinoma of the pancreas.Carry out stdn through PCR with the primer of Actin muscle.Use the variant Auele Specific Primer to take turns amplification and carry out sxemiquantitative PCR with 30.The result shows that v.5 PSCA mainly expresses, and lower in colorectal carcinoma and lung cancer expression level in mammary cancer, metastatic carcinoma and carcinoma of the pancreas.In prostate cancer, bladder cancer, kidney, colorectal carcinoma, lung cancer, ovarian cancer, mammary cancer, metastatic carcinoma and carcinoma of the pancreas, detect PSCA PCR product v.1/v.2/v.4.In healthy tissues, only in prostate gland and stomach, detect PSCA PCR product v.1/v.2/v.4, level is lower in kidney and lung, and in any healthy tissues, does not detect PSCA v.5.In any specimen, do not detect PSCA PCR v.3 and detect product.
Fig. 1 J.PSCA v.4 with PSCA expression v.5.1J (a) design is labeled as B and C shown in arrow among the figure primer distinguish PSCA v.4 with PSCA v.5.PSCA v.4 Auele Specific Primer produced 460bp PCR product and PSCA v.5 Auele Specific Primer produced size and be the PCR product of 945bp.The first chain cDNA of 1J (b) preparation derives from: normal bladder, brain, the heart, kidney, liver, lung, prostate gland, spleen, Skelettmuskel, testis, pancreas, colon, stomach, prostate cancer mixture, bladder cancer and multiple heterograft mixture (prostate cancer, kidney and bladder cancer heterograft).Carry out stdn through PCR with the primer of Actin muscle.Use the variant Auele Specific Primer to take turns amplification and carry out sxemiquantitative PCR with 30.The result shows that v.4 PSCA expresses in prostate cancer, bladder cancer and multiple heterograft mixture, normal kidney and prostate gland.Only in normal prostatic and bladder cancer, detect PSCA v.5.
Fig. 2.The amino acid of psca antibody.Fig. 2 A.The aminoacid sequence of Ha1-4.117VH.The underscore place is a CH.Fig. 2 B.The aminoacid sequence of Ha1-4.117VL.The underscore place is a constant region of light chain.Fig. 2 C.The aminoacid sequence of Ha1-4.120VH.Fig. 2 D.The Ha1-4.120VL aminoacid sequence.The underscore place is a constant region of light chain.Fig. 2 E.The aminoacid sequence of Ha1-5.99VH.The underscore place is a CH.Fig. 2 F.The aminoacid sequence of Ha1-5.99VL.The underscore place is a constant region of light chain.Fig. 2 G.The aminoacid sequence of Ha1-4.121VH.The underscore place is a CH.Fig. 2 H.The aminoacid sequence of Ha1-4.121VLc.5.The underscore place is a constant region of light chain.Fig. 2 I.The aminoacid sequence of Ha1-4.121VLc.26.The underscore place is a constant region of light chain.Fig. 2 J.The aminoacid sequence of Ha1-1.16VH.The underscore place is a CH.Fig. 2 K.The aminoacid sequence of Ha1-1.16VL.The underscore place is a constant region of light chain.Fig. 2 L.The aminoacid sequence of Ha1-4.5VH.The underscore place is a CH.Fig. 2 M.The aminoacid sequence of Ha1-4.5VL.The underscore place is a constant region of light chain.Fig. 2 N.The aminoacid sequence of Ha1-4.40VH.The underscore place is a CH.Figure 20.The aminoacid sequence of Ha1-4.40VL.The underscore place is a constant region of light chain.Fig. 2 P.The aminoacid sequence of Ha1-4.37VH.The underscore place is a CH.Fig. 2 Q.The aminoacid sequence of Ha1-4.37VL.The underscore place is a constant region of light chain.Fig. 2 R.The aminoacid sequence of Ha1-1.43VH.The underscore place is a CH.Fig. 2 S.The aminoacid sequence of Ha1-1.43VL.The underscore place is a constant region of light chain.Fig. 2 T.The aminoacid sequence of Ha1-1.152VH.The underscore place is a CH.Fig. 2 U.The aminoacid sequence of Ha1-1.152VL.The underscore place is a constant region of light chain.
Fig. 3.The Nucleotide of psca antibody and aminoacid sequence.Fig. 3 A.The cDNA of Ha1-4.117VH and aminoacid sequence.The underscore place is a CH.Fig. 3 B.The cDNA of Ha1-4.117VL and aminoacid sequence.The underscore place is a constant region of light chain.Fig. 3 C.The cDNA of Ha1-4.120VH and aminoacid sequence.The underscore place is a CH.Fig. 3 D.The cDNA of Ha1-4.120VL and aminoacid sequence.The underscore place is a constant region of light chain.Fig. 3 E.The cDNA of Ha1-5.99VH and aminoacid sequence.The underscore place is a CH.Fig. 3 F.The cDNA of Ha1-5.99VL and aminoacid sequence.The underscore place is a constant region of light chain.Fig. 3 G.The cDNA of Ha1-4.121VH and aminoacid sequence.The underscore place is a CH.Fig. 3 H.The cDNA of Ha1-4.121VLc.5 and aminoacid sequence.The underscore place is a constant region of light chain.Fig. 3 I.The cDNA of Ha1-4.121VLc.26 and aminoacid sequence.The underscore place is a constant region of light chain.Fig. 3 J.The cDNA of Ha1-1.16VH and aminoacid sequence.The underscore place is a CH.Fig. 3 K.The cDNA of Ha1-1.16VL and aminoacid sequence.The underscore place is a constant region of light chain.Fig. 3 L.The cDNA of Ha1-4.5VH and aminoacid sequence.The underscore place is a CH.Fig. 3 M.The cDNA of Ha1-4.5VL and aminoacid sequence.The underscore place is a constant region of light chain.Fig. 3 N.The cDNA of Ha1-4.40VH and aminoacid sequence.The underscore place is a CH.Figure 30.The cDNA of Ha1-4.40VL and aminoacid sequence.The underscore place is a constant region of light chain.Fig. 3 P.The cDNA of Ha1-4.37VH and aminoacid sequence.The underscore place is a CH.Fig. 3 Q.The cDNA of Ha1-4.37VL and aminoacid sequence.The underscore place is a constant region of light chain.Fig. 3 R.The cDNA of Ha1-1.43VH and aminoacid sequence.The underscore place is a CH.Fig. 3 S.The cDNA of Ha1-1.43VL and aminoacid sequence.The underscore place is a constant region of light chain.Fig. 3 T.The cDNA of Ha1-1.152VH and aminoacid sequence.The underscore place is a CH.Fig. 3 U.The cDNA of Ha1-1.152VL and aminoacid sequence.The underscore place is a constant region of light chain.
Fig. 4.Psca antibody with plant be the V-D-J sequence comparison.Fig. 4 A.The comparison of Ha1-4.117VH (SEQ IDNO:13) and people VH4-31.Fig. 4 B.The comparison of Ha1-4.117VL (SEQ ID NO:14) and people L19.Fig. 4 C.The comparison of Ha1-4.120VH (SEQ ID NO:15) and people VH4-31.Fig. 4 D.Ha1-4.120VL (SEQID NO:16) and people's 02 comparison.Fig. 4 E.The comparison of Ha1-5.99VH (SEQ ID NO:17) and people VH4-34.Fig. 4 F.The comparison of Ha1-5.99VL (SEQ ID NO:18) and people A27.Fig. 4 G.The comparison of Ha1-4.121VH (SEQID NO:19) and people VH4-34.Fig. 4 H.Ha1-4.121c.5VL (SEQ ID NO:20) and people's 08 comparison.Fig. 4 I.The comparison of Ha1-4.121c.26VL (SEQ ID NO:21) and people A3.Fig. 4 J.The comparison of Ha1-1.16VH (SEQ ID NO:22) and people VH6-1.Fig. 4 K.The comparison of Ha1-1.16VL (SEQ ID NO:23) and people B3.Fig. 4 L.The comparison of Ha1-4.37VH (SEQ ID NO:28) and people VH4-31.Fig. 4 M.Ha1-4.37VL (SEQ ID NO:29) and people's 02 comparison.
Fig. 5.The proteic expression of PSCA among recombined small-mouse, rat and the human cell line.Shown mouse, rat and human cell line are with being loaded with the retroviral infection of people PSCA cDNA and neomycin resistance gene or with the contrast virus infection that only is loaded with neomycin resistance gene.The stable recombinant cell lines of screening in the presence of G418.Adopt 1G8 anti--the painted FACS of PSCA MAb (5 μ g/ml) measures the expression of PSCA.Be depicted as the FACS distribution plan in each clone, show that the fluorescence drift only exists in the clone that infected by PSCA, PSCA is at cell surface expression in indication.These clones can be used as immunogenic, MAb screening reagent in MAb is open, and are used in the functional analysis.
Fig. 6.Available from the proteic purifying of colibacillary PSCA.With the amino acid whose pET-21b carrier of coding PSCA cDNA21-94 transformed into escherichia coli bacterial strain BL21 pLysS.Through inducing logarithmic phase to cultivate with IPTG to express PSCA albumen, and from the solubility of cracking bacterium or insoluble part with this albumen of affinity chromatography purifying.Be depicted as the SDS-PAGE Coomassie blue stain gel of wash-out part.This protein can be used as MAb and pAb immunogen and is used as antibody screening reagent.
Fig. 7.The proteic purifying of recombinant glycosylated PSCA by the 293T cell expressing.With being loaded with the amino acid whose psec Tag2 of coding PSCA cDNA28-100 carrier transfection 293T cell.Set up stable reorganization PSCA-secretor type clone through carry out drug screening with HYG.Affinity chromatography purifying through adopting 1G8MAb is present in the PSCA albumen in the conditioned medium.Be depicted as the Coomassie blue stain SDS-PAGE gel of low pH wash-out part.The proteic glycosylation of reorganization PSCA has been indicated in observed wide molecular weight protein hangover in the PSCA of endogenous expression.
Fig. 8.Available from the proteic purifying of colibacillary GST-PSCA.Be blended in the amino acid whose pGEX-2T transformed into escherichia coli of the 18-98 bacterial strain BL21 DE3 of the PSCA of glutathione-S-transferase (GST) with coding.In the logarithmic phase culture, induce GST-PSCA albumen, and from the cracked bacterium, come this albumen of purifying with gsh VISOSE matrix affinity chromatography with isopropyl-(IPTG).Be depicted as the SDS-PAGE Coomassie blue stain gel of the gsh wash-out part that contains GST-PSCA.Show the degraded product that has complete GST-PSCA fusion rotein and contain GST on a small quantity.This protein can be used as MAb and pAb immunogen and is used as the Ab screening reagent.
Fig. 9.Adopt people's psca antibody screening of FACS.Adopt ELISA to measure AC in the supernatant.(pure) 50 μ l/ holes are added in the 96 hole FACS plates, and carry out serial dilution.Add the cell (endogenous or reorganization, 50,000 cells/well) of expressing PSCA, under 4 ℃, hatched this mixture 2 hours.After hatching,, (anti--as to hatch 45 minutes in hIgG-PE) in detecting antibody at 100 μ l under 4 ℃ then with FACS damping fluid washed cell.After hatching end, with FACS damping fluid washed cell, formaldehyde fixed is analyzed with FACScan.Adopt CellQuestPro software analysis data.Solid histogram is represented the data available from negative control cell, and hollow histogram is represented the data available from the PSCA positive cell.
Figure 10.PSCA MAb relative affinity grade through FACS mensuration.Under 4 ℃, with each psca antibody and SW780 cell (50,000 cells/well) incubated overnight (the MAb final concentration is 40nM-0.038pM) of 21 series dilution in 1: 2.After hatching end, washed cell detects antibody in anti--hIgG-PE and hatches jointly.After flush away unconjugated two resists, use the facs analysis cell, adopt CellQuest Pro software to draw the average fluorescent strength of each point.Adopt S type dose response (variable slope) formula, with Graphpad Prism computed in software avidity.Be to describe the representative facs analysis that PSCA MAb 4.121 combines titres shown in the figure.
Figure 11.The expression of mouse and macaque PSCA and the identification of adopting anti-people PSCA MAb in the 293T cell.With pCDNA3.1 carrier that is loaded with mouse PSCA cDNA, ape and monkey PSCA cDNA or empty carrier (neo) transient transfection 293T cell newly.After the transfection 2 days, collecting cell and choose anti-PSCA MAb Ha1-4.117 or mouse MAb 1G8 (5 μ g/ml) dyeing.Be depicted as the FACS distribution plan, shown that the MAbHa1-4.117 that is produced by people PSCA albumen is incorporated into the mouse and the ape and monkey PSCA albumen of in the 293T cell, expressing.Mouse 1G8MAb is incorporated into ape and monkey PSCA, but debond mouse PSCA.This result show can with the ability of the selected MAb of the antigenic cross-reaction of other species.The MAb of cross reaction can be used in the expression and toxicity research of those species.
Figure 12.PSCA internalization after hatching jointly with MAb 4.121.Under 4 ℃, PSCA MAb 4.121 and PC3-PSCA are hatched 90 minutes jointly, so that said antibodies is in said cell surface.Then cell is divided into two equal portions, under 37 ℃ (make antibody internalization) or 4 ℃ (no internalization contrasts), hatches.After hatching under 37 ℃ or 4 ℃, remove the residual PSCA MAb 4.121 that is incorporated into cell surface with pickling.Detection two impermeabilisations subsequently make the PSCA MAb 4.121 of internalization be able to detect with hatching.Adopt the facs analysis cell or under fluorescent microscope, observe.After hatching 2 hours under 37 ℃, about 30% PSCA MAb 4.121 is by internalization.
Figure 13.Psca antibody mediation saporin dependency in the psca expression cell is killed and wounded.In the time of first day, 96 orifice plates are gone in B300.19 cell (750 cells/well) inoculation.In each hole, added in second day the isopyknic indication that contains 2 * concentration one anti-with 2 times of excessive anti-people (Hum-Zap) that are connected with the saporin toxin or anti-goat (polyclonal antibody of goat-Zap) (and Advanced Targeting Systems, San Diego, CA).37 ℃ of following incubated cells 5 days.Incubation period, adds MTS (Promega), and continued to hatch 4 hours after finishing in each hole.Measure the OD of 450nM.Result among Figure 13 (A) is presented at the saporin dependent cellular cytotoxicity that has psca antibody HA1-4.121 and HA1-4.117 mediation in the B300.19-PSCA cell, in the non-specific IgG1 antibody of contrast, does not then have effect.It is anti-that result among Figure 13 (B) shows that adding can not be discerned the saporin bonded two of people Fc, can not the mediated cell toxic action.
Figure 14.The complement-mediated CDCC of PSCA MAb.With RHB damping fluid (RPMI 1640, GibcoLife Technologies, 20mM HEPES) dilution psca antibody (0-50 μ g/ml).The cell of B300.19-PSCA is expressed in washing in the RHB damping fluid, with 10 6The density of individual cell/ml suspends again.In typical test, (Can) cell suspension with 50 μ l joins in flat tissue culture 96 orifice plates rabbit complement serum that 50 μ l psca antibodies, 50 μ l are diluted simultaneously for Cedarlane, Ontario.Mixtures incubated 2 hours is to promote the lysis of complement-mediated in 37 ℃, the incubator of 5%CO2.Blue (Biosource Intl.Camarillo CA) adds in each hole, under 37 ℃, continues to hatch 4-5 hour with the Alamar of 50 μ l.With 96 hole photofluorometers excite at the 530nm place, the fluorescent value in each hole is read in 590nm place emission.The result shows that the psca antibody with IgG1 (HA1-4.121) or IgG2 isotype (HA1-5.99.1) rather than IgG4 isotype (HA1-6.46) can mediate the complement-dependent cracking of target cell.
Figure 15.Produce F (ab ') 2 fragments of MAb Ha1-4.121 through gastric pepsin digestion.With the 20mM sodium-acetate buffer (pH4.5) of the MAb Ha1-4.121 of 20mg with do not hatch the specified time with immobilized stomach en-(Pierce.Rockford IL).Remove the complete MAb and the Fc fragment of digestion through the albumin A chromatography.Be depicted as complete without digestion, the MAb that is not reduced, at the appointed time take out do not reduce equal portions through the material of digestion and the PAGE coomassie dyeing gel of going back raw sample that finally digests F (ab ') 2 products.
Figure 16.The anti-PSCA people MAb of reorganization that measures through flow cytometry is in the combination of PSCA.(16A) the expression constructs transfection 293T cell of usefulness anti-PSCA people MAb heavy chain of coding and light chain.Collect supernatant after 48 hours, analysis combines with PSCA's.(16B) the anti-PSCA people MAb of purifying and be used for the PSCA binding analysis from the hybridoma supernatant.The associativity of following test PSCA.PC3 parent or PC3-PSCA cell were hatched on ice 30 minutes with aforesaid anti-PSCA people MAbs.Washed cell, anti-people Ig was hatched on ice 30 minutes jointly with the PE bonded.Washed cell is analyzed with flow cytometry then.
Figure 17.Through the immunohistochemical methods method to the proteic detection of PSCA.Use antibody HA1-4.117 to detect PSCA albumen available from the expression in cancer patients's the tumor specimen.To be cut into 4 microns section through formalin fixed, paraffin-embedded tissue, place on the glass slide.To section dewax, aquation and reclaim solution (Antigen Retrieval Citra Solution again with antigen; BioGenex, 4600NorrisCanyon Road, San Ramon, CA, 94583) at high temperature handle.Under 4 ℃, section is incubated among the anti-psca antibody Ha1-4.117 of resorcinolphthalein bonded human monoclonal 16 hours then.The said slide glass of washing is 3 times in damping fluid, hatches jointly 1 hour with the anti-resorcinolphthalein of rabbit again, in damping fluid, after the washing, is immersed in DAKO EnVision+ TMPeroxidase-conjugated goat anti-rabbit immunoglobulin two anti-(DAKOCorporation, Carpenteria, CA) in 30 minutes.Washing slice in damping fluid develops with DAB test kit (SIGMA Chemicals) then, uses haematoxylin redyeing, adopts bright visual field microscopy to analyze.The result shows that PSCA expresses in the tumour cell of prostate cancer (figure A, figure B), transitional cell carcinoma of bladder (figure C) and pancreas duct adenocarcinoma (figure D).These results show that PSCA expresses in human cancer, anti-this antigenic antibody can be used as diagnostic reagent.
Figure 18.PSCA MAb Ha1-4.120 is to the inhibition of subcutaneous prostate cancer heterograft growth.With LAPC-9AI tumour cell (2.0 * 10 6Individual cell) it is subcutaneous to be injected into male SCID mouse.With mouse random packet (n=10 in each group), as indicated in the time of the 0th day, through peritoneal injection (i.p.) HA1-4.120 or isotype MAb contrast begin treatment.Weekly animal is carried out 2 treatments, administration was 7 times altogether, until the 28th day that studies.Detected growth of tumor with caliper in every as indicated 3-4 days.The result shows that the anti-PSCA monoclonal antibody of people Ha1-4.120 has suppressed the growth (p＜0.05) of the human prostata cancer heterograft of subcutaneous implantation in the SCID mouse significantly.
Figure 19.The inhibition that PSCA MAb Ha1-5.99 grows to the prostate cancer heterograft of setting up in the SCID mouse.With LAPC-9AI tumour cell (2.0 * 10 6Individual cell) it is subcutaneous to be injected into male SCID mouse.When gross tumor volume reaches 50mm 3The time, with mouse random packet (n=10 in each group), peritoneal injection (i.p.) HA1-5.99.1 or isotype MAb contrast begin treatment as indicated.Weekly animal is carried out 2 treatments, administration was 5 times altogether, until the 14th day that studies.Detected growth of tumor with caliper in every as indicated 3-4 days.The result shows that the anti-PSCA monoclonal antibody of total man Ha1-5.99 has significantly suppressed the growth (p＜0.05) of non-androgen-dependent human prostata cancer heterograft of the foundation of subcutaneous implantation SCID mouse.
Figure 20.The inhibition that PSCA MAb HA1-4.121 grows to the androgen-dependent human prostata cancer heterograft of setting up.With LAPC-9AD tumour cell (2.5 * 10 6Individual cell) it is subcutaneous to be injected into male SCID mouse.When gross tumor volume reaches 40mm 3The time, with mouse random packet (n=10 in each group), the HA1-4.121 of peritoneal injection (i.p.) high density or isotype MAb contrast begin treatment as indicated.Weekly animal is carried out 2 treatments, administration was 7 times altogether, until the 21st day that studies.Detected growth of tumor with caliper in every as indicated 3-4 days.The result of this research shows that Ha1-4.121 has suppressed the growth of people's androgen-dependent prostate cancer heterograft of the foundation in the subcutaneous implantation SCID mouse.The result on statistics for being significantly: the 14th, 17 and 21 days (p＜0.05 of 300 μ g dose groups; The Kruskal-Wallis check; Two tail α=0.05) and 700 μ g dose groups the 10th, 14,17 and 21 days (p＜0.05, Kruskal-Wallis check, two tail α=0.05).
Figure 21.Will be available from patient's androgen-dependent LAPC-9AD tumour cell (2.0 * 10 6Individual cell) is injected into the prostatic episternites of male SCID mouse.Made tumor growth about 10 days, mouse is carried out random packet.Contrast begin treatment with 500 μ g people HA1-4.117, HA1-4.121 or isotype at implantation tumour after 10 days.Send antibody through intraperitoneal 2 times weekly, administration is 7 times altogether.Last administration was put to death animal after 4 days, and the taking-up primary tumo(u)r is also weighed.The result shows that the anti-PSCA monoclonal antibody of people Ha1-4.121 (p＜0.01) and Ha1-4.117 (p＜0.05) have significantly suppressed the growth that normal position (orthotopically) is implanted into the LAPC-9AD prostate cancer heterograft of SCID mouse.
Figure 22.PSCA MAb HA1-4.121 is to the prolongation of SCID mouse survival time of having normal position people's androgen-dependent tumor of prostate of having set up.Will be available from patient's androgen-dependent LAPC-9AD tumour cell (2.0 * 10 6Individual cell) is injected in the prostatic episternites of male SCID mouse.Made tumor growth about 9 days, mouse is carried out random packet.The animal that is randomized into survival group comprises 11 mouse of isotype MAb contrast and 12 mouse of HA1-4.121 treatment group.2 usefulness 1000 μ g Ha1-4.121 or 1000 μ g isotype MAb contrast is treated animal through intraperitoneal weekly, and administration is 9 times altogether.The result shows that HA1-4.121 remarkable (logarithm hierarchical verification: p＜0.01) has prolonged the survival time of the SCID mouse that has people's androgen-dependent tumor of prostate.Treated the last time back 110 days, 2 mouse in the HA1-4.121 treatment group keep the state of no palpable tumour.
Figure 23.HA1-4.21 and TX (taxotere) combination therapy strengthens the tumor of prostate Growth Inhibition.With LAPC-9AI tumour cell (2 * 10 6Individual cell/animal) male SCID mouse is gone in subcutaneous injection.When gross tumor volume reaches 65mm 3The time, as indicated animal is weaved into 4 different groups (n=10 in each group) at random.In the time of the 0th day, begin weekly 2 dosage intraperitoneal with 500 μ g and give Ha1-4.121 or isotype MAb contrast, administration is 6 times altogether.In the time of the 17th day, give last potion.Dosage with 5mg/kg in the time of the 0th, 3 and 7 day gives TX through intravenously.Detected growth of tumor with caliper in every 3-4 days.The result of this research shows with single the treatment with control antibodies and compared in the 28th day, and Ha1-4.121 reaches 45% (ANOVA/Tukey check: p＜0.05) as the medicament of single usefulness to the growth-inhibiting of non-androgen-dependent prostate cancer heterograft in the SCID mouse.Compare with giving the control antibodies treatment separately, give isotype MAb contrast and add that TX reaches 28% to the inhibition of tumor growth, does not have the significance on the statistics.Compare with independent control antibodies, unite and give HA1-4.121 and TX and have the enhanced effect and make the inhibition of tumor growth reach 69% (ANOVA/Tukey check: p＜0.01).When HA1-4.121 is added that with HA1-4.121 or isotype MAb contrast the TX group is compared with the combined group of TX, all demonstrated (the ANOVA/Tukey check: p＜0.05) of the significant difference on the statistics.
Figure 24.The inhibition that people PSCA MAb grows to the carcinoma of the pancreas heterograft in SCID mouse/people HPAC.With pancreatic cancer cell (2 * 10 6Individual/mouse) subcutaneous injection go into immunodeficiency type ICR SCID mouse (TaconicFarm, Germantown, NY).With mouse random packet (n=10 animal/group), in using specified people PSCA monoclonal antibody begin treatment on the same day.Send antibody (500mg/ mouse) through intraperitoneal 2 times weekly, administration is 8 times altogether.The result shows that the anti-PSCA monoclonal antibody of people Ha1-4.121, Ha1-4.117 and Ha1-1.16 have significantly suppressed to be implanted in the growth of the subcutaneous human pancreas cancer heterograft of SCID mouse.Use t check carrying out statistical analysis (two tails, α=0.05).
Figure 25.PSCA MAb HA1-4.121 plants the inhibition of pancreas tumor growth to SCID mouse Chang Weiyi.With HPAC cell (3.0 * 10 6Individual cell) Chang Weiyi implants in the pancreas of SCID mouse.Be 3 groups (n=9 of every group) with the mouse random assignment as indicated.Transplanting the same day with HA1-4.121 (250 μ g or 1000 μ g) or isotype MAb contrast (1000 μ g) begin treatment.2 intraperitoneal give antibody weekly, and administration is 10 times altogether.Last potion was put to death animal after 13 days, and the taking-up primary tumo(u)r is also weighed.The result of this research shows that the HA1-4.121 of two kinds of dosage levels being tested has all significantly suppressed the normal position growth of human pancreas cancer heterograft in the SCID mouse.250 μ g and 1000 μ g AGS-PSCA have suppressed tumor growth respectively and have reached 66% and 70% (Kruskal-Wallis/Tukey check: be respectively p＜0.01 and p＜0.01).
Figure 26.PSCA MAb HA1-4.121 is to the inhibition of MET.In necrotomy, in control antibodies treatment group, observe the MET of visible to lymphoglandula and far-end organ.In two HA1-4.121 treatment groups, all do not observe the visible MET.From all animals, take out lymphoglandula, lung and liver, the existence of dividing a word with a hyphen at the end of a line property of histology tumour.The personnel selection cytokeratin dyes to the section of taking from each animal lung and lymphoglandula, and microscopically is confirmed the quantity of MET.The histologic analysis result shows that lymphoglandula (LN) MET in the animal of treating with HA1-4.121 significantly reduces (p=0.0152 is recorded by the check of Fishers particularity).The incidence that shifts and invade also significantly reduces (p=0.0152 is recorded by the check of Fishers particularity) in the animal with two kinds of concentration HA1-4.121 treatments.Only the lung MET quantity with the HA1-4.121 of 1.0mg dosage treatment mouse significantly reduces (p=0.0498 is recorded by the check of Fishers particularity).
Figure 27.The inhibition that people PSCA MAb grows to the SW780 tumor of bladder in the SCID mouse.With people SW780 transitional cell bladder carcinoma cell line (2 * 10 6Individual/mouse) subcutaneous injection go into immunodeficiency type ICR SCID mouse (TaconicFarm, Germantown, NY).With mouse random packet (n=10 animal/group), the people PSCA MAb begin treatment shown in use on the same day.2 intraperitoneal are sent antibody (250mg/ mouse) weekly, and administration is 7 times altogether.The result shows that HA1-4.117 (p=0.014), HA1-4.37 (p=0.0056), HA1-1.78 (p=0.001), Ha1-5.99 (p=0.0002) and HA1-4.5 (p=0.0008) have significantly suppressed the growth of the SW780 tumor of bladder of subcutaneous implantation in the SCID mouse.With t check carrying out statistical analysis (two tails, α=0.05).
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 genetic abnormality
II.A.2.) antisense instance
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 GAP-associated protein GAP
III.A.) have the proteinic instance of motif
III.B.) expression of PSCA GAP-associated protein GAP
III.C.) modification of PSCA GAP-associated protein GAP
III.D.) purposes of PSCA GAP-associated protein GAP
IV.) psca antibody
V.) PSCA cellullar immunologic response
VI.) PSCA transgenic animal
VII.) method of detection PSCA
VIII.) method of monitoring PSCA-genes involved state 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 as the target of antibody therapy
X.C.) PSCA is as the target of cellullar immunologic 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.) from the vaccine inoculation of treating or prevent purpose
XI.) diagnosis of PSCA and prognosis embodiment
XII.) suppress the proteic function of PSCA
XII.A.) suppress PSCA with intracellular antibody
XII.B.) suppress PSCA with recombinant protein
XII.C.) suppressing PSCA transcribes or translates
XII.D.) the overall consideration of treat-ment
XIII.) evaluation of PSCA regulator, specificity analysis and purposes
XIV.) therepic use of RNAi and siRNA (siRNAs)
XV.) production of test kit/goods
I.) definition:
Except as otherwise noted, all T.T.s used herein, symbol and other scientific term or term have the conventional implication of understanding of those of skill in the art of the affiliated technical field of the present invention.Under the certain situation, have the conventional term of understanding here by definition so that clarification and/or be used for reference, this paper this definition implication that should not be interpreted as the common sense of it and this area essential distinction is arranged.Here the many technology describing or quote and method be understand easily and can use ordinary method to carry out by being proficient in those skilled in the art usually; " molecular cloning laboratory manual " (Molecular Cloning:A Laboratory Manual) (second edition that for example extensively adopts at Sambrook etc.; 1989; Cold SpringHarbor Laboratory Press, cold spring port, New York) described in molecular cloning.Except as otherwise noted, use commercial reagent box and compositions and methods to carry out according to the process and/or the parameter of manufacturers's regulation usually.
Term " advanced prostate cancer ", " local advanced prostate cancer ", " terminal illness " and " local terminal illness " are meant expands the prostate cancer that breaks through capsula prostatica, comprises the C phase disease of American Urological Association (AUA) system definition, the C1-C2 phase disease of Whitmore-Jewett system definition and the T3-T4 phase and the N+ phase disease of TNM (tumour, tubercle, transfer) system definition.Usually do not recommend local terminal illness patient is undergone surgery, compare clinical limitation (organ limitation) patients with prostate cancer, these patients' surgical outcome is all bad basically.Can confirm local terminal illness through the asymmetric or sclerosis in palp sclerosis or prostate gland bottom is arranged on the prostatic lateral border clinically.If tumour is invaded or is seen through capsula prostatica, expand to the operation edge or invade seminal vesicle, local advanced prostate cancer is carried out pathological examination through radical prostatectomy at present and is made diagnosis.
" change Natively glycosylated pattern " and be meant one or more sugar moieties (through removing the potential glycosylation site or deleting glycosylation site) that deletion is found here on natural PS CA sequence, and/or add non-existent glycosylation site in one or more natural PS CA sequences through chemistry and/or enzyme method.In addition, this phrase comprises that the glycosylated character of natural protein changes, and comprises character and the change of ratio of the various sugar moieties of existence.
Term " analogue " is meant and another molecule (for example PSCA GAP-associated protein GAP) similar or the molecule that has similar or respective attributes jointly.For example, the proteic analogue of PSCA can be combined the antibody of PSCA or T cell-specific to combine by specificity.
Except as otherwise noted, term " antibody " uses with broad sense.Therefore, " antibody " can be natural generation or synthetical, like the monoclonal antibody of making through conventional hybridization knurl technology.Anti--psca antibody comprises mono-clonal and polyclonal antibody, and the fragment that contains antigen binding domain and/or one or more complementary determining regions of these antibody.Term used herein " antibody " is meant that specificity combines PSCA and/or has required bioactive any type of antibody or its fragment; This definition (has for example specifically covered monoclonal antibody (comprising full length monoclonal antibodies), polyclonal antibody, multi-specificity antibody; Bi-specific antibody) and antibody fragment, as long as their specificitys combine PSCA and/or show required biological activity.Can use any specific antibody in the method and composition that this paper provided.Therefore, in one embodiment, term " antibody " comprises following molecule: this molecule comprises at least one variable region of the light chain Tegeline that mutually combines and at least one variable region of heavy chain molecule, to form the specific binding site to target antigen.In one embodiment, said antibody is IgG antibody.For example, said antibody is IgG1, IgG2, IgG3 or IgG4 antibody.The antibody that is used for the inventive method and compsn can produce cell culture, phage or various animal, and described animal includes but not limited to: ox, rabbit, goat, mouse, rat, hamster, cavy, sheep, dog, cat, monkey, orangutan, ape.Therefore, in one embodiment, antibody of the present invention is Mammals antibody.The variant that can adopt phage technology separation primary antibody or generation to have the specificity or the avidity characteristic of change.These technology are conventional, and are well-known in the art.In one embodiment, said antibody is to adopt recombination method known in the art to produce.For example, can produce recombinant antibodies through carrier transfection host cell with the dna sequence dna that comprises encoding antibody.The dna sequence dna in available one or more carriers at least one VL of transfection expression and VH district in host cell.Antibody takes place and the exemplary description of production recombination method comprises: Delves, ANTIBODIES PRODUCTION:ESSENTIALTECHNIQUES (Wiley, 1997); Shephard etc., MONOCLONAL ANTIBODIES (OxfordUniversity Press, 2000); Goding, MONOCLONAL ANTIBODIES:PRINCIPLES ANDPRACTICE (Academic Press, 1993); CURRENT PROTOCOLS IN IMMUNOLOGY (JohnWiley & Sons, nearest version).Can modify antibody of the present invention through recombination method, thereby improve the bigger effect of this antibody in the mediation required function.Therefore, adopt recombination method to modify antibody of the present invention also within the scope of the invention through replacing.Usually, said replacement is conservative the replacement.For example, available different residue replaces at least one amino acid in the antibody constant region.Referring to, for example USP 5,624, and 821, USP 6,194,551, patented claim WO 9958572; And Angal etc., Mol.Immunol.30:105-08 (1993).Amino acid whose modification is comprised amino acid whose disappearance, insertion, replacement.In some cases, carry out these variations and be in order to reduce bad activity, for example, CDC.Regular antibody covalently or non-covalently is connected with the substrate that detectable signal is provided.The mark of known many types and interconnection technique, science and technology and patent documentation have been made report widely to them.Can screen these antibody to be used to combine normal or defective PSCA.Referring to for example, ANTIBODY ENGINEERING:APRACTICAL APPROACH (Oxford University Press, 1996).Can discern through following analyzed in vitro method and have required bioactive suitable antibody; These analytical methods include but not limited to: breed, move, stick, soft agar growth, vasculogenesis, cell-cell communication, apoptosis, transhipment, signal transduction; And with lower body inner analysis method, for example tumor growth suppresses.The antibody that this paper provides also can be used for diagnostic uses.As catching or nonneutralizing antibody, can their binding specificity antigen not suppressed receptors bind or this antigenic bioactive ability is screened.As neutralizing antibody, said antibody can be used for competitive binding analysis.They also can be used for the quantitative of PSCA or its acceptor.
" antibody fragment " is defined as the variable region of part at least with its target molecule bonded immunoglobulin molecules, i.e. antigen binding domain.In one embodiment, it particularly including single anti--psca antibody and clone's (comprising excitement, antagonism and neutralizing antibody) thereof with have that multi-epitope is specific to be resisted-the psca antibody compsn.Antibody in the method and composition of this paper can be monoclonal antibody or polyclonal antibody.Antibody can be antigen binding antibody fragment, and said antibody fragment comprises Fab fragment, F (ab ') 2 fragments, strand variable region etc.Can adopt method well known in the art to produce the fragment of complete molecule, comprise enzymic digestion and recombination method.
As used herein, can use any type of " antigen " to produce specific antibody to PSCA.Therefore, induced antigen (eliciting antibody) can be the former epi-position of monoclonal antibody, many epitopes use separately or with the whole protein of one or more immunogenicity toughener couplings known in the art.This induced antigen can be isolating full length protein, cell surface protein (for example, carrying out immunity with antigenic at least a portion cells transfected) or soluble proteins (for example, only using protein extracellular outskirt partial immunity).Can in the cell of genetically engineered transformation, produce said antigen.Encode this antigenic DNA can be genomic or extragenic (for example, cDNA) with the part extracellular region at least of encoding.Term used herein " part " is meant appropriate minimum number amino acid or the nucleic acid that is used to make up immunogenicity of antigens epitope interested.Can adopt any Genetic carrier that is suitable for transforming cells of interest, include but not limited to: adenovirus carrier, plasmid and non-virus carrier, for example cationic-liposome.In one embodiment, the antibodies specific in the method and composition of this paper combines at least a portion of PSCA extracellular region interested.
Antibody that this paper provides or antigen and binding fragment thereof can be incorporated into " biologically active agent ".Term used herein " biologically active agent " is meant conjugated antigen and/or promotion or mediates required biological effect to strengthen the toxic any synthetic or naturally occurring compound of cell killing.
In one embodiment, being used for binding fragment of the present invention is biological active fragment.Term used herein " BA " is meant the antibody or the antibody fragment that can combine required epitope and directly or indirectly give biological effect.Directly effect includes but not limited to: regulate, stimulate and/or the inhibition growth signals; Regulate, stimulate and/or suppress anti-apoptotic signal; Regulate, stimulate and/or inhibition apoptosis or downright bad signal; Regulate, stimulate and/or suppress ADCC cascade reaction and adjusting, stimulation and/or suppress the CDC cascade reaction.
In method and composition of the present invention, also can use " dual specific " antibody.Term used herein " bi-specific antibody " is meant the antibody that at least two kinds of different antigens epi-positions is had binding specificity, is generally monoclonal antibody.In one embodiment, said epi-position is from same antigen.In another embodiment, said epi-position is from two different antigens.The method for preparing bi-specific antibody is known in the art.For example, can produce bi-specific antibody to the method for coexpression through making two kinds of heavy chain immunoglobulin/light chains.Referring to, for example, Milstein etc., Nature 305:537-39 (1983).Perhaps, available chemical connection method prepares bi-specific antibody.Referring to, for example, Brennan etc., Science 229:81 (1985).Bi-specific antibody comprises bispecific antibody fragment.Referring to for example, Hollinger etc., Proc.Natl.Acad.Sci.U.S.A.90:6444-48 (1993), Gruber etc., J.Immunol.152:5368 (1994).
The monoclonal antibody of this paper specifically comprises the fragment of " chimeric " antibody and this antibody; As long as their specificitys combine target antigen and/or have required biological activity; Part heavy chain in this chimeric antibody and/or light chain with derived from specific species or belong to the identical or homology of corresponding sequence of the antibody of antibodies specific family or subfamily; The rest part of chain then with derived from another specific species or belong to the identical or homology (USP 4 of corresponding sequence of the antibody of another antibody family or subfamily; 816,567; With Morrison etc., Proc.Natl.Acad.Sci.USA 81:6851-6855 (1984)).
Term " chemotherapeutics " is meant that all effectively suppress the chemical cpd of tumor growth.The non-limitative example of chemotherapeutics comprises: alkylating agent, for example mustargen, aziridine cpd 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 catharanthus alkaloid and podophyllotoxin derivative, cytotoxic antibiotics, destruction or interference DNA expression.In addition, chemotherapeutics comprises cytotoxic agent (as defined herein), antibody, biomolecules and small molecules.
Term " codon optimized sequence " is meant through displacement frequency of utilization in the particular types host and is lower than the nucleotide sequence that about 20% codon is optimized.Except that the codon optimization, gather the adenosine sequence through removing vacation, remove the exon splicing signal, remove swivel base increment Tumor-necrosis factor glycoproteins and/or optimize GC content and the nucleotide sequence of the expression of optimization in given host is called as " expression enhancement sequences " here.
" combinatorial library " is the set of many chemistry " member " (like reagent) being made up the different compounds that produce through chemosynthesis or biosynthesizing.For example, linear combination chemistry library like polypeptide (like mutain) library, is to be called synthetic given compound length (being amino acid whose number in the polypeptide compound) of amino acid whose chemical component sets and formation with all possible method with one group.Most compounds is the combined hybrid synthetic (Gallop etc., J.Med.Chem.37 (9): 1233-1251 (1994)) through this chemical member.
It is that the technician knows that this area is proficient in the preparation of combinatorial library and screening.This combinatorial chemistry library includes but not limited to; Peptide library (is seen for example USP the 5th; 010; No. 175; Furka, Pept.Prot.Res.37:487-493 (1991), Houghton etc.; Nature; 354:84-88 (1991)), type peptide (PCT announces WO 91/19735), encoded peptide (PCT announces WO 93/20242), biological oligomer (PCT announces WO 92/00091), benzodiazepine
Figure G05817239920061201D000211
(U.S. Patent number 5,288,514), diversomers such as NSC 9226, benzodiazepine
Figure G05817239920061201D000212
and dipeptides (Hobbs etc. at random; Proc.Nat.Acad.Sci.USA90:6909-6913 (1993)), vinylogous polypeptide (Hagihara etc.; J.Amer.Chem.Soc.114:6568 (1992)), have the peptide mimics (Hirschmann etc., J.Amer.Chem.Soc.114:9217-9218 (1992)) of the non-peptide of β-D-glucose support, the similar organic synthesis body library (Chen etc. of little compound; J.Amer.Chem.Soc.116:2661 (1994)), oligocarbarnate (Cho etc.; And/or peptide acyl phosphonic acid ester (Campbell etc., J.Org.Chem.59:658 (1994)) Science261:1303 (1993)).Usually visible Gordon etc., J.Med.Chem.37:1385 (1994), nucleic acid library (is seen for example Stratagene; Corp.), PNAG3 library (seeing for example USP 5,539,083), antibody library (are seen for example Vaughn etc.; Nature Biotechnology 14 (3): 309-314 (1996) and PCT/US96/10287), the carbohydrate library (sees for example Liang etc., Science 274:1520-1522 (1996) and U.S. Patent number 5,593; 853); And for example benzodiazepine
Figure G05817239920061201D000213
(is seen, Baum, C & EN in little organic molecule library; 1-18, the 33rd page (1993); Isoprenoid, U.S. Patent number 5,569,588; A thiazolidone (thiazolidinone) and a thiazan ketone (metathiazanone), U.S. Patent number 5,549,974; Tetramethyleneimine, U.S. Patent number 5,525,735 and 5,519,134; Morpholino compounds, U.S. Patent number 5,506,337; Benzodiazepine ; U.S. Patent number 5; 288,514; Or the like).
The device of preparation combinatorial library can (be seen for example 357NIPS, 390NIPS, Advanced Chem Tech, Louisville KY through commercial the acquisition; Symphony, Rainin, Woburn, MA; 433A, Applied Biosystems, Foster City, CA; 9050, Plus, Millipore, Bedford, NIA).Also develop many machine systems of knowing and solved phase chemistry (phase chemistry) problem.These systems comprise automatic workstation; Like Takeda Chemical Industries; LTD. (Osaka, Japan) automatic DNA synthesizer DNA of developing, and the machine system (Zymate H, the Zymark Corporation that adopt the robotic arm of the manual synthetic operation of simulated scholar; Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.).Above-mentioned any device all is applicable to the present invention.As far as being proficient in those skilled in the art, characteristic and the operation scheme of improving these devices (if necessary) can operate them by described herein.In addition, many combinatorial library self can (be seen for example ComGenex, Princeton, NJ through commercial the acquisition; Asinex, Moscow, RU; Tripos, Inc., St.Louis, MO; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, PA; Martek Biosciences, Columbia, MD; Or the like).
Term used herein " the conservative replacement " is meant aminoacid replacement well known by persons skilled in the art, and carries out the biological activity that this replacement can not change the gained molecule usually.Those skilled in the art recognizes usually that the single amino acids in the polypeptide nonessential region replaces not can the material alterations biological activity (referring to for example; Watson etc.; MOLECULAR BIOLOGY OF THE GENE; The Benjamin/Cummings Pub.Co., the 224th page (the 4th edition, 1987)).Preferably carry out these exemplary replacements according to described in the Table III (a-b).For example, these changes can comprise with Isoleucine (I), Xie Ansuan (V) and leucine (L) and replace other these hydrophobic amino acids; Replace L-glutamic acid (E) with aspartic acid (D), vice versa; Replace l-asparagine (N) with glutamine (Q), vice versa; Replace Threonine (T) with Serine (S), vice versa.Other replacement also can be considered to conservative property, and this depends on the residing environment of concrete amino acid and its effect in this protein three-dimensional structure.For example, glycocoll (G) and L-Ala (A) are often interchangeable, and be just also interchangeable like L-Ala (A) and Xie Ansuan (V).Hydrophobic relatively methionine(Met) (M) Chang Keyu leucine and Isoleucine exchange, and also can exchange with Xie Ansuan sometimes.Methionin (K) and l-arginine (R) are that the difference pK of its electric charge and this two seed amino acids residue is often not interchangeable on the remarkable position in the notable feature of amino-acid residue.Other variation that under particular environment, can be considered to " conservative property " in addition is (referring to the Table III (a) of for example this paper; " Biochemistry " the 2nd edition 13-15 page or leaf.Lubert Stryer compiles (StanfordUniversity); Henikoff etc., PNAS 1992, volume 89,10915-10919; Lei etc., J Biol Chem1995 May 19; 270 (20): 11882-6).Other replacement also is allowed to, and can determine by rule of thumb or according to known conservative replacement.
Term " cytotoxic agent " is meant the material that suppresses or stop cell expressing activity, cell function and/or cause cytoclasis.This term comprises the ri chemotherapeutic, and toxin, and small molecules toxin or enzyme activity toxin like bacterium, fungi, plant or animal-origin comprise its fragment and/or variant.The example of cytotoxic agent includes but not limited to Jin Tating (auristatin), duomycin, maytansinol (maytansinoid), yttrium, bismuth, ricin, ricin A-chain, Kang Letading (combrestatin), a Ka-7038 (duocarmycin), many Luo Tating (dolostatins), Zorubicin, daunorubicin, taxol, cis-platinum, cc 1065, ethidium bromide, MTC, etoposide, VM-26, vincristine(VCR), vinealeucoblastine(VLB), NST-757, dihydroxyl anthracin diketone, NSC-3053, diphtheria toxin, PE (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α-sarcina, gelonin, mitogillin, restrictocin (retstrictocin), phenomycin, enomycin, Curie's rhzomorph (curicin), crotin, calicheamycin, Sapaonaria officinalis suppressor factor and glucocorticosteroid and other chemotherapeutic; And ri; Ri like At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32 and Lu comprises Lu177.Antibody also can with the anticancer prodrug activating enzymes coupling that can prodrug be changed into its activity form.
Term used herein " binary " is meant the little antibody fragment with two antigen binding sites, and this fragment packet is contained in the variable region of heavy chain (VH) that is connected in variable region of light chain (VL) in the same polypeptied chain (VH-VL).Be as short as through use and can not make two interregional paired joints in the same chain, said zone compelled with another chain in the complementary region pairing, and produced two antigen binding sites.Binary has in following document more comprehensively to be described: for example, and European patent 404,097; WO 93/11161; With Hollinger etc., Proc.Natl.Acad.Sci.USA 90:6444-48 (1993).
" gene product " in this article refers to peptide/protein or mRNA.For example, " gene product of the present invention " is meant " aminoacid sequence of cancer ", " cancer protein ", " protein of the listed cancer of Table I ", " mRNA of cancer ", " mRNA of the listed cancer of Table I " or the like in this article sometimes.In one embodiment, cancer protein is by the nucleic acid encoding of Fig. 1.Said cancer protein can be a fragment, perhaps can be the fragment by the full length protein of the nucleic acid encoding of Fig. 1.In one embodiment, the cancer aminoacid sequence is used to confirm the homogeny or the similarity of sequence.In another embodiment, this sequence is to have proteinic allelic variant by the nucleic acid encoding of Fig. 1 natural.In another embodiment, this sequence is the sequence variants that this paper further describes.
In method and composition of the present invention, can use " allos coupling " antibody.Term used herein " allos coupling antibody " is meant two covalently bound antibody.Known method prepares these antibody in the available synthetic protein chemistry, comprises the use linking agent.Referring to for example, USP 4,676,980.
Be used for measuring specific nucleic acid or protein existence, do not exist, quantitatively or " high flux screening " of other character test be proficient in well-known to those having ordinary skill in the art.Similarly, also know in conjunction with test and reporter gene test.Therefore, for example U.S. Patent number 5,559, and 410 have disclosed proteinic high flux screening method; U.S. Patent number 5,585,639 have disclosed detection nucleic acid bonded high flux screening method (promptly using array detection); And U.S. Patent number 5,576,220 and 5,541,061 has disclosed the high flux screening method of detector ligand/antibodies.
In addition, high throughput screening system can (be seen for example Amersham Biosciences, Piscataway, NJ through commercial the acquisition; Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc.Fullerton, CA; Precision Systems, Inc., Natick, MA; Or the like).These systems accomplish all processes usually automatically, comprise that the absorption of all samples and reagent, liquid distribute, timing is cultivated and are being fit to read microplate on the detector that detects at last.But these compounding systems provide high-throughput and can start fast, and are high flexible and custom IC.The manufacturers of this system provides the detailed protocol explanation of various high throughput system.Therefore, for example Zymark Corp. provides and has described the technical specification pph that is used for detecting the screening system that genetic transcription, part combination etc. is regulated.
Term " homologue " is meant through for example have chemical property sequence identical or similar residue in the corresponding position and shows and another molecule homologous molecule.
In one embodiment, the antibody that provides of this paper is " people's antibody ".The whole sequence that term used herein " people's antibody " is meant light chain and sequence of heavy chain (comprising complementary determining region (CDR)) is basically all from the antibody of people's gene.In one embodiment, through trioma technology, human B cell technological (referring to for example, Kozbor etc.; Immunol.Today 4:72 (1983)); EBV transformation technology (referring to for example, Cole etc., MONOCLONAL ANTIBODY AND CANCER THERAPY 77-96 (1985)) or use phage display (referring to for example; Marks etc., J.Mol.Biol.222:581 (1991)) the preparation human monoclonal antibodies.In a certain concrete embodiment, said people's antibody produces in transgenic mice.The technology for preparing these groups of people's antibody to total length people antibody is well known in the art, and any this type of technology all can be used.According to one preferred embodiment, through engineered with the transgenic mice of expressing human heavy chain and light chain antibody gene in preparation total length human antibody sequence.Patented claim WO 02/43478 and USP 6,657,103 (Abgenix) and subsequent application thereof are seen in an exemplary description of preparation transgenic mice.Can merge the transgenic mice B cell source, that can produce required antibody then to prepare the hybridoma cell line of continuous this antibody of generation.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 etc., J.Exp.Med.188:483-95 (1998).
" human leucocyte antigen (HLA) " or " HLA " be people I type or the main histocompatibility complex of II type (MHC) albumen (referring to for example, Stites etc., IMMUNOLOGY, the 8th edition, Lange Publishing, Los Altos, CA (1994).
Term used herein " humanized antibody " is meant the antibody formation that contains from the antibody sequence of inhuman (for example, mouse and people's antibody).These antibody are to contain the chimeric antibody that is derived from the non-human immunoglobulin minmal sequence.Usually; Said humanized antibody can comprise at least one whole basically with common two variable regions, in these variable regions corresponding to the whole of those non-human immunoglobulin or basically all the hypermutation ring with all or whole basically FR district be in the human normal immunoglobulin sequence those.Said humanized antibody also can randomly comprise the part at least of constant region for immunoglobulin (Fc), and said Fc is generally the Fc of human normal immunoglobulin.Referring to for example, the USP 4,816,567 of Cabilly; Queen etc. (1989) Proc.Nat ' l Acad.Sci.USA 86:10029-10033; With ANTIBODY ENGINEERING:A PRACTICAL APPROACH (Oxford University Press 1996).
Term " hybridization " etc. is meant the conventional hybridization condition when being used for polynucleotide; Preference is as hybridizing in 50% methane amide/6 * SSC/0.1%SDS/100mg/ml ssDNA; Wherein, hybridization temperature is higher than 37 ℃, uses the temperature of 0.1 * SSC/0.1%SDS washing to be higher than 55 ℃ simultaneously.
Phrase " isolating " or " biological pure " are meant basically or the composition of the material of following usually when not being contained in native state fully.Therefore, isolating peptide of the present invention preferably is not contained in the material of following this peptide in the natural surroundings usually.For example, a kind of polynucleotide when with psca gene outside the corresponding or complementary contaminative polynucleotide of the gene of group or the polypeptide outside coding psca gene product or its fragment then to be called when separating basically be " isolating ".Those skilled in the art can adopt the separate nucleic acid method to obtain isolating PSCA polynucleotide easily.For example, when adopting physics, machinery or chemical process to remove the protein of following usually in the PSCA albumen in the cellular constituent, then being called PSCA albumen is " isolating ".Those skilled in the art can adopt the standard purification method to obtain isolating PSCA albumen easily.Perhaps can prepare isolating protein through chemical process.
" mark " that suits comprises radionuclide, enzyme, substrate, cofactor, suppressor factor, fluorescence part, chemiluminescent moiety, magnetic-particle etc.The patent that the use of these marks is stated in religion comprises: USP 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; With 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 etc., Nat.Biotechnol.21:1473-79 (2003).
Term " Mammals " is meant any mammiferous biology that is categorized as, and comprises mouse, rat, rabbit, dog, cat, ox, horse and people.In one embodiment of the invention, said Mammals is a mouse.In another embodiment of the invention, said Mammals is the people.
Term " metastatic prostate cancer " and " metastatic disease " refer to be diffused into regional nodes or are diffused into the prostate cancer at position at a distance, comprise the D phase disease of AUA system and the TxNxM+ phase disease of TNM system.For local advanced prostate cancer case, the metastatic disease patient is not undergone surgery usually, and hormone (male sex hormone melts) therapy is preferred treat-ment.The metastatic prostate cancer patient finally can develop into male sex hormone refractory state in 12-18 month after the treatment beginning.Almost there is half can be after developing into this stage dead in 6 months among these male sex hormone refractory patients.The common site that prostate cancer shifts is a bone.The prostate cancer bone shifts normally skeletonization property rather than molten bone property (promptly causing os purum to form).Bone shifts the most common in backbone, is femur, basin then, rib frame, skull and humerus.Other common metastasis site comprises lymphoglandula, lung, liver and brain.Metastatic prostate cancer is diagnosed through surgical incision or peritoneoscope pelvic lymphadenectomy, body radioactivity radioisotope scanning, bone sctinography and/or the examination of living tissue of osteopathy kitchen range usually.
Term " regulator " or " detection compound " or " drug candidates " or phraseological equivalent are described the ability with the expression with the phenotype of understanding direct or indirect change cancer or cancer sequence (for example nucleic acid or protein sequence) to be detected here; Or any molecule of the effect of cancer sequence (for example producing signal, genetic expression, protein interaction etc.), for example protein, oligopeptides, little organic molecule, polysaccharide, polynucleotide etc.On the one hand, the regulator effect of cancer protein of the present invention that will neutralize." neutralization " be meant activity of proteins and subsequently the effect of pair cell be suppressed or check.On the other hand, regulator makes can the neutralize effect of gene of the present invention and respective egg white matter thereof of said protein level normalizing.In preferred embodiments, regulator changes expression characteristic, or nucleic acid that provides here or protein expression characteristic, or changes the sub-approach of downstream effect.In one embodiment, said regulator suppresses cancerous phenotype, for example healthy tissues fingerprint.In another embodiment, regulator is induced cancerous phenotype.Usually detect a plurality of mixtures to obtain difference responsing reaction with different reagent concentrations is parallel to different concns.Usually, be that zero-dose or the concentration that is lower than detection level are as negative control with one of these concentration.
Regulator, drug candidate or detection compound comprise the chemical reagent of plurality of classes, but their organic molecules normally, preferred molecular weight greater than 100 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 500D.Candidate agent comprises structurally essential with protein interaction functional group, is hydrogen bond specifically, and comprises at least one amido, carbonyl, hydroxyl or carboxyl usually, preferably at least two chemical functional groups.Said candidate agent comprises carbocyclic ring or heterocycle structure and/or usually by substituted aromaticity structure of one or more above-mentioned functional groups or aromaticity paradigmatic structure.Regulator also comprises following biomolecules, like peptide class, carbohydrate, lipid acid, steroid, purine, pyrimidine, verivate, analog or its combination.Particularly preferably be peptide.The regulator of one kind is a peptide, for example contains 5-35 the amino acid of having an appointment, and preferably contains 20 amino acid of having an appointment, and more preferably contains 7-15 the amino acid whose peptide of having an appointment.Preferred cancer modulatory protein is soluble, comprises that one is non-ly striden the film district and/or the terminal Cys of N-separates with hydrotropy.In one embodiment, segmental C-end be retained as free acid simultaneously the N-end be that free amine is to help coupling, promptly with the halfcystine coupling.In one embodiment, cancer protein of the present invention and immunogenic agents described here are puted together.In one embodiment, said cancer protein and BSA put together.Peptide of the present invention, the peptide of preferred length for example can mutually combine or combines to form long peptide/protein with other amino acid.This adjusting peptide can be digested the protein (as indicated above) of natural generation, peptide or " biasing (biased) " peptide at random at random.In an embodiment preferred, be antibody and the fragment thereof that defines here based on peptide/proteinic regulator.
The regulator of cancer also can be a nucleic acid.The nucleic acid regulator can be nucleic acid, random nucleic acid or the " biasing " random nucleic acid of natural generation.For example, the digestion product of protokaryon or eukaryotic gene group can be used in the above-mentioned proteinic corresponding analogs.
Term among this paper " monoclonal antibody " refers to available from a group antibody of homogeneous antibody basically, promptly comprises the identical antibody of a group but possibly have the antibody of the two mutants of a small amount of natural generation.Monoclonal antibody is a high degree of specificity, and it is directly to single epitope.On the contrary, conventional (polyclone) antibody preparation generally includes the multiple antibody to (or specificity is directed against) different epi-positions.In one embodiment, a plurality of monoclonal antibodies of said polyclonal antibody, the single antigen that they have containing a plurality of epitopes has different epitope specificities, avidity or avidity.Modifier " monoclonal " is meant that the proterties of said antibody is available from homologous antibody population basically, and is not to be interpreted as to produce this antibody through any special method.For example, be used for monoclonal antibody of the present invention and can adopt by Kohler etc., the hybridoma technology that Nature 256:495 (1975) describes first prepares, and maybe can pass through recombinant DNA method (referring to for example, USP 4,816,567) and prepare.Also can adopt for example Clackson etc., Nature 352:624-628 (1991) and Marks etc., the described technology of J.Mol.Biol.222:581-597 (1991) is isolated " monoclonal antibody " from phage antibody library.These monoclonal antibodies combine with the Kd at least about 1 μ M usually, more generally are at least about 300nM, are at least about 30nM usually, preferably are at least about 10nM, more preferably are at least about 3nM or better, measure through ELISA usually.
" motif "; Like any aminoacid profile of the biology motif middle finger constitutive protein matter primary sequence of a PSCA GAP-associated protein GAP part, this pattern is with certain concrete function (for example protein-protein interaction, protein-DNA interaction etc.) or modify (for example phosphorylation, glycosylation or amidation are modified) or location (for example secretion sequence, nuclear localization sequence etc.) or with to produce the sequence that body fluid or cellular immunity be correlated with relevant.Motif can be adjoin maybe can be arranged in usually certain position relevant with certain function or characteristic.In the HLA motif, " motif " is meant the pattern of residue in the peptide of confirming length, and as far as the normally about 8-13 of I class HLA motif amino acid whose peptide, as far as the normally about 6-25 of II class HLA motif amino acid whose peptide, it can be by specific HLA molecular recognition.Peptide motif by each combination of proteins HLA of separately people HLA allelotrope coding is different usually, and main (primary) is also different with the pattern of less important (secondary) anchor residues.Common motif is listed in the Table V.
" pharmaceutical excipient " comprises materials such as adjuvant, carrier, pH regulator agent and buffer reagent, tension regulator, wetting agent, sanitas.
" pharmaceutically acceptable " be meant nontoxic, inert and/or with the compsn of people or other Mammals physical compatibility.
Term " polynucleotide " expression length is at least the polymerized form of two kinds of Nucleotide types of ribonucleotide or the deoxynucleoside or the modified forms of 10 bases or base pair, refers to comprise strand and the double chain form of DNA and/or RNA.In the art, this term exchanges with " oligonucleotide " usually and uses.Polynucleotide can comprise the nucleotide sequence that discloses here, and as shown in Figure 1, thymus pyrimidine wherein (T) also can be a uridylic (U); This definition relates to the difference between DNA and the RNA chemical structure, and specifically, one of four main bases are uridylic (U) rather than thymus pyrimidine (T) among the RNA.
Term " polypeptide " expression is at least about 4,5,6,7 or 8 polymer of amino acid.In this specification sheets, use amino acid whose standard trigram or one-letter symbol.In the art, this term exchanges with " peptide " or " protein " usually and uses.
HLA " main anchor residues (primary anchor residue) " is to think that along an amino acid on the peptide sequence specific position it can provide the point of contact between immunogenic peptide and the HLA molecule.Confirm in the peptide of length 1-3, normally 2 main anchor residues define " motif " of immunogenic peptide.These residues are considered to be fit to closely contact with the combination ditch of HLA molecule, and the side chain with them inserts in the specific pocket that combines ditch simultaneously.For example, in one embodiment, the main anchor residues of HLA I quasi-molecule is positioned at position 2 (counting from the N-terminal position) and is positioned on peptide epitopes C-terminal the 8th, 9,10,11 according to the invention or the 12 residue positions.Perhaps, in another embodiment, in conjunction with the main anchor residues space of the peptide of HLA II quasi-molecule rather than at the end of peptide, the length of the peptide here has 9 amino acid usually at least.The main anchor position of each motif and hyper-base preface (supermotif) is put and is listed in Table IV (a).For example, can produce the analogue peptide through existence or the disappearance that changes the specific residue during main and/or less important anchor position put shown in the Table IV.Binding affinity and/or cell mass that this analogue is used to regulate the peptide that comprises specific HLA motif or hyper-base preface cover (population coverage) scope.
" ri " includes but not limited to also list nonrestrictive exemplary application in following these (at Table IV (I))).
" at random " or grammer equivalent are used for nucleic acid and protein here, represent that every kind of nucleic acid all is made up of Nucleotide that (is connected) basically at random and amino acid respectively with peptide.These at random peptide (or here discuss nucleic acid) can mix any Nucleotide or amino acid in any position.Can design compound method and produce protein or nucleic acid at random, on sequence length, forming all or most of possible combination, thereby form candidate's biological activity protein reagent library at random.
In one embodiment, the library is " completely randomization ", does not all have preferred sequence or constant sequence in any position.In another embodiment, the library is " biasing at random " library.Be some positions in the sequence or keep constant, or be selected from the limited possible residue of number.For example; Nucleotide or amino-acid residue can be at classification (for example hydrophobic amino acid, the wetting ability residues of regulation; Space biasing (little or big) residue) selects at random to form the halfcystine of crosslinked usefulness, the proline(Pro) that the SH-3 structural domain is used in to form nucleic acid binding domain; The Serine of usefulness such as phosphorylation site, Threonine, tyrosine or Histidine also are like this for purine etc. perhaps.
" recombinant chou " DNA or RNA molecule are at the DNA of external making or RNA molecule.
Term used herein " strand Fv " or " scFv " or " strand " antibody are meant the antibody fragment that comprises antibody VH and VL district, and wherein these zones are present in the polypeptied chain.Usually, the Fv polypeptide also comprises the peptide linker between VH and the VL district, and this joint makes said sFv can form the required structure of antigen combination.For sFv, can be referring to Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODY, volume 113, Rosenburg and Moore compile Springer-Verlag, New York, 269-315 page or leaf (1994).
The non-limitative example of " small molecules " comprises and combining with PSCA or interactional compound, part (comprise hormones, neuropeptide, chemokine, taste-additive, phosphatide and combine and its function equivalent of the preferred PSCA of inhibition protein function).This non-limiting micromolecular molecular weight should be less than about 10kDa, is more preferably less than about 9, about 8, about 7, about 6, about 5 or about 4kDa.In certain embodiments, small molecules ability physical property is associated with PSCA albumen or is combined PSCA albumen; In the pathways metabolism of natural generation, do not come to light; And/or compare non-aqueous solution solution more soluble in water.
Term used herein " specificity " is meant the selective binding of antibody to target epitope.Can under given condition, antibody be compared with the combination of irrelevant antigen or antigen mixture with antibody with suitable antigenic combination, come the binding specificity of test antibody.If said antibody combines to exceed 2,5,7 and more preferably 10 times with suitable antigenic combine at least than its nothing to do with antigen or antigen mixture, think that then it is specific.In one embodiment, specific antibody is a kind ofly only to combine PSCA antigen and the irrelevant antigenic antibody of debond.In another embodiment, specific antibody is a kind of people of combination PSCA antigen but does not have with this PSCA antigen and combine 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or the inhuman PSCA antigen bonded antibody of amino acids homology more.In another embodiment, specific antibody is a kind of people of combination PSCA antigen and combines mouse PSCA antigen but the antibody higher with human antigen's combination degree.In another embodiment, specific antibody is a kind of people of combination PSCA antigen and combines primates PSCA antigen but the antibody higher with human antigen's combination degree.In another embodiment, said specific antibody combine people's PSCA antigen and any inhuman PSCA antigen but with human antigen or the higher antibody of its any built up section degree.
" severity " of hybridization is prone to confirmed by one of ordinary skill in the art, and can by virtue of experience calculates according to probe length, wash temperature and salt concn usually.Usually, probe is long more, and the temperature that suitable annealing needs is high more, and short probe needs lower temperature.The ability that is present in the nucleotide sequence reannealing of time variation under the environment that is lower than melting temperature(Tm) when complementary strand is depended in hybridization usually.Then can use higher relatively temperature but required homology degree is high more between probe and the hybridization sequences.Consequently, higher relatively temperature makes reaction conditions stricter, and the strict degree of the low more then reaction conditions of temperature is low more.Other details of hybridization severity is explained visible Ausubel etc., " molecular biology fresh approach " (Current Protocolsin Molecular Biology), Wiley Interscience Publishers, (1995).
" stringent condition " or " high stringent condition " of this paper definition can be identified through following characteristic; But be not limited thereto; They are: in (1) when washing, adopted LIS and high temperature, 0.015M sodium-chlor/0.0015M Trisodium Citrate/0.1% sodium lauryl sulphate for example, and temperature is 50 ℃; (2) use denaturing agent such as methane amide in the crossover process; For example 50% (v/v) methane amide and 0.1% bovine serum albumin/0.1%Ficoll/0.1% Vinylpyrrolidone polymer/50mM sodium phosphate buffer (pH6.5) and 750mM sodium-chlor, 75mM Trisodium Citrate, temperature is 42 ℃; Or 50% methane amide, 5x SSC (0.75M NaCl are used in (3); 0.075M Trisodium Citrate), salmon sperm DNA (50mg/ml), 0.1%SDS and 10% T 500 of 50mM sodium phosphate (pH6.8), 0.1% trisodium phosphate, 5x denhardt solution, supersound process; Temperature is 42 ℃; 42 ℃ with 0.2x SSC (sodium chloride/sodium citrate) washing and 55 ℃ with the washing of 50% methane amide, be used in 55 ℃ then and wash with high severity with the 0.1x SSC that contains EDTA." medium stringent condition " is described in; But be not limited to; Sambrook etc. are at " molecular cloning laboratory manual " (Molecular Cloning:A LaboratoryManual; New York:Cold Spring Harbor Press, 1989) description in, and comprise washing soln and the hybridization conditions (for example temperature, ionic strength and %SDS) of using strict degree to be lower than above-mentioned condition.An example of medium stringent condition is in 65 ℃ of solution that containing following composition, to cultivate a night: 1% Ox blood serum, 0.5% sodium phosphate pH7.5,1.25mM EDTA and 7%SDS 5 * SSC (150mM NaCl; The 15mM trisodium citrate), then about 50 ℃ with 2 * SSC/1%SDS and at 50 ℃ with 0.2 * SSC/0.1%SDS washing filter membrane.How those skilled in the art will understand attemperation, ionic strength etc. to adapt to factors such as probe length.
HLA " hyper-base preface " is the peptide binding specificity of being shared by the HLA molecule of two or more HLA allelotrope codings.All phenotypic frequencies of the ultra type of HLA-are listed in Table IV (f) among the different ethnic populations.The non-limiting composition of various ultra types (supertype) 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)
Crowd's fraction of coverage that the ultra type combination calculation of different HLA-goes out is listed in the Table IV (g).
Term relevant on " treatment " or " treatment " and the grammer in this article refers to any improvement to any disease consequence, reduces and/or relieving side effects like prolonged survival period, sickness rate, and these are sub products of changing the treatment pattern; Be readily appreciated that in the art though do not require complete radical curing of disease, can eradicate disease fully then is preferred result.
" transgenic animal " (for example mouse or rat) is to have the animal that contains genetically modified cell, the ancestors that said transgenic is introduced into animal or (for example embryo stage) is introduced into animal in utero." transgenic " is the interior DNA of genome that is integrated into cell, to produce transgenic animal.
HLA or cellullar immunologic response " vaccine " are here represented to contain or the compsn of one or more peptides of the present invention of encoding.The instance that many these type of vaccines are arranged is like the mixture of one or more peptides; One or more peptides of the present invention that multi-epitope (polyepitopic) peptide is contained; Or the encode this independent peptide or the nucleic acid of polypeptide, the minigene of the multi-epitope peptide of for example encoding." one or more peptides " can comprise 1-150 or more in any integer, for example 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 or peptide more how of the present invention.Said peptide or polypeptide can be chosen wantonly and modified, as passing through fatization, adding target sequence or other sequence.HLA I class peptide of the present invention can mix or be connected with HLA II class peptide be easy to the activation of Kiwi poison T lymphocyte and helper T lymphocyte.The HLA vaccine can comprise the antigen presenting cell of peptide-pulse, for example dendritic cell.
Term " variant " is meant the molecule that shows the variation that is different from said type or normal type, as on the corresponding position of the protein of clearly describing (PSCA albumen as shown in Figure 1), containing the protein of one or more different aminoacids residues.An example of variant proteins is an analogue.Montage isotype and SNP (SNP) are other examples of variant.
" PSCA GAP-associated protein GAP " of the present invention comprises special those that describe here; And allelic variant, conservative variant, analogue and the homologue of replacing, that they can need not undo experimentation according to the method that the method for listing or this area obtain easily here is just separable/and produce and make characteristic and identify.Also comprise the fusion rotein of different PSCA albumen or its segmental part combination results and the fusion rotein of PSCA albumen and heterologous polypeptide formation.This PSCA albumen is referred to as PSCA GAP-associated protein GAP, protein of the present invention or PSCA.Term " PSCA GAP-associated protein GAP " has been meant 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 or 25 with upper amino acid; Or at least 30,35,40,45,50,55,60,65,70,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,225,250,275,300,325,330,335,339 or the more polypeptide fragment or the psca protein sequence of amino acids.
II.) PSCA polynucleotide
One aspect of the present invention provides corresponding with all or part psca gene, mRNA and/or encoding sequence or the complementary polynucleotide; Preferably with unpack format, comprise coding PSCA GAP-associated protein GAP and segmental polynucleotide, DNA, RNA, DNA/RNA heterozygote and relevant molecule, with psca gene or mRNA sequence or its part mutually complementary polynucleotide or oligonucleotide and with psca gene, mRNA or the polynucleotide or the oligonucleotide of hybridizing with the polynucleotide (being referred to as " PSCA polynucleotide ") of coding PSCA.Under all situations that chapters and sections are mentioned, T also can be U among Fig. 1.
The instance of PSCA polynucleotide comprises: have the PSCA polynucleotide of sequence shown in Figure 1, the nucleotide sequence of PSCA shown in Figure 1 wherein changes T into U; At least 10 contiguous nucleotides with polynucleotide of sequence shown in Figure 1; Or have sequence shown in Figure 1 but T changed at least 10 contiguous nucleotides of the polynucleotide of U.
The polynucleotide of the long relatively part of coding PSCA albumen also within the scope of the invention.For example, about amino acid/11 that can be through multiple technique for generating well known in the art coding PSCA albumen shown in Figure 1 or shown in Figure 3 or " variant " (or 20 or 30 or 40 etc.) arrives the polynucleotide of about amino acid 20 (about 30 or 40 or 50 etc.).These polynucleotide passages can comprise any part in the sequence of PSCA shown in Fig. 1.
II.A.) purposes of PSCA polynucleotide
II.A.1. the monitoring of genetic abnormality
Polynucleotide recited above have multiple different purposes.The chromosomal localization figure of people's psca gene lists among the embodiment that is entitled as " karyomit(e) of PSCA is drawn ".For example, because this chromosomal psca gene figure, the polynucleotide of coding PSCA albumen different zones are used to confirm that the cytogenetics of this chromosome region is unusual, for example are accredited as unusual with various related to cancer.In some gene, comprise that multiple chromosome abnormalty in being rearranged in has been accredited as that the cytogenetics that often occurs in many various cancers is unusual (for example sees Krajinovic etc., Mutat.Res.382 (3-4): 81-83 (1998); Johansson etc., Blood86 (10): 3905-3914 (1995) and Finger etc., P.N.A.S.85 (23): 9158-9162 (1988)).Therefore, the polynucleotide of the coding PSCA albumen specific region unusual ratio of cytogenetics that most likely is used for describing to encode in the chromosomal region of the PSCA that may cause the malignant phenotype originally maybe more accurate new tool.In the literary composition, these polynucleotide satisfied this area to the sensitivity that improves the karyomit(e) screening with the demand that identifies meticulousr and more uncommon chromosome abnormalty (for example seeing Evans etc., Am.J.0bstet.Gynecol171 (4): 1055-1057 (1994)).
In addition, proved PSCA high expression level in prostate gland and other cancer, the PSCA polynucleotide are used to evaluate the method for psca gene product situation in healthy tissues and the cancerous tissue.Usually, the polynucleotide of coding PSCA albumen specific region are used to evaluate the psca gene specific region zone of one or more motifs (as contain) and whether have disorder (like disappearance, insertion, point mutation or cause the variation of antigenicity forfeiture).Exemplary detection method comprises that RT-PCR measures with single strand conformation polymorphism (SSCP) analysis and (for example sees Marrogi etc.; 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 relevant with embodiment of the present invention described here is genomic dna, cDNA, ribozyme and antisense molecule; And based on replaceable main chain or comprise the nucleic acid molecule of replaceable base; They can be from natural origin or synthetic, and comprises and can suppress the molecule that RNA or psca protein are expressed.For example, antisense molecule can be RNA or other molecule, comprises PNAG3 (PNA) or non-nucleic acid molecule, as relying on the phosphorothioate derivative that the mode specificity combines DNA or RNA with base pair.Those skilled in the art can use PSCA polynucleotide described here and polynucleotide sequence to obtain the nucleic acid molecule of these types easily.
Antisense technology need use the external source oligonucleotide that combines target polynucleotide in the cell.Term " antisense " is meant target (for example PSCA) complementation in this oligonucleotide and their born of the same parents.See for example Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRCPress, 1989; With Synthesis 1:1-5 (1988).PSCA antisense oligonucleotide of the present invention comprises verivate, and like S-oligonucleotide (phosphorothioate derivative or S-oligo see Jack Cohen, and be the same), this material has the activity of enhanced anticancer growth.S-oligo (nucleosides thiophosphatephosphorothioate) is the analogue such as electronics such as grade of oligonucleotide (O-oligo), and wherein the non-bridge joint Sauerstoffatom of phosphate is substituted by sulphur atom.S-oligo of the present invention can use the sulphur atom transfer agent 3H-1,2-benzo two mercaptan-3-ketone-1,1-dioxide are handled corresponding O-oligo and are prepared.See for example Iyer, R.P. etc., J.Org.Chem.55:4693-4698 (1990); And Iyer, R.P. etc., J.Am.Chem.Soc.112:1253-1254 (1990).Other PSCA antisense oligonucleotide of the present invention comprises morpholino antisense oligonucleotide known in the art (see for example Partridge etc., 1996, Antisense & Nucleid Acid Drug Development 6:169-175).
PSCA antisense oligonucleotide of the present invention can be and preceding 100 the 5 ' codons of psca gene group sequence and the RNA or the DNA of back 100 3 ' codons or corresponding mRNA complementation or stable hybridization usually.Although complementary degree height is preferred, do not require complete complementation.Use with the oligonucleotide of this regional complementarity and can not hybridize with PSCA mRNA selective cross with mRNA of other adjusting subunit of protein kinase.In one embodiment, PSCA antisense oligonucleotide of the present invention is the fragment that contains with 15 to 30 residues of the antisense DNA molecule of the sequence of PSCA mRNA hybridization.Randomly, the PSCA antisense oligonucleotide is the oligonucleotide with preceding 10 5 ' codons of PSCA or 30-residue of back 10 3 ' codon complementary.Perhaps, said antisense molecule is modified so that suppress psca expression with ribozyme, sees for example L.A.Couture & D.T.Stinchcomb; Trends Genet 12:510-515 (1996).
II.A.3. primer and primer are right
Other specific embodiments of these Nucleotide of the present invention comprise can specific amplification polynucleotide of the present invention or the primer and the primer of its any specific part right, and with the probe of nucleic acid molecule of the present invention or its any part selectivity or specific hybrid.Probe can identify with detectable label, like ri, fluorescent chemicals, bioluminescent compound, chemiluminescence compound, metal chelator or enzyme labelling.This probe and primer are used to whether there are the PSCA polynucleotide in the test sample and are used as the instrument of expressing the proteic cell of PSCA that detects.
The example of this probe comprises and contains all or part of people PSCA cDNA polypeptide of sequence shown in Figure 1.The right example of primer that can specific amplification PSCA mRNA is also described in an embodiment.Those skilled in the art will understand, and can prepare a large amount of different primers and probe and they are used for effectively amplification and/or detection PSCA mRNA based on the sequence that provides here.
PSCA polynucleotide of the present invention can be used for many purposes, comprising but be not limited to them as amplification and/or detect psca gene, mRNA or its segmental probe and primer; Reagent as diagnosis and/or prediction prostate cancer and other cancer; As the encoding sequence that can guide the PSCA expression of polypeptides; As the instrument of regulating or suppress psca gene expression and/or the translation of PSCA transcript; And as therapeutical agent.
The present invention includes and use any probe evaluation described here and separate PSCA or PSCA associated nucleic acid sequences from natural origin (like people or other Mammals); And isolated nucleic acid sequences itself, said sequence can be included in whole or most of sequences of finding in the used probe.
The separation of II.A.4.PSCA-nucleic acid molecules encoding
PSCA cDNA sequence described here can be separated other polynucleotide of coding psca gene product; And separate coding psca gene product homologue, alternately shear the polynucleotide of isotype, allelic variant and psca gene product mutant form and the polynucleotide of coding PSCA GAP-associated protein GAP analogue.Known and variously be used for separating the encode molecular cloning method of full-length cDNA of psca gene and (see for example Sambrook, J. etc., " molecular cloning laboratory manual " (second edition), Cold Spring Harbor Press, New York, 1989; " the molecular biology fresh approach " that Ausubel etc. compile, Wiley and Sons, 1995).For example, (for example Lambda ZAP Express Stratagene) carries out the lambda particles phage PCR cloning PCR to available commercially available cloning system easily.The PSCA cDNA of serviceable indicia or its fragment are identified the phage clone that contains psca gene cDNA as probe.For example, in one embodiment, can synthesize PSCA cDNA (for example Fig. 1) or its part and it is used as probe and retrieve and the corresponding overlapping and full-length cDNA of psca gene.Can be through waiting and separate psca gene itself with PSCA dna probe or primer screening genome dna library, bacterium artificial chromosome library (BAC), yeast artificial chromosome library (YAC).
II.A.5. recombinant nucleic acid molecules and host-vector system
The present invention also provides recombinant DNA or the RNA molecule that contains PSCA polynucleotide, its fragment, analogue or homologue; Include but not limited to phage, plasmid, phagemid, clay, YAC, BAC and various virus well known in the art and non-virus carrier, and transformed or cells transfected by this recombinant DNA or RNA molecule.Produce that the method for this molecule can know (see for example Sambrook etc., 1989, the same).
The host-vector system that the present invention also provides contain can be in suitable protokaryon or eukaryotic host cell the recombinant DNA that contains PSCA polynucleotide, its fragment, analogue or homologue of (expression).The example of suitable eukaryotic host cell comprises that yeast cell, vegetable cell protect zooblast, like mammalian cell or insect cell (for example can by the cell of baculovirus infection, like Sf9 or HighFive cell).The example of suitable mammalian cell comprises various prostate cancer cell lines; Like DU145 and TsuPr1; But other transfection or transducible prostate cancer cell line; Primary cell (PrEC), and many mammalian cells (like COS, CHO, 293,293T cell) that is commonly used to express recombinant protein.More particularly, available any this area is used usually and by the host-vector system of extensively understanding, is utilized the polynucleotide that contain PSCA encoding sequence or its fragment, analogue or homologue to produce PSCA protein residues fragment.
Can obtain many suitable expression PSCA albumen or its segmental host-vector system, see for example Sambrook etc., 1989, the same; " molecular biology fresh approach ", 1995, the same).Be used for preferred carrier that Mammals expresses include but not limited to pcDNA 3.1myc-His-tag (Invitrogen) and retrovirus vector pSR tkneo (Muller etc., 1991, MCB11:1785).Use these expression vectors, can in some prostate cancers and non-prostate cell line, express PSCA, comprising for example 293,293T, rat-1, NIH 3T3 and TsuPr1 cell.Host-vector system of the present invention can be used to make PSCA albumen or its fragment.This host-vector system can be used to study the functional performance of PSCA and PSCA two mutants or analogue.
Recombinant human PSCA albumen or its analogue or homologue or fragment can be made with the construction mammalian cells transfected of the PSCA-associated nucleotide that is encoded.For example; Can the be encoded expression plasmid transfection of PSCA or its fragment, analogue or homologue of 293T cell; Thereby the PSCA GAP-associated protein GAP is expressed in the 293T cell, and available standards purification process (for example with anti--psca antibody affinity purification) separates reorganization PSCA albumen.In another embodiment, the white subclone of PSCA encoding sequence is gone into retrovirus vector pSR MSVtkneo and is used for infecting various mammal cell lines, like NIH 3T3, TsuPr1,293 and rat-1, to set up psca expression clone.Also available various other expression systems well known in the art.The expression constructs of leading peptide that coding is connected to PSCA encoding sequence frame can be used to produce the reorganization PSCA albumen of secreted form.
As discussed here, the redundancy of genetic code allows to exist in the psca gene sequence and changes.Specifically, the host of particular types known in the art has the preference of specific cryptosystem usually, therefore can adopt the sequence of known required host's preference.For example, preferred analogue codon sequence is usually with high frequency codon replacement rare codon (promptly in required host's known array frequency of utilization less than 20% codon).For example capable of using the codon that obtains on the Internet use table (URL is dna.affrc.go.jp/ ~ nakamura/cocon.html) can calculate the preferred codon of particular types.
Known other sequence modification can strengthen the protein expression in the cell host.These modifications comprise that the vacation of deletion coding gathers adenosine signal, exon splice site signal, swivel base increment repeating sequences, and/or other this type of fully understand to the deleterious sequence of genetic expression.The GC content of sequence can be adjusted to the M.L. of given cell host, and this level can be calculated with reference to the known of in cell host, expressing.If possible, can modify mRNA hair clip secondary structure to sequence to avoid predicting.Other useful modification is included in ORF and begins place's adding translation startup consensus sequence, and is of Kozak, Mol.Cell Biol., 9:5073-5080 (1989).Those skilled in the art know following rule; Be that the eucaryon rrna is bar none from beginning translation near the AUG codon of 5 ' end; This principle only lost efficacy under few situation and (saw for example Kozak PNAS 92 (7): 2662-2666; (1995) and Kozak NAR 15 (20): 8125-8148 (1987)).
III.) PSCA GAP-associated protein GAP
Others of the present invention provide PSCA GAP-associated protein GAP.The proteic specific embodiments of PSCA comprises having Fig. 1, the let others have a look at polypeptide of all or part of aminoacid sequence of PSCA of preferred Figure 1A.Perhaps, the proteic embodiment of PSCA is included in variant, homologue or the analogue polypeptide that changes in the PSCA aminoacid sequence shown in Figure 1.
The embodiment of PSCA polynucleotide comprises: have the PSCA polypeptide of sequence shown in Figure 1, the peptide sequence of PSCA shown in Figure 1 wherein changes T into U; At least 10 contiguous nucleotides with polypeptide of sequence shown in Figure 1; Or have sequence shown in Figure 1 but T changed at least 10 contiguous nucleotides of the polypeptide of U.
Table II provides amino acid abbreviations.Usually the conservative amino acid replacement be can in protein, carry out and proteinic conformation or function do not changed.Protein of the present invention can comprise 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 conservative property and replace.
Embodiment of the present invention as herein described comprise proteic variant of the acceptable PSCA in a large amount of this areas or analogue, as have aminoacid insertion, disappearance and substituted polypeptide.The PSCA variant can be used methods known in the art, and for example site-directed mutagenesis, L-Ala scanning and PCR mutagenesis are made.Site-directed mutagenesis (Carter etc., Nucl.Acids Res., 13:4331 (1986); Zoller etc.; Nucl.Acids Res.; 10:6487 (1987)), mutagenesis (Wells etc. are selected in cassette mutagenesis (Wells etc., Gene, 34:315 (1985)), restriction; Philos.Trans.R.Soc.London SerA, 317:415 (1986)) or other known technology can on clone's DNA, produce PSCA modification D NA.
Scanning amino acid analysis also can be used to identify on the contiguous nucleotide sequence and the active one or more amino acid relevant like protein-protein interaction of particular organisms.Preferred scanning amino acid is less relatively neutral amino acids.This seed amino acid comprises L-Ala, glycocoll, Serine and halfcystine.L-Ala normally should group in preferred scanning amino acid because do not have side chain on its β carbon atom and unlikely change the main chain conformation of variant.L-Ala also is preferred usually, because it is modal amino acid.In addition, it be typically found at the position that buried and be exposed (Creighton, Proteins, (W.H.Freeman & Co., N.Y.); Chothia, J.Mol.Biol., 150:1 (1976)).If L-Ala replaces and not produce an amount of variant, structure amino acid such as then can use.
Like the definition of this paper, the identifiable attribute of at least one of PSCA variant, analogue or homologue is to have an epi-position can play " cross reaction " with the PSCA albumen that contains Fig. 1 aminoacid sequence.In this sentence, " cross reaction " be meant antibody or T cell that specificity combines the PSCA variant also specificity combine to have the PSCA albumen of aminoacid sequence shown in Figure 1.When a polypeptide no longer contains anyly can be combined the epi-position of proteic antibody of initial PSCA or T cell recognition by specificity the time, this polypeptide no longer is a proteinic variant shown in Figure 1.Be proficient in the epi-position of the different sizes of antibodies that those skilled in the art will appreciate that identification of protein, think typical amino acid number the minimum epi-position from about 4 or 5 amino acid that adjoin or do not adjoin of collection.See for example Nair etc., J.Immunol 2,000 165 (12): 6949-6955; Hebbes etc., Mol Immunol (1989) 26 (9): 865-73; Schwartz etc., J Immunol (1985) 135 (4): 2598-608.
The PSCA GAP-associated protein GAP variant of other type and the aminoacid sequence of Fig. 1 or its fragment have 70%, 75%, 80%, 85% or 90% or higher similarity.The PSCA protein variant of other particular type or analogue comprise one or more described here or biological motifs of the present known PSCA in this area.Therefore, the present invention includes and compare the segmental analogue of PSCA (nucleic acid or amino acid) that initial fragment function (for example immunogenicity) characteristic has changed.Know that also this motif or motif known in the art will be used to nucleic acid or the aminoacid sequence of Fig. 1.
As discussed here, embodiment of the present invention comprise the polypeptide that contains less than PSCA albumen full length amino acid sequence shown in Figure 1.For example, representative instance of the present invention comprises the peptide/protein that contains any 4,5,6,7,8,9,10,11,12,13,14,15 or more contiguous amino acids of PSCA albumen shown in Figure 1.
The PSCA GAP-associated protein GAP produces with the standard peptide synthetic technology or with chemical montage method well known in the art.Perhaps available recombination method produces the nucleic acid molecule of coding PSCA GAP-associated protein GAP.In one embodiment, the available core acid molecule produces the proteic regulation fragment of PSCA (or its variant, homologue or analogue).
III.A.) have the proteinic embodiment of motif
Other exemplary embodiment of present invention disclosed herein comprises the PSCA polypeptide of the amino-acid residue that contains one or more biological motifs contained in the PSCA peptide sequence shown in Figure 1.Various motif known in the art, and can (following URL:pfam.wustl.edu/ for example be arranged through many internet sites that can openly obtain; 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, BrownUniversity, brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; And BIMAS, bimas.dcrt.nih.gov/.) assess the situation that exists of this motif.
In Table V-XVIII and XXII-LI, list and identified and have the subsequence that all PSCA variant proteins have motif.
Table IV (h) has been listed some the common motifs based on pfam retrieval (seeing URL pfam.wustl.edu/).The hurdle (1) of Table IV (h) has been listed: the abbreviation of (1) motif title, the homogeny per-cent between (2) this motif family different members, (3) motif title or description, and (4) modal function; Also comprise positional information if this motif is relevant with the location.
And growth is lacked of proper care relevant and owing to PSCA over-expresses in some cancer (seeing for example Table I), the polypeptide that contains above-mentioned one or more PSCA motifs is useful for the concrete characteristic of illustrating the malignant phenotype in view of above-mentioned PSCA motif.For example, known casein kinase i I, cAMP and the protein kinase that depends on camp and protein kinase C are that the enzyme relevant with malignant phenotype's development (seen for example Chen etc., Lab Invest., 78 (2): 165-174 (1998); Gaiddon etc., Endocrinology 136 (10): 4331-4338 (1995); Hall etc., Nucleic Acids Research 24 (6): 1119-1126 (1996); Peterziel etc., Oncogene 18 (46): 6322-6329 (1999) and O ' Brian, Oncol.Rep.5 (2): 305-309 (1998)).In addition, glycosylation and Semen Myristicae acidylate also are to modify with cancer and cancer development proteins associated matter that (for example see Dennis etc., Biochem.Biophys.Acta 1473 (1): 21-34 (1999); Raju etc., Exp.Cell Res.235 (1): 145-154 (1997)).Amidation is that another kind is also modified (for example seeing Treston etc., J.Natl.Cancer Inst.Monogr. (13): 169-175 (1992)) with cancer and cancer development proteins associated matter.
In another embodiment, protein of the present invention comprises one or more immunoreactivity epi-positions that identify with the acceptable method in this area (like the method for listing among Table V-XVIII and the XXII-LI).Available specific algorithm identifies in the PSCA albumen can the allelic peptide of best combination specific HLA confirms CTL epi-position (Table IV for example; Epimatrix TMAnd Epimer TM, Brown University, URLbrown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.htm l; 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, and need not undo experimentation and just can carry out.In addition, identify that the method for the peptide become the immunogenicity epi-position is well known in the art, and need not undo experimentation and just can in external or body, carry out.
This area knows that also the analogue that produces this epi-position is to regulate immunogenic principle.For example, we can begin from the epi-position that has CTL or HTL motif (seeing for example the HLA I class and the HLA II class motif/hyper-base preface of Table IV).Amino acid and use through replacing a specified location replace it can produce similar epi-position for specified another amino acid in this position.For example, based on the base of Table IV definition, we can use any other residue, and for example preferred residue replaces deleterious residue; Replace not too preferred residue with preferred residue; Or with the initial preferred residue that produces of another preferred residue replacement.Replacement can occur in the main anchor station of certain peptide or other position in the peptide; See for example Table IV.
Many bibliographys have all reflected the technology of identifying and produce the epi-position in protein of interest matter and the analogue thereof.See the for example WO 97/33602 of Chesnut etc.; Sette, Immunogenetics 1,999 50 (3-4): 201-212; Sette etc., J.Immunol.2001 166 (2): 1389-1397; Sidney etc., Hum.Immunol.1997 58 (1): 12-20; Kondo etc., Immunogenetics 1,997 45 (4): 249-258; Sidney etc., J.Immunol.1996 157 (8): 3480-90; With Falk etc., Nature 351:290-6 (1991); Hunt etc., Science 255:1261-3 (1992); Parker etc., J.Immunol.149:3580-7 (1992); Parker etc., J.Immunol.152:163-75 (1994)); Kast etc., 1994152 (8): 3904-12; Borras-Cuesta etc., Hum.Immunol.2000 61 (3): 266-278; Alexander etc., J.Immunol.20001 64 (3); 164 (3): 1625-1633; Alexander etc., PMID:7895164, UI:95202582; O ' Sullivan etc., J.Immunol.1991 147 (8): 2663-2669; Alexander etc., Immunity 1,994 1 (9): 751-761, and Alexander etc., Immunol.Res.1998 18 (2): 79-92.
Of the present inventionly, embodiment contains the different motifs that Table IV (a), IV (b), IV (c), IV (d) and IV (h) list about comprising; And/or the CTL epi-position of the prediction of one or more Table V-XVIII and XXII-LI; And/or the HTL epi-position of the prediction of one or more Table X LVIII-LI, and/or the peptide of the combination of one or more T cell binding motifs known in the art.Embodiment preferred does not contain insertion, disappearance or replaces in the intervening sequence of motif or polypeptide.In addition, also can consider on the either side of these motifs, to have the instance (major part that for example, comprises the polypeptide structure that contains motif) of a plurality of N-ends and/or C-terminal amino acid residue.Usually, the number of N-end and/or C-terminal amino acid residue is about 1-100 amino-acid residue on the motif either side, preferred 5-50 amino-acid residue.
The instance of PSCA GAP-associated protein GAP can have many forms, the preferably separation form.The PSCA protein molecular of purifying will be substantially free of and can weaken PSCA and antibody, T cell or other other protein of part bonded or molecule.The type and the degree of separation and purifying will depend on required purposes.The instance of PSCA GAP-associated protein GAP comprises the PSCA GAP-associated protein GAP of purifying and the solubility PSCA GAP-associated protein GAP of functionalization.In one embodiment, the solubility PSCA albumen of functionalization or its fragment keep by 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.This protein has the proteic characteristic of original PSCA, for example can induce generation specificity to combine the antibody of original PSCA albumen associated epitope to produce; Can be by this antibodies; Can induce the activation of HTL or CTL; And/or can be combined the HTL of urporotein or CTL to discern by same specificity.
The PSCA related polypeptide that contains special structures of interest can be predicted and/or identifies with various analytical technologies well known in the art; These technology comprise; For example; The analytical procedure of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf, or based on immunogenic method.The fragment that contains this structure is for generation subunit's specific anti-psca antibody or T cell, or evaluation combines the cytokine of PSCA particularly useful.For example, available Hopp, T.P. and Woods, the method for K.R. (1981, Proc.Natl.Acad.Sci.U.S.A.78:3824-3828) generate wetting ability collection of illustrative plates and identify the immunogenicity peptide fragment.Available Kyte, J. and Doolittle, the method for R.F. (1982, J.Mol.Biol.157:105-132) generate wetting ability collection of illustrative plates and identify the immunogenicity peptide fragment.The method of available Janin J. (1979, Nature 277:491-492) but generate contact residues per-cent (%) collection of illustrative plates and identify the immunogenicity peptide fragment.Use Bhaskaran R., the method for Ponnuswamy P.K. (1988, Int.J.Pept.Protein Res.32:242-255) generates average flexibility collection of illustrative plates and identifies the immunogenicity peptide fragment.Available Deleage, G., the method for Roux B. (1987, Protein Engineering1:289-294) generates β-corner collection of illustrative plates and identifies the immunogenicity peptide fragment.
Thereby available special algorithm confirm the CTL epi-position identify in the PSCA albumen can the allelic peptide of the specific HLS of best combination (for example, through using the SYFPEITHI website, URL address, its World Wide Web is syfpeithi.bmi-heidelberg.com/; The algorithm of listing of Table IV (A)-(E); Epimatrix TMAnd Epimer TM, Brown University, URL (brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); And BIMAS, URL bimas.dcrt.nih.gov/).Measurable with above-mentioned algorithm at the described peptide epitopes of this scholar MHC I quasi-molecule part from PSCA, for example HLA-A1, A2, A3, A11, A24, B7 and B35 (seeing for example Table V-XVIII, XXII-LI).Specifically; The relative section of the proteic whole aminoacid sequence of PSCA and other variant; Promptly with the HLA I class point mutation of the corresponding prediction of this variant or 9 side joint residues on the either side of exon both sides; With the HLAII class point mutation of prediction or 14 side joint residues on the either side of exon both sides, be used to hla peptide motif searching algorithm, this algorithm can find in above-mentioned Bioinformatics and Molecular Analysis Section (BIMAS) network address; Also have the SYFPEITHI website in addition, its URL is syfpeithi.bmi-heidelberg.com/.
Hla peptide motif searching algorithm be by Ken doctor Parker according to HLA I quasi-molecule, what especially the combination of the particular peptide sequence in the HLA-A2 groove was set up (for example sees Falk etc., Nature 351:290-6 (1991); Hunt etc., Science 255:1261-3 (1992); Parker etc., J.Immunol.149:3580-7 (1992); Parker etc., J.Immunol.152:163-75 (1994)).This algorithm can position 8-peptide, 9-peptide and the 10-peptide from the whole protein sequence with classification so that predictability ground combines HLA-A2 and many other HLA I quasi-molecules (ability).Many HLA I class binding peptides are 8-, 9-, 10 or the 11-peptide.For example, as far as I class HLA-A2, this epi-position preferably contains leucine (L) or methionine(Met) (M) and contains Xie Ansuan (V) or leucine (L) (seeing for example Parker etc., J.Immunol.149:3580-7 (1992)) at the C-end at 2.The binding peptide result of the PSCA prediction of selecting is shown in here Table V-XVIII and XXII-LI.Each family member's who selects candidate's (9-peptide and 10-peptide) and their position, the aminoacid sequence of each particular peptide and the bonding force scoring of estimation in Table V-XVIII and XXII-XLVIII, have been shown.Candidate's 15 peptides and their position that in Table X LVIII-LI, have shown each family member who selects, the aminoacid sequence of each concrete peptide and the scoring of the bonding force of estimation.The bonding force scoring is corresponding to containing peptide complex body dissociated expectation transformation period under the condition of 37 ℃ of pH6.5.The HLAI quasi-molecule of estimating to have the polypeptide cell surface of the highest combination scoring combine the most closely and the transformation period the longest, best immunogenicity target is provided therefore for the T cell recognition.
Peptide and HLA are allelic actually to be combined to go up the stable case that HLA expresses through antigen manufacturing deficiency clone T2 and assess (for example seeing Xue etc., Prostate 30:73-8 (1997) and Peshwa etc., Prostate 36:129-38 (1998)).The immunogenicity of concrete peptide can be assessed through external activation at antigen presenting cell (like dendritic cell) CD8+ CTL (CTL) when existing.
Should be with each through BIMAS website, Epimer TMAnd Epimatrix TMThe epi-position of website prediction; Or will " be used for " PSCA albumen of the present invention by HLA I class or the specified epi-position of II class motif; Said HLAI class or II class motif can obtain or become the part of said technology with said technology; As list in Table IV (or with Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/ confirms)." be used for " expression in the text and estimate PSCA albumen, for example, be proficient in those skilled in the relevant art and can carry out easily through visual evaluation or through computer based pattern method for searching.PSCA proteic each have the subsequence of 8,9,10 or 11 amino-acid residues of HLA I class motif, or have 9 of HLA II class motif or the subsequence of more a plurality of amino-acid residues all belongs within the scope of the invention.
III.B.) expression of PSCA GAP-associated protein GAP
In the embodiment that embodiment below describes; PSCA can express in commercial expression vector cells transfected (like the 293T cell) easily; The coding that the commodity expression vector for example has CMV-to drive contains the expression vector (pcDNA3.1/mycHIS of the PSCA of terminal 6 Histidines of C-and MYC mark; Invitrogen or Tag5, GenHunter Corporation, Nashville TN).The Tag5 carrier provides an IgGK secretion signal, can be used for promoting cells transfected to produce excretory PSCA albumen.For example, can adopt standard technique to be secreted into the PSCA of the HIS-mark in the substratum with ni-sepharose purification.
III.C.) modification of PSCA GAP-associated protein GAP
The modification of PSCA GAP-associated protein GAP is included within the scope of the invention like covalent modification.A kind of type of covalent modification comprises the target amino acid residue that makes the PSCA polypeptide and can react with organic derivating agent of the proteic selected side chain of PSCA or N-or the reaction of C-terminal residue.The another kind of type that PSCA polypeptid covalence in the scope of the invention is modified comprises the proteinic Natively glycosylated pattern of change the present invention.The another kind of type of PSCA covalent modification comprises with USP 4,640,835; 4,496,689; 4,301,144; 4,670,417; The mode of enumerating in 4,791,192 or 4,179,337 is received one of multiple nonprotein type polymer with the PSCA polypeptide chain, these polymkeric substance such as polyoxyethylene glycol (PEG), W 166 or polyoxyalkylene.
PSCA GAP-associated protein GAP of the present invention can be modified the chimeric molecule that contains the PSCA that is fused to other heterologous polypeptide or aminoacid sequence with formation.This chimeric molecule can be synthetic through chemistry or recombination method.Chimeric molecule can contain and is fused to other taa or its segmental protein of the present invention.Perhaps, protein of the present invention can contain the segmental fusions of PSCA sequence (amino acid or nucleotide sequence), has so just formed on its length not and amino acid shown in Figure 1 or the direct homologous molecule of nucleotide sequence.This chimeric molecule can contain a plurality of identical PSCA subsequences.Chimeric molecule can comprise the PSCA GAP-associated protein GAP that has polyhistidine epi-position mark (providing fixed nickel alternative bonded epi-position) and cytokine or with the fusions of growth factor.This epi-position mark places the proteic amino of PSCA-or carboxyl-end usually.In another embodiment, chimeric molecule can comprise the fusions of PSCA GAP-associated protein GAP and Tegeline or Tegeline specific region.For the bivalent form (also becoming " immunoadhesin ") of this chimeric molecule, this fusions can be the Fc zone of IgG molecule.The Ig fusions should comprise the replacement that replaces at least one variable region of Ig molecule with solvable (membrane spaning domain is deleted or inactivation) form of PSCA polypeptide.In an embodiment preferred, shown in the Tegeline fusions comprise hinge, CH2 and the CH3 district of IgG1 molecule or hinge, CHI, CH2 and CH3 district.Be to make the Tegeline fusions, can referring to, for example, the U.S. Patent number 5,428,130 that deliver June 27 nineteen ninety-five.
III.D.) purposes of PSCA GAP-associated protein GAP
Protein of the present invention has many different purposes.Because PSCA expresses at prostate cancer and other cancer camber, thereby the PSCA GAP-associated protein GAP is used to assess the definite malignant phenotype's of state of psca gene product in healthy tissues and the cancerous tissue method.Usually, be used to assess in those zones zone of one or more motifs (as contain) whether have entanglement (like disappearance, insertion, point mutation etc.) from the polypeptide of PSCA albumen specific region.Exemplary test utilizes target to contain the antibody or the T cell of PSCA GAP-associated protein GAP of the amino-acid residue of one or more biological motifs contained in the PSCA peptide sequence; With estimate in healthy tissues and the cancerous tissue should the zone characteristic, or initiation is to the immunne response of this epi-position.Perhaps, the PSCA GAP-associated protein GAP that contains the amino-acid residue of the one or more BA motifs of PSCA albumen is used to screen the factor with this regional interaction of PSCA.
PSCA protein fragments/subsequence for produce or signature analysis structural domain specific antibody (for example discerning the antibody of epi-position in the outer or born of the same parents of PSCA albumen born of the same parents) to identify that to combine the own or cytokine in PSCA or its ad hoc structure territory particularly useful; And particularly useful in various treatments and diagnostic use, include but not limited to the method for diagnostic assay, Theratope and this vaccine of preparation.
The protein of psca gene or its analogue, homologue or segment encoding has many purposes, includes but not limited to produce antibody and is used for identifying the part and other reagent and cellular constituent that combines the psca gene product.The anti-PSCA albumen that produces or its segmental antibody can be used for diagnosis to be measured with prognosis, and is used to the image formation method that the people is those cancers of listing in the Table I of characteristic with expression PSCA albumen.This cancer patients's method can expressed and be used to treat to this antibody in born of the same parents.PSCA-associated nucleic acid or protein also are used to produce HTL or CTL replys.
Used various for detecting the effective immunity test of PSCA albumen, comprising but be not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assay (ELISA), enzyme linked immunological fluorometric assay (ELIFA), immunocytochemical method or the like.Antibody can be labeled and be used as the immune photographic developer (for example in radiation flicker imaging method) that can detect the cell of expressing PSCA.PSCA albumen is also particularly useful for producing cancer vaccine, and this will further describe in the text.
IV.) psca antibody
Another aspect of the present invention provides the antibody that combines the PSCA GAP-associated protein GAP.The preferred antibodies specificity combines the PSCA GAP-associated protein GAP but not relevant with PSCA proteinic peptide or the protein of debond under physiological condition (or weak combination).In the literary composition, the example of physiological condition comprises: 1) PBS; 2) contain the Tris BS of 25mM Tris and 150mM NaCl; Or saline water (0.9%NaCl); 4) animal serum such as human serum; Or 5) each combination 1)-4); These reactions should take place under pH7.5, or in the scope of pH7.0-8.0, take place, or in the scope of pH6.5-8.5, take place; Simultaneously, these are reflected under 4-37 ℃ the temperature and take place.For example, can combine the PSCA GAP-associated protein GAP in conjunction with the antibody of PSCA, for example its homologue or analogue.
Psca antibody of the present invention is particularly useful for cancer (seeing for example Table I) diagnosis and prognosis detection and image formation method.Similarly, this antibody also is useful for treatment, diagnosis and/or prediction prostate gland and other cancer, as long as PSCA also expresses or over-expresses in these other cancers.The antibody (for example single-chain antibody) of expressing in the born of the same parents in addition, also has therapeutic action in handling the cancer (for example late period or metastatic prostate cancer) relevant with the expression of PSCA or other late period or metastatic cancer.
The present invention also provides the various immunity tests that are used for detecting and quantizing OSCA and mutant PSCA GAP-associated protein GAP.This test can comprise the psca antibody that one or more could suitably discern and combine the PSCA GAP-associated protein GAP.These are tested available various immunoassay patterns well known in the art and carry out, comprising various types of radioimmunoassays, enzyme-linked immunosorbent assay (ELISA), enzyme linked immunological fluorometric assay (ELIFA) or the like.
Non-antibody immunoassay of the present invention can comprise that also the T cell immunogenicity is measured (suppressing or stimulation) and mhc (MHC) combines to detect.
In addition, the present invention also provides and can detect prostate cancer and express the immune imaging method of the cancer of PSCA with other, comprising but be not limited to the radiation flicker imaging method of the psca antibody of applying marking.This detection method is used to express detection, the monitor and predict of the cancer (like prostate cancer) of PSCA clinically.
Psca antibody also is used to purifying PSCA GAP-associated protein GAP and the method for separating PSCA homologue and associated molecule.For example, the method for purifying PSCA GAP-associated protein GAP is included in and allows lysate or other solution that psca antibody combines will to contain under the condition of PSCA GAP-associated protein GAP the PSCA GAP-associated protein GAP with hatching with solid substrate link coupled psca antibody; Wash this solid substrate to remove impurity; And from link coupled antibody elution PSCA GAP-associated protein GAP.Other purposes of psca antibody according to the invention comprises proteic resisting-idiotype antibody of generation simulation PSCA.
The method of various manufacturing antibody is well known in the art.For example; Can prepare antibody (" antibody laboratory manual " (Antibodies:A Laboratory Manual) through PSCA GAP-associated protein GAP, peptide or the fragment immunity mammalian hosts of puting together form with unpack format or immunity; CSH Press, Harlow and Lane edit (1988); Harlow, " antibody " (Antibodies), Cold Spring Harbor Press, NY (1989)).In addition, also can use the fusion rotein of PSCA, like the PSCAGST-fusion rotein.In one embodiment, make the gst fusion protein that comprises the whole of Fig. 1 or Most amino-acids sequence, then it had been produced suitable antibody originally as immunity.In another embodiment, synthesized the PSCA GAP-associated protein GAP and it has been used as immunogen.
In addition, naked DNA immunological technique known in the art be used to (being with or without the PSCA GAP-associated protein GAP or the psca expression cell of purifying) produce immunogenic immunne response to coding (referring to Donnelly etc., 1997, Ann.Rev.Immunol.15:617-648).
But the proteic aminoacid sequence of the PSCA shown in the analysis 1 is to select the proteic specific region of PSCA that produces antibody.For example, the hydrophobicity of PSCA aminoacid sequence capable of using and hydrophilicity analysis are identified the hydrophilic region in the PSCA structure.Have that PSCA albumen zone and other of immunogenic structure is regional can identify that with various other methods known in the art these methods are the analytical method of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf for example easily with structural domain.Available Hopp, T.P. and Woods, the method for K.R. (1981, Proc.Natl.Acad.Sci.U.S.A.78:3824-3828) generate the wetting ability collection of illustrative plates.Available Kyte, J. and Doolittle, the method for R.F. (1982, J.Mol.Biol.157:105-132) generate the wetting ability collection of illustrative plates.The method of available JaninJ. (1979, Nature 277:491-492) but generate contact residues per-cent (%) collection of illustrative plates.Use Bhaskaran R., the method for Ponnuswamy P.K. (1988, Int.J.Pept.Protein Res.32:242-255) generates average flexibility collection of illustrative plates.Available Deleage, G., the method for Roux B. (1987, Protein Engineering 1:289-294) generates β-corner collection of illustrative plates.Therefore, any each zone that identifies in these programs of usefulness or the method is within the scope of the invention.This paper has also further set forth the preferred method that produces psca antibody through the mode of embodiment.Preparation is well known in the art as the method for immunogenic protein or polypeptide.The method of the immunogenic conjugate of preparation protein and carrier (like BSA, KLH or other carrier proteins) is also known in this area.Under the certain situation, adopt directly to combine, for example use carbodiimide reagent; Under other situation, for example (Rockford, the binding reagents that IL) provides are effective by Pierce Chemical Co..Know as this area, often use the PSCA immunogen through injecting with suitable adjuvant between in due course.In immunologic process, can determine whether to form enough antibody through the titre of antibody.
The PSCA monoclonal antibody can be made with various methods well known in the art.For example, well-known, with the standard hybridoma technology of Kohler and Milstein or the technology of improving the B cell that produces antibody is not extremely changed and prepared the not dead cell system of secreting required monoclonal antibody.The not dead cell system of secreting required antibody is through screening as antigenic immunoassay with the PSCA GAP-associated protein GAP.When identifying suitable not dead cell culture, extendible cell and from the vitro culture thing or from ascites fluid, make antibody.
Antibody of the present invention or fragment can be passed through the recombination method manufacturing.Specificity combines the zone in required PSCA albumen zone also can produce in chimeric or complementary determining region (CDR) the grafted antibody form of many source of species.Also can make humanization or people's psca antibody, and they should be used for treatment.Through the method that replaces corresponding human antibody sequence to prepare humanization murine antibody or other non-human antibody with one or more non-human antibody CDR be know (see for example Jones etc., 1986, Nature 321:522-525; Riechmann etc., 1988, Nature332:323-327; Verhoeyen etc., 1988, Science 239:1534-1536).Also can be referring to Carter etc., 1993, Proc.Natl.Acad.Sci.USA 89:4285 and Sims etc., 1993, J.Immunol.151:2296.
Making completely, the method for human monoclonal antibodies comprises phage display and transgenic method (referring to for example Vaughan etc., 1998, Nature Biotechnology 16:535-539).The big combinatorial library of people PSCA monoclonal antibody people Ig capable of using gene produces (being phage display) (Griffiths and Hoogenboom, " setting up external immunity system: from people's antibody of phage display library " (Building an in vitro immune system:human antibodies from phagedisplay libraries) through clone technology completely.Draw certainly: Clark; M. " protein engineering that is used for the antibody molecule of male sex's diagnosis and treatment " (the Protein Engineering of Antibody Molecules forProphylactic and Therapeutic Applications in Man) that writes; Nottingham Academic; The 45-64 page or leaf, (1993); Burton and Barbas, " from people's antibody of combinatorial library " (HumanAntibodies from combinatorial libraries).The same, the 65-82 page or leaf).The also available transgenic mice that contains human immunoglobulin gene's seat through the engineering structure of people PSCA monoclonal antibody is made completely; As on December 3rd, 1997 disclosed Kucherlapati and Jakobovits etc. PCT patented claim WO 98/24893 said (also can be referring to Jakobovits; 1998, Exp.Opin.Invest.Drugs7 (4): 607-614; The USP 6,162,963 that on December 19th, 2000 delivered; Delivered on November 12nd, 2000 6,150,584; With delivered on September 5th, 2000 6,114598).This method has been avoided the external manipulation of display technique of bacteriophage requirement, and can effectively make the real people's antibody of high-affinity.
The reactivity of psca antibody and PSCA GAP-associated protein GAP can be set up through many methods of knowing, comprising using suitable PSCA GAP-associated protein GAP, the cell of expressing PSCA or Western trace, immunoprecipitation, ELISA and the facs analysis of its extract.Psca antibody or its fragment can be puted together with the detectable label substance markers or with second molecule.Suitable detectable includes but not limited to ri, fluorescent chemicals, bioluminescent compound, chemiluminescence compound, metal chelator or enzyme.In addition, the method for understanding usually with this area produces the bi-specific antibody to two or more PSCA epi-positions.All dimerization (Homodimeric) antibody also can make (for example, Wolff etc., Cancer Res.53:2560-2565) through crosslinking technological known in the art.
One preferred embodiment in; The monoclonal antibody of the Ha1-1.16 of being called provided by the present invention, Ha1-5.99, Ha1-4.117, Ha1-4.20, Ha1-4.121, Ha1-4.37 is to be sent to American type culture collection (ATCC) on May 4th, 2005, and is given preserving number PTA-6698, PTA-6703, PTA-6699, PTA-6700, PTA-6701 and PTA-6702 respectively.
V.) PSCA cellullar immunologic response
The antigenic mechanism of T cell recognition is illustrated.Effective peptide epitopes vaccine composition of the present invention can be in the most of crowds in the whole world inductive treatment property and preventative immunne response.For understanding the value and the effect of conjugate inducing cell immunne response of the present invention, the brief overview to the immunology correlation technique is provided.
The restricted T cell recognition of HLA-HLA molecule and as the antigenic complex body of the peptide of part (Buus, S. etc., Cell 47:1071,1986; Babbitt, B.P. etc., 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 sequencing of the endogenous binding peptide through the research substituted antigen analogues of single amino acids and natural process; Identified the corresponding Key residues of the required motif of specificity conjugated antigen molecule with HLA; Listing in Table IV (also can be referring to for example; Southwood etc., J.Immunol.160:3363,1998; Rammensee etc., Immunogenetics 41:178,1995; Rammensee etc., SYFPEITHI, the URL website (134.2.96.221/scripts.hlaserver.dll/home.htm) of visit World Wide Web; 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. and Hammer, J.Curr.Biol.6:52,1994; Ruppert etc., Cell74:929-937,1993; Kondo etc., J.Immunol.155:4307-4312,1995; Sidney etc., J.Immunol.157:3480-3490,1996; Sidney etc., people Immunol.45:79-93,1996; Sette, A. and Sidney, J.Immunogenetics 1999-11; 50 (3-4): 201-12, summary).
In addition, the x-ray crystal analysis of HLA-peptide complex body shows, the peptide of HLA molecule combines the pocket in crack/ditch can the allele-specific pattern to hold the residue of peptide part; The peptide that these residues have determined to have them again combines the ability of HLA.(see for example Madden, D.R.Annu.Rev.Immunol.13:587,1995; Smith etc., Immunity 4:203,1996; Fremont etc., Immunity 8:305,1998; Stern etc., Structure 2:245,1994; Jones, E.Y.Curr.Opin.Immunol.9:75,1997; Brown, J.H. etc., Nature 364:33,1993; Guo, H.C. etc., Proc.Natl.Acad.Sci.USA 90:8053,1993; Guo, H.C. etc., Nature 360:364,1992; Silver, M.L. etc., Nature 360:367,1992; Matsumura, M. etc., Science 257:927,1992; Madden etc., Cell 70:1035,1992; Fremont, D.H. etc., Science 257:919,1992; Saper, M.A., Bjorkman, P.J. and Wiley, D.C., J.Mol.Biol.219:277,1991).
Therefore, confirm I class and II class allele-specific HLA binding motif, perhaps I class or II class hyper-base preface can identify in the protein and specific HLA antigen bonded relevant range.
Therefore, be tested and appraised the HLA motif and can identify vaccine candidate object based on epi-position; Also can combine to detect and further estimate this material standed for to confirm the binding affinity and/or the combination cycle relevant with its corresponding HLA molecule with epi-position through the HlA-peptide.Can carry out other confirmation work in these vaccine candidate objects, to be chosen in the epi-position that has preferable characteristic on colony's covering and/or the immunogenicity.
Available several different methods is estimated the immunogenicity of cell, comprising:
1) the former generation T cell culture of estimating normal individual (is seen for example Wentworth, P.A. etc., Mol.Immunol.32:603,1995; Celis, E. etc., Proc.Natl.Acad.Sci.USA 91:2105,1994; Tsai, V. etc., J.Immunol.158:1796,1997; Kawashima, I. etc., HumanImmunol.59:1,1998).This process comprises when having antigen presenting cell peripheral blood lymphocyte (PBL) several weeks in external use test peptides stimulated healthy.The specific T-cells of this peptide will be activated during this period, the lymphokine of available for example peptide sensitization target cell-or the 51Cr-release test detect.
2) immune HLA transgenic mice (is seen for example Wentworth, P.A. etc., J.Immunol.26:97,1996; Wentworth, P.A. etc., Int.Immunol.8:651,1996; Alexander, J. etc., J.Immunol.159:4753,1997).For example, in the method, the peptide that the subcutaneous Hla of giving transgenic mice is joined with Freund's incomplete adjuvant.Immunization obtains splenocyte after several weeks, about 1 week of vitro culture when test peptides exists.With for example peptide sensitization target cell and the 51Cr-release test detection of peptides specific T-cells of expressing the antigenic target cell of endogenous generation.
3) memory T cell among effective vaccinated immune body of confirmation and/or the chronic is replied and (is seen for example Rehermann, B. etc., J.Exp.Med.181:1047,1995; Doolan, D.L. etc., Immunity 7:97,1997; Bertoni, R. etc., J.Clin.Invest.100:503,1997; Threlkeld, S.C. etc., J.Immunol.159:1648,1997; Diepolder, H.M. etc., J.Virol.71:6011,1997).Therefore, can be through cultivating because therefore the contacted antigen of disease have produced " natural " immunne response, or the PBL that inoculated this antigenic vaccine person detects memory response.With patient's PBL test peptides and antigen presenting cell (APC) when existing vitro culture 1-2 week to activate " memory " T cell, make comparisons with " originally " T cell.When during cultivation finishes, use the tests such as 51Cr release, T cell proliferation or lymphokine release that comprise with peptide sensitization target cell to detect the T cytoactive.
VI.) PSCA transgenic animal
Coding PSCA GAP-associated protein GAP also can be used to produce transgenic animal or " knocking out " animal, this animal can be used for exploitation then and screens medicative reagent.According to prior art, the cDNA of coding PSCA can be used to the genomic dna of clones coding PSCA.Available then cloned genes group sequence produces the transgenic animal of the cell that contains the DNA that expresses coding PSCA.Make transgenic animal, the animal of mouse or rat and so on especially for example, method become the ordinary method of this area, and be described in, for example, the USP of delivering on April 12nd, 1988 on September 26th, 4,736,866 and 1989 delivered 4,870,009.Usually, specific cell will become the target spot that the PSCA transgenic that has tissue-specific enhancer is mixed.
The transgenic animal that comprise the transgenic copy of the PSCA that encodes can be used to check that the DNA of coding PSCA expresses the enhanced effect.This animal also can be used as the laboratory animal of medicament, and said medicament is considered to provide the protection of for example relevant with its over-expresses pathological state.According to this aspect of the invention, compare and have this and genetically modifiedly do not treat animal, the sickness rate of the animal pathological state of with medicament treatment reduces, and this this medicament of explanation has potential therapeutic action to this pathological state.
Perhaps; The inhuman homologue of available PSCA makes up PSCA and " knocks out " animal, said animal owing to the foreign gene of coding PSCA and introduce homologous recombination between the genomic dna of coding PSCA of change of animal embryo cell cause encoding PSCA genetic flaw or be changed.For example, according to prior art, the cDNA of available code PSCA comes the genomic dna of clones coding PSCA.The encode part of genomic dna of PSCA of part can or be substituted with another gene (for example coding can be used to monitor the gene of the selectable marker of integration) by deletion.Usually, the unaltered side joint DNA (simultaneously at 5 ' and 3 ' end) (see for example Thomas and Capecchi, Cell, 51:503 (1987) is about the description of homologous recombination vector) that comprises thousands of bases in the carrier.Carrier be introduced into embryonic stem cell line (for example passing through electroporation) and the DNA that select to introduce with the cell of endogenous dna homology reorganization (for example seeing Li etc., Cell, 69:915 (1992)).Then the injection cell of selecting (is seen for example Bradley to the blastocyst of animal (for example mouse or rat) to form aggregation chimera; Draw " teratocarcinoma and embryonic stem cell actual development " (Teratocarcinomas andEmbryonic Stem Cells:A Practical Approach) (IRL that writes from E.J.Robertson; Oxford; 1987), 113-152 page or leaf).Can chimeric embryo be implanted the female raising animal of suitable false pregnancy then, make said embryo's full-term pregnancy then to produce " knocking out " animal.The offspring who in its sexual cell, has homologous recombination DNA can identify and be used for breeding the animal that all zooblasts all contain homologous recombination DNA through standard technique.But the ability of their resist the disease of characterized knock-out animal is perhaps fallen ill owing to lacking the PSCA polypeptide.
VII.) be used to detect the method for PSCA
Another aspect of the present invention relates to the method that detects PSCA polynucleotide and PSCA GAP-associated protein GAP, and the method for identifying the cell of expressing PSCA.The expression characteristic of PSCA makes it become the diagnostic marker that shifts disease.Therefore, the state of psca gene product just comprises that for prediction the various factorss such as susceptibility, advance rate and/or tumor invasion property that terminal illness takes place provide useful information.Go through like this paper; The state of psca gene product can be analyzed with the whole bag of tricks well known in the art in patient's sample, analyzes (for example on laser capture micro-dissections sample, carrying out RT-PCR), Western engram analysis and tissue array analysis comprising immunohistochemical analysis, the various Northern engram technologies that comprise in situ hybridization, RT-PCR.
More particularly, the invention provides the test of the PSCA polynucleotide in the detection of biological sample (like serum, bone, prostate gland and other tissue, urine, seminal fluid, cell product etc.).Detectable PSCA polynucleotide comprise, for example, and recombinant DNA or RNA molecule that psca gene or its fragment, PSCA mRNA, PSCA mRNA replace splice variant and contain the PSCA polynucleotide.The method that many amplifications and/or detection PSCA polynucleotide exist is well known in the art and can be used for the practice of this aspect of the present invention.
In one embodiment, the method for PSCA mRNA comprises with at least a primer and makes cDNA through rt from sample in the detection of biological sample; The PSCA cDNA of cDNA that so makes as justice and antisense primer amplification with the PSCA polynucleotide to increase wherein; And whether detection exists the PSCA cDNA of amplification.Can choose the PSCA cDNA sequence of confirming amplification wantonly.
In another embodiment, the method for psca gene comprises the at first genomic dna of sample separation in the detection of biological sample; With the PSCA polynucleotide as justice and the antisense primer isolating genomic dna that increases; And whether detection exists the psca gene of amplification.Can design the suitable justice of arbitrary number and the combination of antisense probe from PSCA nucleotide sequence (seeing for example Fig. 1), and be used for this purpose.
The present invention also provides detection tissue or the middle PSCA albumen of other biological sample (like serum, seminal fluid, bone, prostate gland, urine, cell product etc.) to have the test of situation.Detect the method for PSCA GAP-associated protein GAP and also know, comprise that for example immunoprecipitation, immunohistochemical analysis, Western engram analysis, molecule combine detection, ELISA, ELIFA etc.For example, there is the method for situation to comprise at first to make the reactive fragment of PSCA-of sample contact psca antibody, antibody in the PSCA GAP-associated protein GAP or contains the recombinant protein of psca antibody antigen binding domain in the detection of biological sample; The combination of PSCA GAP-associated protein GAP in the test sample then.
The method of identifying the cell of expressing PSCA is also within the scope of the invention.In one embodiment, the detection method of the cell of evaluation expression psca gene comprises detecting in the cell whether have PSCA mRNA.Detecting the method for specific mRNA in the cell knows; Comprising; For example; Use complementary DNA probe to carry out cross experiment (like the in situ hybridization of PSCA riboprobe, Northern trace and correlation technique) and various nucleic acid amplification test (the specificity complementary primer as with PSCA carries out RT-PCR, and the augmentation detection method of other type, like for example branched DNA, SISBA, TMA or the like) with mark.Perhaps, test that identify to express the cell of psca gene comprise detect in the cell or the PSCA GAP-associated protein GAP of emiocytosis have a situation.Various detection method of protein are well known in the art, and are used to detect PSCA GAP-associated protein GAP and the cell of expressing the PSCA GAP-associated protein GAP.
The psca expression analysis also is used as the instrument of identifying and estimating the reagent of regulating the psca gene expression.For example, psca expression significantly raises in prostate cancer, and in the cancer of the listed tissue of Table I, expresses.Identify that inhibition PSCA expresses or the molecule or the biological reagent of over-expresses have therapeutic value in cancer cells.For example, can adopt the screening method that quantizes psca expression through RT-PCR, nucleic acid hybridization or antibodies to identify this reagent.
VIII.) method of monitoring PSCA-genes involved and product state thereof
It is a rapid process of multistep that known cancer forms; Wherein, Cell growth progressively imbalance simultaneously cell develop into precancerous condition by the normal physiological state and (for example see Alers etc. to cancerous state then; Lab Invest.77 (5): 437-438 (1997) and Isaacs etc., Cancer Surv.23:19-32 (1995)).In the literary composition, the evidence (like the unusual psca expression in the cancer) of inspection biological sample dysregulated cellular growth can be discovered this abnormal physiology state in early days before pathological state (like cancer) develops into the treatment stage that option is more limited or prognosis is worse.In this embodiment, can the state of the interior PSCA of the state of PSCA in the interested biological sample and for example corresponding normal specimens (as from individual or do not receive other individual sample of pathology effects) be compared.The change of PSCA state (comparing normal specimens) provides the evidence of dysregulated cellular growth in the biological sample.Remove to use not receive biological sample that pathology influences as normal specimens, we also can use predetermined standard value, and for example predetermined normal mRNA expression level (is for example seen Grever etc., J.Comp.Neurol.1996-12-9; 376 (2): 306-14 and USP 5,837,501) the PSCA state with sample compares.
In the literary composition, term " state " has the acceptable implication in this area, is meant the circumstance or state of gene and product thereof.Usually, those skilled in the art can use many parameters to estimate the circumstance or state of gene and product thereof.The biological activity of the gene product (like PSCA mRNA, polynucleotide and polypeptide) of the location (location that comprises the psca expression cell) of the gene product that these parameters include but not limited to express and level, expression.Usually, the change of PSCA state comprises the variation of PSCA and/or psca expression cellular localization, and/or PSCA mRNA and/or protein expression increase.
The PSCA state of sample can be analyzed with many methods well known in the art, includes but not limited to immunohistochemical analysis, in situ hybridization, on laser capture micro-dissections sample, carries out RT-PCR analysis, Western engram analysis and tissue array analysis.The typical method of estimating psca gene and gene product state can find in following document; For example: " the molecular biology fresh approach " that Ausubel etc. compile; 1995, the 2 (Northern traces), 4 (Southern traces), 15 (immunoblottings) and 18 (pcr analysis) unit.Therefore; The PSCA state that those skilled in the art can in all sorts of ways and estimate biological sample, comprising but be not limited to that Northern that genome Southern analyzes (be used for test example such as psca gene in disorder), PSCA mRNA analyzes and/or pcr analysis (be used for test example such as polynucleotide sequence or PSCA mRNA changes of expression level) and Western and/or immunohistochemical analysis variation and/or the PSCA albumen of variation, polypeptide localized variation in sample, PSCA protein expression level and combining of polypeptide binding partners of test example such as peptide sequence (be used for).Detectable PSCA polynucleotide comprise, for example, and psca gene or its fragment, PSCA mRNA, alternately splice variant, PSCA mRNA and the recombinant DNA or the RNA molecule that contain the PSCA polynucleotide.
The expression characteristic of PSCA makes its diagnostic flag that becomes limitation and/or shift disease, and information is provided for the growth of biological sample or oncogenic potential.Specifically, the state of PSCA provides useful information for predicting susceptibility, progress and/or tumor invasion property to the specified disease stage.The invention provides method and the test of confirming PSCA state and the cancer (like the cancer of the listed tissue of Table I) of diagnosis expression PSCA.For example; Owing to compare normal prostate tissue; PSCA mRNA expresses in prostate gland and other cancer so to heavens; The test of estimating interior PSCA mRNA transcript of biological sample or protein level can be used to diagnosis and PSCA imbalance diseases associated, and can when confirming suitable treat-ment, provide the information of forecasting of usefulness.
The information that the expression status of PSCA provides comprises existence, state and the location of heteroplasia cell, precancerous cell and cancer cells; Prediction is to the susceptibility in various diseases stage and/or the aggressive of measurement tumour.In addition, this expression characteristic makes it can be used as the photographic developer that shifts disease.Therefore, one aspect of the present invention relates to the various molecules predictions and the diagnostic method of PSCA state in the detection of biological sample (as from the sample of individuality of suffering from or be suspected to have with the dysregulated cellular growth pathology (like cancer) that is characteristic).
As stated, the PSCA state of biological sample can detect through many methods well known in the art.For example, the PSCA state of taking from the biological sample of body privileged site can detect through there being or not existing psca expression cell (as expressing PSCA mRNA or the proteic cell of PSCA) in the assessment sample.For example; When finding this cell in the biological sample (like lymphoglandula) that does not under normal circumstances contain the cell of expressing PSCA; Because this variation of PSCA state is relevant with dysregulated cellular growth usually in the biological sample, therefore this detection can provide the evidence of dysregulated cellular growth.Specifically, dysregulated cellular growth index is cancer cells is transferred to health from original organ (like prostate gland) other zone (like lymphoglandula).In the literary composition, the evidence of dysregulated cellular growth is importantly to be, this be because; For example; Can in a certain proportion of patients with prostate cancer, detect recessive nodus lymphoideus transferring rate, and this transfer is relevant with known disease progression predictor, and (see for example Murphy etc., Prostate 42 (4): 315-317 (2000); Su etc., Semin.Surg.Oncol.18 (1): 17-28 (2000) and Freeman etc., J Urol 1995-8154 (2 Pt 1): 474-8).
On the one hand; The invention provides the method for monitoring psca gene product; This method is through confirming the state of the psca gene product that individual cells is expressed; Said individuality is suspected to have and dysregulated cellular growth diseases associated (like hyperplasia or cancer), and the state that will so confirm then compares with the state of the psca gene product of corresponding healthy tissues.Healthy tissues exists in the specimen in unusual this individual cell of psca gene product explanation and has dysregulated cellular growth relatively.
On the other hand; The invention provides for having the useful test of cancer in definite individuality, this method comprises that detection test cell or the interior PSCA mRNA of tissue sample or protein expression significantly raise with respect to the normal cell or the in-house expression level of correspondence.Can be such as but not limited to the listed existence of organizing inner evaluation PSCA mRNA of Table I.Because corresponding healthy tissues is not expressed PSCA mRNA or expressed with lower level, reproduction, existence and/or seriousness that significant psca expression can be used to explain cancer appear in any of these tissue.
In relevant embodiment, the PSCA state is confirmed at protein level rather than nucleic acid level.For example, this method comprises the proteic level of PSCA of the cell expressing of confirming the test organization sample, and the level that will confirm like this compares with the interior PSCA level of expressing of corresponding normal specimens.In one embodiment, for example assessed the proteic existence of PSCA with immunohistochemical method.The psca antibody or the binding partners that can detect the PSCA protein expression are used to the various mode determinations that are used for this purpose well known in the art.
In another embodiment, we can estimate the state of PSCA Nucleotide and aminoacid sequence in the biological sample, to identify the disorder in these molecular structures.These disorders can comprise insertion, disappearance, replacement etc.This evaluation is useful because in a large amount of and growth imbalance phenotype proteins associated matter, observe Nucleotide and aminoacid sequence disorder (for example see Marrogi etc., 1999, J.Cutan.Pathol.26 (8): 369-378).For example, the sudden change in the PSCA sequence can be explained the existence or the promotion of tumour.Therefore, the sudden change of PSCA shows potential loss function or tumor growth quickening, and this mensuration has diagnosis and predictive value.
Test disorderly in many observation Nucleotide and the aminoacid sequence is well known in the art.For example, observe the size and the structure of psca gene product nucleic acid or aminoacid sequence through Northern described here, Southern, Western, PCR and dna sequencing method.In addition, observe other method of disorder in Nucleotide and the aminoacid sequence, like single-strand conformation polymorphism analysis, be well known in the art (see the USP of for example delivering on September 7th, 1,999 5,382,510 and deliver January 17 nineteen ninety-five 5,952,170).
In addition, but the methylation state of psca gene in our the detection of biological sample.The unusual demethylation and/or the supermethylation on gene 5 ' regulatory region CpG island often come across not dead cell and transformant, and can cause heterogeneic expression to change.For example; π-class glutathione S-transferase (a kind of in normal prostatic expressed protein; But in the prostate cancer more than 90%, do not express) the promotor supermethylation permanent silence is transcribed this gene; And be that the most frequent detected genome changes (De Marzo etc., Am.J.Pathol.155 (6): 1985-1992 (1999)) in the prostate cancer.In addition, this variation at least 70% senior prostatic intraepithelial neoplasm appearance pathology (PIN), occur (Brooks etc., Cancer Epidemiol.Biomarkers Prev., 1998,7:531-536).In another embodiment; Deoxidation-azacytidine is induced LAGE-I tumour-specific gene (this gene is not expressed in normal prostatic in the lymphocytoblast like cell; But in the prostate cancer of 25-50%, express) expression; Explain tumour express be since demethylation cause (Lethe etc., Int.J.Cancer 76 (6): 903-908 (1998)).The test of many detection gene methylation states is well known in the art.For example, we can use the methylation state of the Restriction Enzyme that methylates sensitivity but can not cut the sequence that contains the methylated CpG site being understood the CpG island in the Southern hybridizing method.In addition, MSP (methylation status of PTEN promoter) can show the methylation state in all the CpG sites that exist in the gene CpG island of giving rapidly.This process comprises that elder generation with sodium sulfite anhy 96 (it will change into uridylic to all unmethylated cytosine(Cyt)s) modifying DNA, increases with the Auele Specific Primer that methylates with non-not methylate DNA then.Disorderly method also can find in " molecular biology fresh approach " Unit (1995) the 12nd of volumes such as for example Frederick M.Ausubel about methylating.
Gene amplification is the method for the another kind of PSCA of acquisition state.The directly gene amplification of working sample; (the Thomas that transcribes that quantizes mRNA of Southern trace or the Northern trace through routine for example; 1980; Proc.Natl.Acad.Sci.USA, 77:5201 5205), Dot blot (DNA analysis) or in situ hybridization, use probe here according to the appropriate flags of the sequence that provides.Perhaps, can use the antibody of the specific duplex of identification, comprising DNA duplex, RNA duplex and DNA RNA heterozygote duplex or DNA protein duplex.Antibody is labeled and measures then, and wherein duplex is attached to the surface, thereby forms duplex on the surface, therefore can detect whether there is the antibody that is attached to duplex.
Usually can detect biopsy or peripheral blood to understand existing of cancer cells, for example use Northern trace, Dot blot or RT-PCR to analyze and detect psca expression.The PSCA mRNA that exists RT-PCR to increase has shown the existence of cancer.The RT-PCR test is well known in the art.Whether the RT-PCR test of having estimated the peripheral blood tumour cell at present can be used for diagnosis and handles multiple human solid tumor.In the prostate cancer field, these comprise RT-PCR test (Verkaik etc., 1997, the Urol.Res.25:373-384 that detects the cell of expressing PSA and PSM; Ghossein etc., 1995, J.Clin.Oncol.13:1195-2000; Heston etc., 1995, Clin.Chem.41:1687-1688).
Another aspect of the present invention is the individual susceptibility to cancer of assessment.In one embodiment, prediction comprises PSCA mRNA or PSCA albumen in the test set tissue samples to the method for cancer susceptibility, and it exists explanation to the cancer susceptible, and wherein the expression degree of PSCA mRNA is corresponding with the susceptibility degree.In with specific embodiments, detected the situation that exists of PSCA in prostate gland or other tissue, exist the PSCA explanation to prostate cancer susceptible (or occur or exist tumor of prostate) in the sample.Similarly, we can assess PSCA Nucleotide and aminoacid sequence in the biological sample integrity to identify the disorder in these molecular structures, said disorder such as insertion, disappearance, replacement etc.Having one or more disorders in the sample P SCA gene product is indexs of cancer susceptibility (or occur or exist tumour).
The present invention also comprises the method for measuring tumor invasion property.In one embodiment; The method of measuring tumor invasion property comprises PSCA mRNA or the proteic level of PSCA of confirming tumor cells expression; With the level of so confirming with take from PSCAmRNA or the proteic level of PSCA expressed in the corresponding healthy tissues of same individuality or healthy tissues reference sample and compare; Wherein, relative normal specimens, the proteic expression degree of PSCA mRNA or PSCA has been explained the aggressive degree in the tumor sample.In one embodiment, the aggressive of tumour is to estimate through the expression degree of confirming PSCA in the tumour cell, and the high more explanation of expression level is invasive tumor.Other embodiment has been estimated the integrity of PSCA Nucleotide and aminoacid sequence in the biological sample, identifying the disorder in these molecular structures, said disorder such as insertion, disappearance, replacement etc.Having one or more disorderly explanations is the stronger tumours of aggressive.
Another embodiment of the invention relates to the method for interior malignant tumour of for some time in the development of individuality of observing.In one embodiment; Observe interior malignant tumour of for some time and comprise PSCA mRNA or the proteic level of PSCA of confirming that the tumor sample inner cell is expressed in the method for the development of individuality; PSCA mRNA or the proteic level of PSCA expressed in homologue's sample that the level of so confirming and different time are taken from same individuality compare; Wherein, the information that PSCA mRNA that expresses in the tumor sample in for some time or the proteic degree of PSCA provide cancer development.In one embodiment, through measuring the development that the PSCA that expresses in for some time inner tumour cell estimates cancer, expressing in for some time raises shows cancer development.Simultaneously, we can estimate the integrity of PSCA Nucleotide and aminoacid sequence in the biological sample, and to identify the disorder in these molecular structures, said disorder such as insertion, disappearance, replacement etc. exist one or more disorders that cancer development has been described.
Above-mentioned diagnostic method also can with any associating in multiple prediction known in the art and the diagnostic method.For example; Another embodiment of the invention relates to the expression (or the disorder in psca gene and the psca gene product) and the corresponding to method of malignant tumour correlation factor of observing psca gene and psca gene product, and this method is used as the means of diagnosis and prediction group tissue samples state.Can adopt the multiple factor relevant with malignant tumour; Like (the for example expression of the PSA of prostate cancer, PSCA and PSM of malignant tumour Expression of Related Genes; Or the like) and total cytological observation (see for example Bocking etc., 1984, Anal.Quant.Cytol.6 (2): 74-88; Epstein, 1995, Hum.Pathol.26 (2): 223-9; Thorson etc., 1998, Mod.Pathol.11 (6): 543-51; Baisden etc., 1999, Am.J.Surg.Pathol.23 (8): 918-24).For example; Owing to have one group and the corresponding to specificity factor of disease; Therefore this provides important information for diagnosis and prediction group tissue samples state, and the expression disorder of psca gene and psca gene product (or to) and the corresponding to method of other malignant tumour correlation factor of observing psca gene and psca gene product are useful.
In one embodiment; Observe the expression (or the disorder in psca gene and the psca gene product) and the corresponding to method of other malignant tumour correlation factor interior PSCA mRNA of essential test set tissue samples or proteinic over-expresses of psca gene and psca gene product; PSA mRNA or proteinic over-expresses (the perhaps expression of PSCA or PSM) in the test set tissue samples, and the consistence of observing PSCA mRNA or protein and PSA mRNA or proteinic over-expresses (the perhaps expression of PSCA or PSM).In one embodiment; Detected the expression of PSCA and PSA mRNA in the prostata tissue, the over-expresses consistent existence that prostate cancer is described of interior PSCA of sample and PSA mRNA, to the recurrence or the state of the susceptibility or the tumor of prostate of prostate cancer.
Here described the method that detects and quantize PSCA mRNA or protein expression, standard nucleic acid and protein detection and quantification technique are well known in the art.The standard method of detection and quantification PSCA mRNA comprises that the RT-PCR of the in situ hybridization of the PSCA riboprobe of applying marking, the Northern trace that uses the PSCA polynucleotide probes and correlation technique, use PSCA Auele Specific Primer analyzes and the detection method of other amplification type, and branched DNA, SISBA, TMA or the like are for example arranged.In one embodiment, adopt sxemiquantitative RT-PCR to detect and quantize the expression of PSCA mRNA.Can use the primer of any amount of PSCA that can increase for this purpose, include but not limited to specifically described various primer sets here.In one embodiment, in the immunohistochemistry of biopsy is measured from using polyclone or the monoclonal antibody with wild-type psca protein specific reaction.
IX.) with the evaluation of PSCA interacting molecule
Any through in the acceptable several different methods in this area, those skilled in the art can identify and PSCA interacting proteins, small molecules and other reagent with the PSCA albumen and the nucleotide sequence that disclose here.For example, the system of our a kind of so-called interaction trap capable of using (also becoming " double cross test ").In this system, molecule and transcription factor interaction that instructs reporter gene expression and reconstruction, thereby detectable reporter expression of gene.The reconstruction of other system through the eukaryotic transcription activator be identification of protein-protein interaction in vivo, sees the USP of for example delivering on September 21st, 1,999 5,955; Delivered on July 20th, 280,1999 5,925; Delivered on December 8th, 523,1998 5,846; Delivered on December 21st, 722 and 1999 6,004,746.This area also exist based on the algorithm of genomic predicted protein matter function (see for example Marcotte etc., Nature 402:41999 November 4,83-86).
Perhaps, we can screen peptide library and identify and the interactional molecule of psca protein sequence.In the method, through coding in the screening library at random or the incompatible evaluation of controlled amino acid set combine the peptide of PSCA.The peptide of this library coding is used as the expressing fusion protein of phage coat protein, can select the proteic phage particle of anti-PSCA then.
Therefore, the structure to part or acceptor molecule before need not has any understanding just to identify the peptide that serves many purposes, and for example treats, predicts or diagnose peptide.Can be used to identify that typical peptide library and screening method with the interactional molecule of psca protein sequence are described in, for example, the USP of delivering on March 3rd, 1998 on March 31st, 5,723,286 and 1998 delivered 5,733,731.
Perhaps, the clone of expression PSCA is used to identify the protein-protein interaction of PSCA mediation.The available immunoprecipitation technology of this interaction detects (sees for example Biochem.Biophys.Res.Commun.1999 such as Hamilton B.J., 261:646-51).Available resisting-psca antibody immunoassay PSCA albumen from the clone of expressing PSCA.Perhaps, anti-His-tag antibody can in the clone of engineered expression PSCA and His-tag (above-mentioned carrier) fusions, adopted.Can detect the protein bound of the complex body of immunoprecipitation through following method, for example Western trace, 35S-methionine(Met) labelled protein, protein micrometering preface, silver dyes and two dimensional gel electrophoresis.
Can identify and interactional small molecules of PSCA and part through relevant embodiment with this screening assay.For example, can identify the small molecules of disorderly protein function, comprise disorderly PSCA mediation phosphorylation and dephosphorylation ability, with the molecule of DNA or RNA interaction of molecules, as adjusting cell cycle, second messenger's signal transduction or tumorigenic index.Similarly, can identify the small molecules of regulating PSCA-relevant ions passage, protein pump or cell communication function, and be used for treating patient and (see for example Hille with cancer of expressing PSCA; B., " ionic channel of excitable membrane " (Ionic Channels of ExcitableMembranes), second edition; Sinauer Assoc.; Sunderland, MA, 1992).In addition, the part of adjusting PSCA function can combine the ability of PSCA and activated receptor construction to identify based on it.Typical method is described in the USP of for example delivering on July 27th, 1,999 5,928,868, and comprises the rare method that part is micromolecular hybrid ligand that is formed up to.In an exemplary embodiment, utilize and come coexpression hybrid ligand/micromolecular fusion rotein and cDNA library transcription activating protein through the engineered cell that can express PSCA and the conjugated protein fusion rotein of DNA-.This cell also contains reporter gene, and the expression of reporter gene makes first and second fusion roteins approaching each other, just can take place near having only when hybrid ligand combines two target sites of hybridizing on the albumen each other.Select to express those cells of reporter gene, and identified unknown small molecules or unknown part.This method can be identified the regulator that activates or suppress PSCA.
One embodiment of the invention comprises screening and the interactional method of PSCA aminoacid sequence shown in Figure 1, this method comprises makes a group molecule contact PSCA aminoacid sequence, make this group molecule and PSCA aminoacid sequence be easy to interact, confirm to exist under the interactional condition with the interactional molecule of PSCA aminoacid sequence, will get along well then interactional molecule of PSCA aminoacid sequence and and the step of the interactional molsep of PSCA aminoacid sequence.In one embodiment, this method also comprises purifying, signature analysis and evaluation and the interactional molecule of PSCA aminoacid sequence.The molecule that identifies can be used to regulate the function of PSCA.In an embodiment preferred, the PSCA aminoacid sequence links to each other with peptide library.
X.) Treatment and composition for
Evaluation normal expression but the PSCA that also in the listed cancer of Table I, expresses will start this type of method for cancer of many treatments in limited tissue.
Notice that the targeting anti-tumor therapy also was effective when promptly convenient target protein was expressed in healthy tissues (or even normal vital organ tissue).Vital organ is the essential organ that earns a bare living, like heart or colon.Non-vital organ is can excise but the individual organ that still can survive.The example of non-vital organ has ovary, mammary gland and prostate gland.
For example; Herceptin
Figure G05817239920061201D000591
is the medicine of a kind of FDA approval, and it is taken place by the protein of the various HER2 of being called, HER2/neu and erb-b-2, and immunoreactive antibody forms.It is sold by Genentech and is a kind of antitumour drug that obtains business success.2002 Herceptin
Figure G05817239920061201D000592
sales volume reach about 400,000,000 dollars.Herceptin
Figure G05817239920061201D000593
is used to treat the positive metastatic breast cancer of HER2.Yet the expression of HER2 is not limited to this tumour.This protein is expressed in many healthy tissuess.Specifically; Known HER2/neu is present in normal kidney and the heart, and these tissues are present in the middle of all people that accept Herceptin
Figure G05817239920061201D000594
.Latif, Z. etc. also confirm to exist in the normal kidney HER2/neu (B.J.U.International (2002) 89:5-9).Shown in the document (it has estimated the preferred indication whether renal cell carcinoma should become anti--HER2 antibody such as Herceptin), the benign renal tissue is made this albumen and mRNA.Especially, HER2/neu albumen strong over-expresses in the benign renal tissue.
Although HER2/neu expresses in vital tissues such as heart and kidney, Herceptin be a kind of very effective, obtain the FDA approval and obtain the medicine of business success.Herceptin is to the influence of heart tissue, and promptly " cardiac toxic (cardiotoxicity) " only is a kind of treatment spinoff.When separately with Herceptin treatment patient, only in few patient remarkable cardiac toxic can take place.For cardiac toxic is minimized, has more strict access requirement for the treatment of carrying out with HER2/neu.Before can treating, need assess the factor of hereditary defect for example to the cardiac condition influence.
Show normal expression although it should be noted that nephridial tissue especially, even possibly be higher than the expression of heart tissue, any valuable Herceptin spinoff does not appear in kidney.In addition, in the various healthy tissuess of expressing HER2, have only seldom spinoff can take place.Have only heart tissue valuable spinoff to occur.Express tissue especially significantly at HER2/neu,, any spinoff do not occur like nephridial tissue.
In addition, find with EGF-R ELISA (EGFR); Erbitux (ImClone) has good therapeutic action for the antitumor therapy of target spot.EGFR also expresses in many healthy tissuess.Spinoff after the use anti-EGFR therapy in the healthy tissues is very limited.Common spinoff is in 100% the patient who receives treatment, all to observe serious fash in the EGFR treatment.
Therefore, in healthy tissues even the normal vital tissues expression of target protein can not destroy with this protein be that the targeting preparation of target is in the effect of some this albumen also being crossed in the tumour of expressing as therapeutical agent.For example, the expression in the vital organ itself is not deleterious.In addition, but can remove the organ (for example prostate gland and ovary) that is considered to administration and can not cause death.At last, some vital organ does not receive the influence of normal organ expression owing to have immunity privilege (immuno-privilege).Organ with immunity privilege is to avoid the blood influence and be not subject to the organ that immunotherapy influences thus through blood-organ barrier.Example with organ of immunity privilege is brain and testis.
Therefore, the treat-ment of inhibition PSCA protein-active can be used for suffering from the patient of the cancer of expressing PSCA.These treat-ment are divided into 3 types usually.The first kind is because of PSCA is relevant with growth of tumour cell, makes growth of tumour cell suppressed or growth retardation or induce killing and wounding it through adjustment PSCA function.Second type comprises various inhibition PSCA albumen and its binding partners or other protein bound or associating method.The 3rd type comprises that various inhibition psca genes are transcribed or the method for PSCA mRNA translation.
X.A.) anti-cancer vaccine
The invention provides the cancer vaccine that contains PSCA GAP-associated protein GAP or PSCA-associated nucleic acid.With regard to the expression of PSCA, cancer vaccine can prevent and/or treat the cancer of expressing PSCA, and the very little or not influence to the influence of non-target tissue.In vaccine, using the tumour antigen that produces cell-mediated HI is well known in the art with the method for carrying out anticancer therapy; And be used to people PSMA and rodent PAP immunogen are used for treatment of prostate cancer (Hodge etc.; 1995, Int.J.Cancer 63:231-237; Fong etc., 1997, J.Immunol.159:3113-3117).
Nucleic acid molecule through using PSCA GAP-associated protein GAP or coding PSCA and can express and present the immunogenic recombinant vectors of PSCA (comprising many t cell epitopes or antibody usually) and be not difficult to implement this method.For example Heryln etc. (sees, Ann Med 1999-2 31 (1): 66-78 in the vaccine system that one skilled in the art will appreciate that many conveying immunoreactivity epi-positions known in the art; Maruyama etc., Cancer ImmunolImmunother 2000-649 (3): 123-32).In brief, this method that in Mammals, produces immunne response (for example cell-mediated and/or HI) may further comprise the steps: make mammiferous immunity system contact certain immunoreactivity epi-position (epi-position on PSCA albumen for example shown in Figure 1 or its analogue or the homologue) thus make Mammals produce the specific immune response (for example producing the antibody of this epi-position of specific recognition) of this epi-position.
Complete PSCA albumen, its immunogenicity zone or epi-position can make up through the whole bag of tricks carries.This vaccine composition can comprise, for example, and lipopeptid (Vitiello for example; A. etc., J.Clin.Invest.95:341,1995), be wrapped in the peptide combinations that gathers in (DL-lactide-co-glycolide) (" PLG ") microsphere and (see for example Eldridge etc.; Molec.Immunol.28:287-294; 1991:Alonso etc., Vaccine 12:299-306,1994; Jones etc., Vaccine 13:675-681,1995), be contained in peptide combinations in the immunostimulating complex (ISCOM) and (for example see Takahashi etc., Nature344:873-875,1990; Hu etc., Clin Exp Immunol.113:235-243,1998), multiple antigenic peptide system (MAP) (sees 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; (Perkus, M.E. etc. draw certainly: " vaccine development general introduction " (the Coneepts in vaccine development) that Kaufmann, S.H.E. compile, the 379th page, 1996 to be used for the peptide (normally crystalline peptide) of trajectory delivery system and the peptide of viral delivery vehicles; Chakrabarti, S. etc., Nature 320:535,1986; Hu, S.L. etc., Nature 320:537,1986; Kieny, M.-P. etc., AIDS Bio/Technology 4:790,1986; Top, F.H. etc., J.Infect.Dis.124:148,1971; Chanda, P.K. etc., Virology 175:535,1990), particle (for example, Kofler, N. etc., J.Immunol.Methods.192:25,1996 in virus or synthetic source; Eldridge, J.H. etc., Sem.Hematol.30:16,1993; Little Falo, L.D. etc., 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. etc., Vaccine 11:293,1993), liposome (Reddy, R. etc., J.Immunol.148:1585,1992; Rock, K.L., Immunol.Today 17:131,1996), cDNA (Ulmer, J.B. etc., Science 259:1745,1993 that perhaps naked cDNA or particle absorb; Robinson, H.L., Hunt, L.A. and Webster, R.G., Vaccine 11:957,1993; Shiver, J.W. etc. draw certainly: " vaccine development general introduction " (the Concepts in vaccine development) that Kaufmann, S.H.E. compile, the 423rd page, 1996; Cease, K.B. and Berzofsky, J.A., Annu.Rev.Immunol.12:923,1994, and Eldridge, J.H. etc., Sem.Hematol.30:16,1993).Also can use the conveying technology of targeting toxins, also become receptor-mediated target-seeking, like Avant Immunotherapeutics, Inc. (Needham, those technology Massachusetts).
In the patient who suffers from the PSCA associated cancer, vaccine composition of the present invention also can be treated for example operation of method for cancer, chemotherapy, pharmacological agent, radiotherapy etc. with other and combined, and comprises with immunological adjuvants such as IL-2, IL-12, GM-CSF uniting use.
Cell vaccine:
Available specific algorithm is identified and is combined the allelic peptide of corresponding HLA (to see for example Table IV to confirm the CTL epi-position in the PSCA albumen; Epimer TMAnd Epimatrix TM, Brown University (URLbrown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.htm l); And BIMAS (URL bimas.dcrt.nih.gov/; SYFPEITHI, URLsyfpeithi.bmi-heidelberg.com/).In an embodiment preferred; The PSCA immunogen contains the aminoacid sequence of one or more usefulness technical evaluation well known in the art; Sequence shown in Table V-XVIII and XXII-LI, or 8,9,10 or 11 HLA I class motif/hyper-base prefaces (for example Table IV (A), Table IV (D) or Table IV (E)) peculiar amino acid whose peptide is arranged and/or have the peptide of 9 the amino acid whose HLA of comprising II class motif/hyper-base prefaces (for example Table IV (B) or Table IV (C)) at least.Known in the art, HLA I class combines the ditch end to close basically, therefore has only the peptide of certain size range could conform to ditch to merge with it and combines, and the length of HLA I class epi-position has 8,9,10 or 11 amino acid usually.On the contrary, it is open basically that HLA II class combines the ditch end; Therefore have an appointment 9 or more the peptide of amino acids can be combined by HLA II quasi-molecule.Because the combination ditch of HLA I and II class is different, so HLA I class motif is that length is special, and promptly the position 2 of I class motif is second amino acid that peptide amino arrives the carboxyl direction.The amino acid position of II class motif is just relative each other, rather than with respect to whole peptide, the amino and/or the C-terminal that promptly have the sequence of motif can combine other amino acid.HLA II class epi-position is that length has 9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 or 25 amino acid usually, or 25 more than the amino acid.
Many methods (for example producing first step of hybridoma) that in Mammals, produces immunne response known in the art.The method that in Mammals, produces immunne response comprises makes mammiferous immunity system be exposed to proteinic immunogenicity epi-position (for example PSCA albumen) to produce immunne response.A concrete embodiment is included in the method that produces in the host to the immunne response of PSCA, and this method is to make the host contact at least a PSCA B cell or cytotoxic t cell epitope or its analogue of capacity; And certain at least after this practice makes the host contact PSCA B cell or cytotoxic t cell epitope or its analogue more at interval.A concrete embodiment comprises the method for the immunne response that produces anti-PSCA GAP-associated protein GAP or artificial multi-epitope peptide: administration of human or other Mammals are contained in the PSCA immunogen (for example PSCA albumen or its peptide fragment, PSCA fusion rotein or analogue etc.) in the vaccine product.Usually, this vaccine product also contains suitable adjuvant (seeing for example USP 6,146,635) or general auxiliary epi-position, like PADRETM peptide (Epimmune Inc., San Diego, CA; See for example Alexander etc., J.Immunol.2000 164 (3); 164 (3): 1625-1633; Alexander etc., Immunity 1,994 1 (9): 751-761 and Alexander etc., Immunol.Res.1998 18 (2): 79-92).Another kind method is included in the immunogenic immunne response of the individual interior anti-PSCA of generation; Method is following: in the muscle of mammalian organism or skin, comprise the dna molecular of the immunogenic dna sequence dna of coding PSCA in the body, this dna sequence dna operability is connected in the adjusting sequence that the control dna sequence dna is expressed; Wherein said dna molecular is by cellular uptake, and dna sequence dna is at cell inner expression, and anti-this immunogenic immunne response (seeing for example U.S. Patent number 5,962,428) of generation.Also can choose wantonly and give gene vaccine agonist (facilitator), like the negatively charged ion lipid; Saponin(e; Lectin; Estrogens compounds; The hydroxylation low alkyl group; DMSO 99.8MIN.; And urea.In addition, the antiidiotypic antibody that can simulate PSCA is to produce the reaction for target antigen.
Nucleic acid vaccine:
Vaccine composition of the present invention comprises the form of nucleic acid-mediation.Can give patient's code book invention protein DNA or RNA.Can adopt the genetic immunization method to produce preventative or the therapeutic body fluid and the cellullar immunologic response of the cancer cells of the anti-PSCA of expression.Can be injected directly in the individual muscle or skin with the suitable construction of regulating sequence containing coding PSCA GAP-associated protein GAP/immunogenic DNA, thus the PSCA albumen/immunogen of muscle or skin cells this construction of picked-up and expression coding.Perhaps comprise the PSCA GAP-associated protein GAP in the vaccine.The immunogenic expression of PSCA GAP-associated protein GAP causes producing anti-preventative or therapeutic body fluid and the cellular immunity that has the proteic T cell of PSCA.Can use various preventative and therapeutic genes immunization technology (going up information and the bibliography of announcing) known in the art referring to for example internet address genweb.com.Conveying based on nucleic acid is described in, for example, and Wolff etc., Science 247:1465 (1990) and USP 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720.Comprise that based on the example of the conveying technology of DNA " naked DNA ", the short effect (bupivacaine (bupivicaine), polymkeric substance, peptide-mediation) conveying, cation lipid complex body and the conveying (" particle gun ") of particle-mediation or pressure-mediated conveying (see for example USP 5; 922,687).
From the purpose of therapeutic or preventative immunization, protein of the present invention can be expressed through virus vector or bacteria carrier.The various viral gene delivery system that can be used for the present invention practice includes but not limited to that cowpox, fowl pox, canary pox, adenovirus, influenza virus, poliovirus, adeno associated virus, slow virus and sindbis alphavirus (see for example Restifo; 1996, Curr.Opin.Immunol.8:658-663; J.Natl.Cancer Inst.87:982-990 (1995) such as Tsang).Also can use non-viral delivery system, method is in the patient, to introduce the naked DNA (for example at intramuscular or intradermal) of coding PSCA GAP-associated protein GAP with the inducing antitumor responsing reaction.
For example, vaccinia virus can be used as carrier to express the nucleotide sequence of coding peptide of the present invention.Introduce after the host, recombined vaccinia virus marking protein immunogenic peptide, thereby cause host's immunne response.The method that cowpox carrier and immunization are used for example is described in the USP 4,722,848.Another kind of carrier is BCG (Bacille Calmette Guerin).The BCG carrier is described in Stover etc., Nature351:456-460 (1991).Through the description here; Those skilled in the art give or immunization peptide of the present invention up to many other carrier available treatment property, for example adenovirus and adeno-associated virus vector, retrovirus vector, Salmonella typhi (Salmonella typhi) carrier, toxicide anthrax toxin carrier etc.
Therefore, gene delivery system is used to transport PSCA-associated nucleic acid molecule.Used total length people PSCA cDNA in one embodiment.Used the PSCA nucleic acid molecule of coding specific cytotoxic T lymphocyte (CTL) and/or antibody epitope in another embodiment.
Vaccine exsomatizes
Also available various stripped method produces immunne response.A kind of method comprises uses antigen presenting cell (APC), like dendritic cell (DC), so that patient's immune system is arrived in the PSCA antigen presentation.Dendritic cell express MHC I and II quasi-molecule, B7 are total to stimulator and IL-12, are highly single-minded antigen presenting cells therefore.In prostate cancer, PSMA (PSMA) pulse be used to the I clinical trial phase to stimulate immunity system (Tjoa etc., 1996, the Prostate 28:65-69 of patients with prostate cancer from the body dendritic cell; Murphy etc., 1996, Prostate 29:371-380).Therefore, dendritic cell can be used to the PSCA peptide is presented to the MHC I or the II quasi-molecule of T cell.In one embodiment, can be attached to the PSCA peptide pulse of MHC I class and/or II quasi-molecule from the body dendritic cell.In another embodiment, dendritic cell are by complete PSCA albumen pulse.Another embodiment comprises with various execution carriers known in the art makes psca gene over-expresses in dendritic cell; Said carrier is adenovirus (Arthur etc., 1997, Cancer GeneTher.4:17-25), retrovirus (Henderson etc. for example; 1996; Cancer Res.56:3763-3770), slow virus, adeno associated virus, DNA transfection (Ribas etc., 1997, Cancer Res.57:2865-2869) or tumour derived RNA transfection (Ashley etc.; 1997, J.Exp.Med.186:1177-1182).Also can be engineered the cell of expression PSCA express immunomodulator, like GM-CSF, and with it as immunizing agent.
X.B.) with PSCA as the target of treatment that with antibody is the basis
PSCA is very attractive target in the therapeutic strategy based on antibody.Known many kinds are used for the outer antibody strategy (referring to for example, complement and ADCCmediated killing as well as the use of intrabodies) with the interior molecule of born of the same parents of target born of the same parents in this area.Because the corresponding normal cell of PSCA is expressed by various cancerous cell lines; Prepared the PSCA-immunoreactivity compsn that general gives; Said composition has good susceptibility, and can be because of this immunoreactivity compsn and non-target organ and tissue combine toxigenicity, non-specific and/or non-target property effect.Can be used for the cancer that systemic treatment is expressed PSCA with the antibody of PSCA structural domain generation specific reaction, its can with toxin or therapeutical agent coupling, or as the exposed antibody that can suppress cell proliferation or function.
Can psca antibody be imported the patient, thereby make this antibodies in PSCA and adjust following function: for example with combine reacting to each other and the destruction tumour cell of mediation thereafter and/or suppress growth of tumour cell of object.The mechanism that this antibody-like has result of treatment can comprise: the cytolysis of complement-mediated, ADCC, to the adjusting of PSCA physiological function, part is combined or the inhibition of signal transduction pathway, the change and/or the apoptosis that distribute to the adjusting of tumour cell differentiation, to tumor angiogenesis factor.Examples include: for non-Hodgkin's lymphoma Rituxan?
Figure G05817239920061201D000651
for metastatic breast cancer, Herceptin?
Figure G05817239920061201D000652
and for colorectal cancer, Erbitux?
Figure G05817239920061201D000653
It will be understood by those skilled in the art that antibody can be used for selectively targeted and binding immunoassay originality molecule, the immunogenicity zone of PSCA sequence for example shown in Figure 1.In addition, those skilled in the art will appreciate that with antibody and cytotoxic agent coupling be very conventional (referring to for example, Slevers etc., Blood 93:113678-3684 (on June 1st, 1999)).With cytotoxic agent and/or therapeutical agent when for example directly sending the method for molecule (for example PSCA) the specificity bonded antibody coupling of they and this cell expressing to cell; Said cytotoxic agent will be to those cell performance its known biological effects (that is cytotoxicity).
Many compsns and the method that is used for antibody-cytotoxic agent conjugate with killer cell known in the art.Aspect cancer, method commonly used comprises the conjugate of the zoobiology significant quantity that has tumour, and said conjugate comprises and target agent (for example anti-psca antibody) selected cytotoxic agent of bonded and/or therapeutical agent, is easy to combine or navigate to cell surface.Typical embodiment is the method for giving the cell of expressing PSCA with cytotoxic agent and/or therapeutic agent delivery; Said method comprises makes cytotoxic agent and immunologic opsonin be incorporated into the antibody coupling of PSCA epi-position, and said cell is contacted with this antibody-drug conjugates.Another exemplary embodiment is the doubtful method of suffering from the individuality of metastatic cancer of treatment, said method comprising the steps of: parenteral route gives said individuality and comprises treatment significant quantity and pharmaceutical composition cytotoxic agent and/or therapeutical agent link coupled antibody.
Can according in the treatment of other types of cancer the various approach of successful Application adopt the cancer immunotherapy of anti-PSCA, the cancer of said other type includes but not limited to: colorectal carcinoma (Arlen etc., 1998; Crit.Rev.Immunol.18:133-138), multiple myeloma (Ozaki etc., 1997, Blood 90:3179-3186; Tsunenari etc., 1997, Blood 90:2437-2444), cancer of the stomach (Kasprzyk etc.; 1992, Cancer Res.52:2771-2776), B cell lymphoma (Funakoshi etc., 1996; J.Immunother.Emphasis Tumor Immunol.19:93-101), white blood disease (Zhong etc., 1996, Leuk.Res.20:581-589), colorectal carcinoma (Moun etc.; 1994, Cancer Res.54:6160-6166; Velders etc., 1995, Cancer Res.55:4398-4403) and mammary cancer (Shepard etc., 1991, J.Clin.Immunol.11:117-127).Some treatment approach relate to exposed antibody and toxin or ri coupling, for example respectively with Y 91Or I 131With anti-CD20 antibodies (for example, ZevalinTM, IDEC Pharmaceuticals Corp. or Bexxar TM, CoulterPharmaceuticals) coupling, other then relates to and gives antibody and other therapeutical agent jointly, for example give jointly HerceptinTM (trastuzumab) and taxol (Genentech, Inc.).Can be with said antibody and therapeutical agent coupling.For treatment of prostate cancer, for example can give psca antibody and radiotherapy, chemotherapy simultaneously and protect the hormone removal.Also can be with antibody and following material coupling: toxin (for example, Mylotarg TM, Wyeth-Ayerst, Madison, NJ; A kind of and antitumor antibiotics calicheamycin link coupled recombinant human IgG4 κ antibody) or maytansinol (maytansinoid, for example based on the tumor promotion prodrug of Taxan, TAP, platform; ImmunoGen, Cambridge, MA also can be referring to for example; USP 5,416,064) or AuristatinE (Nat Biotechnol.2003 July; 21 (7): 778-84.(Seattle?Genetics))。
Though the psca antibody treatment can be used for all stages of cancer, Antybody therapy particularly is suitable in late period or the metastatic carcinoma.Can be accepted one or the patient that takes turns chemotherapy more specify and to carry out Antybody therapy of the present invention.Perhaps, for the patient who does not accept chemotherapy, can Antybody therapy of the present invention be combined to carry out with chemotherapy or radiotherapy scheme.In addition, Antybody therapy can make the coupling chemotherapeutics that adopt to reduce dosage become possibility, especially in that toxic tolerance is not among the good patient to chemotherapeutics.(Cancer Res.53:4637-4642 such as Fan; 1993), (International J.of Onco.9:217-224 such as Prewett; 1996) and described the common use of multiple antibody and chemotherapeutics among the Hancock etc. (Cancer Res.51:4575-4580,1991).
Though the psca antibody treatment can be used for all stages of cancer, Antybody therapy particularly is suitable in late period or the metastatic carcinoma.Can be accepted one or the patient that takes turns chemotherapy more specify and to carry out Antybody therapy of the present invention.Perhaps, for the patient who does not accept chemotherapy, can Antybody therapy of the present invention be combined to carry out with chemotherapy or radiotherapy scheme.In addition, Antybody therapy can make the coupling chemotherapeutics that adopt to reduce dosage become possibility, especially in that toxic tolerance is not among the good patient to chemotherapeutics.
Cancer patients's psca expression or level be can estimate, the immunohistochemistry evaluation of tumor tissues, quantitative PSCA imaging or the existence of other reliable indication psca expression and the technology of degree preferably adopted.The immunohistochemical analysis of tumor biopsy or specimens from pri is preferred for this purpose.The immunohistochemical analysis method of tumor tissues is well known in the art.
Anti--PSCA the monoclonal antibody of treatment prostate cancer and other cancer comprises those and can imitate the antibody of immunne response by force or directly cause Cytotoxic antibody by inducing antitumor.Just in this point; Anti--PSCA monoclonal antibody (MAb) can cause oncolysis through cell cytotoxicity (ADCC) mechanism complement-mediated or that antibody relies on, the effector cell Fc acceptor site interaction on the complete Fc part that these two kinds of mechanism all require immunoglobulin molecules and the complement proteins.In addition, the anti--PSCAMAb that tumor growth is brought into play direct biological effect can be used for treating the cancer of expressing PSCA.The direct mechanism of action of cell toxicant MAb comprises: the characteristic of cell growth inhibiting, adjusting cytodifferentiation, the adjusting tumor vessel formation factor and apoptosis-induced.With the in vitro tests of multiple evaluation necrocytosis estimated specific anti--mechanism of PSCA MAb performance antitumor action, these tests are normally known in the art, like cytolysis of ADCC, ADMMC, complement-mediated or the like.
Among some patients, use muroid or other non-human monoclonal antibodies or the chimeric MAb of people/mouse can induce medium immunne response to strong anti-non-human antibody.This can cause from circulation, removing antibody and reducing effect.When the most serious, this immunne response can cause forming in a large number immunocomplex, and this species complex possibly cause renal failure.Therefore, the monoclonal antibody that preferably is used for treat-ment of the present invention be long fully the people's or humanized, and combine target PSCA antigen, but low antigenicity arranged in the patient or do not have antigenicity with the high-affinity specificity.
Treat-ment of the present invention comprises the anti--PSCA MAb that gives separately and compsn or the mixture of other MAb.This MAb mixture has some advantage, but this is the different epi-positions of their MAb targets of containing, adopt different effect mechanisms or cell toxicant MAb and the MAb that depends on the immunological effect function directly made up.The MAb of this combination can bring into play synergistic therapeutic action.In addition, anti--PSCA MAb can give with other form of therapy, includes but not limited to various chemotherapeutics, male sex hormone blocker, immunomodulator (for example IL-2, GM-CSF), operation or radiotherapy.Anti--PSCA MAb " exposes " form with it or non-binding form gives, perhaps can combine with therapeutical agent.
Anti--psca antibody preparation can give the approach that antibody is transported to tumour cell through any.Route of administration includes but not limited in the intravenously, intraperitoneal, intramuscular, knurl, intradermal or the like.Treatment generally comprises through acceptable route of administration such as intravenous injection (IV) and repeats to resist-the psca antibody preparation, and its dosage range generally is about every kg body weight 0.1 .2 .3 .4 .5 .6 .7 .8 .9., 1,2,3,4,5,6,7,8,9,10,15,20 or 25 milligram.Usually, the dosage of 10-1000mg MAb is effectively and is to tolerate weekly.
Based on using Herceptin TMThe clinical trial of treatment metastatic breast cancer, a kind of acceptable dosage is, initial load dosage is about every kg of patient body weight intravenous injection 4mg, then with the dosage of about 2mg/kg weekly intravenous injection once resist-PSCA MAb preparation.Preferably, initial load dosage was inculcated and is given with 90 minutes or longer time.Regularly maintenance dose gives with 30 minutes or inculcating of longer time, but as long as initial dose well tolerable.Be proficient in those skilled in the art and all know, in concrete pathology, many factors can influence the ideal dosage.These factors comprise; For example, expression degree in the patient of the binding affinity of used Ab or MAb and transformation period, PSCA, circulation get rid of the antigenic degree of PSCA, required steady state AC level, therapeutic frequency, unite chemotherapeutics or the influence of other reagent and concrete patient's the healthy state of use with treat-ment of the present invention.
Randomly, but in the given sample of evaluate patient the level (the for example level of cycle P SCA antigen and/or psca expression cell) of PSCA to help to confirm the most effectively dosage etc.This evaluation also can be used to monitor the purpose of whole treatment; And assessment (the for example urinary cytology in the bladder cancer treatment and/or the immunocytochemistry level that can combine other parameter; Or through analogizing the Serum PSA level in the prostate cancer therapy) weigh result of treatment.
Anti--idiotype is anti--and vaccine element that psca antibody also can be used as anti-cancer therapies induces the immunne response to the cell of expressing the PSCA GAP-associated protein GAP.Specifically, the generation of anti--idiotype antibody is well known in the art; This method be fit to produce anti--idiotype of the epi-position on the simulation PSCA GAP-associated protein GAP anti--psca antibody (for example see Wagner etc., 1997, Hybridoma 16:33-40; Foon etc., 1995, J.Clin.Invest.96:334-342; Herlyn etc., 1996, Cancer Immunol.Immunother.43:65-76).This resisting-idiotype antibody can be used for the cancer vaccine method.
An object of the present invention is to provide the psca antibody that suppresses or delay to express the growth of tumour cell of PSCA.Another object of the present invention provide in Mammals, (be preferably the mankind) thus suppress the method that vasculogenesis and other biological function reduce tumor growth, said method adopts psca antibody, especially with these psca antibodies and radiotherapy or chemotherapeutics or the two coupling.
In one embodiment, when coupling psca antibody and chemotherapeutics or radiotherapeutic agents or their combined therapy comprise the tumour of people's tumour, has synergy.In other words, psca antibody to the restraining effect of tumor growth when coupling chemotherapeutics or radiotherapy or their combination, than improving manyly in the anticipation.Synergy can through for example with single with the psca antibody treatment or with the adduction comparison of psca antibody and chemotherapeutics or radiotherapy, combination therapy shows more greatly during the restraining effect ratio of tumor growth is envisioned.Preferably, adopt alleviating of cancer to prove synergy, this adduction treatment that alleviates with respect to the treatment of adopting exposed psca antibody or employing psca antibody and chemotherapeutics or radiotherapy is beyond thought.
Adopt the psca antibody and the combination of chemotherapy or radiotherapy or the method that adopts both to suppress growth of tumour cell simultaneously to comprise: before beginning chemotherapy or the radiotherapy, during or give psca antibody afterwards; And their any combination (that is, before beginning chemotherapy and/or the radiotherapy with during, before and afterwards or before, during and afterwards).For example, before beginning radiotherapy and/or chemotherapy, between 1-60 days, between preferred 3-40 days, more preferably give said psca antibody between 5-12 days usually.Yet, can be according to the needs of treatment record and particular patients ', so that can being provided, efficacious therapy implements this method with the mode of final prolongation patient life.
The giving and can carry out in several ways of chemotherapeutics comprises that outer and approach carries out the general administration through gi tract.In one embodiment, with said psca antibody and chemotherapeutics as the molecule administration that separates.In another embodiment, said psca antibody is to adhere to (for example, through coupling) in chemotherapeutics.(referring to the embodiment of title) and (referring to the part of title) for " with PSCA as the target that with antibody is basic treatment " for " through people's clinical trial of treatment of the anti-psca antibody of end user in the body and diagnosis people cancer ".The specific examples of chemotherapeutics or chemotherapy comprises: cis-platinum, dicarbazine (DTIC), dactinomycin, mechlorethamine (mustargen), streptozocin, endoxan,, carmustine (BCNU), lomustine (CCNU), Dx (Zorubicin), daunorubicin, Procarbazine, MTC, cytosine arabinoside, VP, Rheumatrex, 5 FU 5 fluorouracil, vinealeucoblastine(VLB), vincristine(VCR), bleomycin, taxol (safe plain), docetaxel (TX), rIL-2, asparaginase, busulfan, carboplatin, CldAdo, dicarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprorelin, megestrol, melphalan, mercaptopurine, Plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, Plicamycin, streptozocin, Tamoxifen, teniposide, testolactone, Tioguanine, plug be for group, uracil mustard, vinorelbine, TV, taxol or their combination.
Be used for radioactive source with the psca antibody coupling and can be positioned at the outside or inner of the patient that receives treatment.When said radioactive source is positioned at patient's outside, this treatment is called as external beam radiotherapy (EBRT).When said radioactive source is positioned at patient's inside, this treatment is called as brachytherapy (BT).
Adopt the standard equipment of making for this purpose (for example AECL theratron and Varian clinac) to give this radiation according to the standard technique of knowing.Radiation dose depends on multiple factor known in the art.These factors comprise that the organ of receiving treatment, the healthy organ that in the radiation path, may be adversely affected, patient are to the tolerance of radiotherapy and the body region that need treat.This dosage is generally 1-100Gy, more specifically is 2-80Gy.Some dosage of having reported comprise: spinal cord is bestowed 35Gy, kidney is bestowed 15Gy, liver is bestowed 20Gy and prostate gland is bestowed 65-80Gy.Yet what should stress is that the present invention is not limited to any concrete dosage.Can be by the treatment doctor according to confirming dosage to the material elements under the stable condition (comprising above-mentioned factor).
External radiation source and get into distance between patient's the point and can be and to make killing and wounding and spinoff is minimized and reach the suitable any distance of equilibrated to target T cell.Usually the distance between external radiation source and entering patient's the point is 70-100cm.
Brachytherapy is normally through placing the patient to carry out radioactive source.Usually, radioactive source is placed on about 0-3cm place of the organ that will treat of distance.Known technology comprises in a matter, the chamber and surperficial brachytherapy.Can permanent implanted or temporary implantation radioactive seed.Some have been used for permanent implanted typical radioactive atom and have comprised iodine-125 and radon.Some typical radioactive atoms that have been used for temporary implantation comprise radium, caesium-137 and iridium-192.Some other radioactive atoms that have been used for brachytherapy comprise americium-241 and gold-198.The radiation dose that is used for brachytherapy can be identical with the radiocurable dosage of above-mentioned external beam.Be used for confirming when confirming the dosage of brachytherapy, also will considering the character of used radioactive atom the factor of external beam radiotherapy dosage except above-mentioned.
X.C.) PSCA is as the target of cellullar immunologic response
Vaccine is another embodiment of the present invention with the method for preparing vaccine, and said vaccine contains one or more HLA-binding peptides described here of immune significant quantity.In addition, vaccine of the present invention comprises the compsn of one or more said peptides.Peptide can separately exist in the vaccine.Perhaps, said peptide can be used as the homopolymer that contains a plurality of copies of same peptide or exists as the heteropolymer of different peptides.The advantage of polymkeric substance has been the enhanced immunoreation, and owing to having used different epi-positions to make polymkeric substance have extra inducing and the antibody of the different antigenic determinants reactions of the causal organism of immunne response target-seeking or tumour-related peptides and/or the additional capabilities of CTL.Said compsn can be that the antigen of natural generation is regional or this can pass through for example reorganization or chemical synthesis process prepares.
Can be well known in the art with the carrier that vaccine of the present invention uses, comprising, for example: Thyroprotein; BSA such as human serum albumin; Toxoid,tetanus, polyamino acid is as gathering L-Methionin, L-glutamic acid, influenza virus, hepatitis B virus nucleoprotein or the like.Vaccine can contain on the physiology can anti-(can accept) thinner, like water or salt solution, and the preferably phosphoric acid BS.Usually also can contain adjuvant in the vaccine.Adjuvants such as incomplete Freund's adjuvant, Tai-Ace S 150, white lake or alum all are examples of material well known in the art.In addition, as described herein, peptide of the present invention is combined to cause CTL with lipid like three palmityls-S-glyceryl cysteinyl Serine-Serine (P3CSS) reply.In addition, find adjuvant, as the synthetic oligonucleotide that contains synthesizing cytimidine-thiophosphoric acidization-guanine (CpG) can make CTL reply to improve 10-100 doubly (seeing for example Davila and Celis, J.Immunol.165:539-547 (2000)).
Through injection, aerosol, oral, transdermal, pass through in mucous membrane, the pleura, in the sheath or other suitable pathways with after the peptide combinations immunity of the present invention, CTL that host's immunity system can be through producing a large amount of required antigen-specifiies and/or HTL produce vaccine and react.Afterwards, the host has partial immunity power at least to the expression of generation subsequently or the cell of over-expresses PSCA, perhaps can produce some therapeutic actions at least to the relevant tumour of this antigen.
In some embodiments, should with I class peptide compsn with induce or promote to produce to the neutralizing antibody of this target antigen with or the helper cell combination of components of replying.An embodiment preferred of this compsn comprises I class of the present invention and II class epi-position.Another embodiment of this compsn comprises I class of the present invention and/or II class epi-position and cross reactivity HTL epi-position; Like
Figure G05817239920061201D000701
(Epimmune; San Diego; CA) molecule (for example be described in USP 5,736,142 in).
Vaccine of the present invention also can comprise antigen presenting cell (APC), like dendritic cell (DC), as the carrier of presenting peptide of the present invention.Can be at external generation vaccine composition, movable then and gather in the crops dendritic cell, thereby at external loading dendritic cell.For example, dendritic cell can be used minigene transfection for example of the present invention or use the peptide pulse.Then these dendritic cell are given the patient and cause immunne response in vivo.But the vaccine composition based on DNA or peptide is also used in the body, movable dendritic cell of while, thus load dendritic cell in vivo.
When selecting the epi-position array of multi-epitope compsn, preferably adopt following principle, said multi-epitope compsn to be used to vaccine or be used for selecting in the vaccine and comprised and/or by the discontinuous epi-position of nucleic acid (like minigene) coding.For carrying out the suitable following principle of balance of this kind selection.Add a plurality of epi-positions in the given vaccine composition and can be but need not be the epi-position of adjoining in the natural antigen sequence that produces this epi-position.
1.) be chosen in the epi-position of using the back simulation immunne response relevant with removing tumour.For HLA I class, comprise 3-4 epi-position from least one taa (TAA).Adopted similar principle for HLA II class; From at least one TAA, selected 3-4 epi-position (seeing for example Rosenberg etc., Science 278:1447-1450) again.From the epi-position of a TAA can with can target tumor and change the vaccine of common TAA expression pattern from the epi-position combination of one or more other TAA to produce.
2.) select to have the epi-position of the essential binding affinity relevant with immunogenicity: to HLA I quasi-molecule, IC50 is 500nM or lower, is generally 200nM or lower; To the II quasi-molecule, IC50 is 1000nM or lower.
3.) select to have the peptide of enough hyper-base prefaces, or have the peptide array of enough allele-specific motifs, to obtain colony's fraction of coverage of broad.For example preferably have colony's fraction of coverage of at least 80%.Available statistical computation method known in the art-Monte Carlo analyzes and estimates width or the redundancy that colony covers.
4.) when selecting the cancer associated antigens epi-position, often select analogue, this is because the patient possibly set up the tolerance to natural epi-position.
5.) particularly suitable epi-position is called " nested epi-position (nested epitope) ".Nested epi-position is shown at least two epi-position overlappings of a certain given peptide sequence.Nested peptide sequence can comprise B cell, HLA I class and/or HLA II class epi-position.When nested epi-position was provided, general purpose provided the maximum epi-position number of each sequence.Therefore, be to avoid providing on the one hand than the N-terminal length of peptide N-terminal epi-position or the peptide longer than the C-terminal of peptide C-terminal epi-position.When the multi-epitope sequence is provided, when for example containing the sequence of nested epi-position, to cause a disease or other harmful organism performance for guaranteeing not have, the screening sequence is normally important.
6.) if produced multi-epitope protein or when producing minigene, a purpose is the peptide that produces the minimum that comprises epi-position interested.This principle is identical or similar with the principle of the peptide of selecting to comprise nested epi-position.Yet if artificial multi-epitope peptide, the minimized purpose of length is to make to be incorporated into that the intervening sequence between each epi-position balances each other in this multi-epitope protein.For example, can introduce amino-acid residue at interval and link to each other, thereby or be convenient between each epi-position, cut off and strengthen epi-position and present with the epi-position of avoiding linking to each other (can be but do not exist in the target antigen and can only produce through artificial epi-position side by side epi-position) by immune system recognition.Because acceptor can produce the immunne response to the non-natural epi-position, so need avoid epi-position to link to each other usually.When continuous epi-position is to pay particular attention in " advantage epi-position " time.The advantage epi-position can cause other is weakened or be suppressed zero of epi-position replying.
7.) when having the sequence of a plurality of variants of same target protein, also can select possible peptide epitopes according to their confidentiality.For example, the standard of confidentiality can stipulate that the whole 9-peptide core of the whole sequence of HLA I class binding peptide or II class binding peptide is guarded certain its percentage of concrete proteantigen appointed sequence evaluation.
X.C.1. minigene vaccine
The many different methods that can carry a plurality of epi-positions are simultaneously arranged.The nucleic acid of peptide of the present invention of encoding is a useful especially instance of the present invention.Guide according to above chapters and sections are set preferably is contained in the epi-position in the minigene.A method that preferably gives the nucleic acid of code book invention peptide uses coding to contain the minigene construction of the peptide of one or more epi-positions of the present invention.
Hereinafter and following document description the application of multi-epitope minigene: Ishioka etc., J.Immunol.162:3915-3925,1999; An, L. and Whitton, J.L., J.Virol.71:2292,1997; Thomson, S.A. etc., J.Immunol.157:822,1996; Whitton, J.L. etc., J.Virol.67:348,1993; Hanke, R. etc., Vaccine 16:426,1998.For example; Can be through a kind of Polyepitope DNA plasmid of engineered generation, this plasmid-encoded PSCA that has hyper-base preface and/or motif derive a plurality of HTL epi-positions (seeing for example Table V-XVIII and XXII-LI) and the endoplasmic reticulum-encoding transport signals sequence of epi-position, general helper T cell epitope or PSCA.Vaccine also can comprise the epi-position derived from other TAA.
The immunogenicity that can in transgenic mice, verify the multi-epitope minigene is with the order of magnitude of assessment to test epi-position inductive CTL responsing reaction.In addition, the interior immunogenicity of the body of dna encoding epi-position can be directed against with specific CTL strain and is associated by the vitro responses of the target cell of this DNA plasmid transfection.Therefore, these experiments can show two kinds of effects of this minigene: 1.) produce that CTL replys and 2.) induce the epi-position of the cell expressing coding of CTL identification.
For example, for the dna sequence dna that produces the selected epi-position of coding (minigene) in the human cell, to express, can instead translate the aminoacid sequence of this epi-position.The codon table of can choosing is selected each amino acid whose codon.The dna sequence dna of these coding epi-positions can directly adjoin, and can produce the successive peptide sequence when therefore serving as interpreter.Express and/or the immunogenicity optimum for making, can when the design minigene, add other element.Can be comprised by the example of the aminoacid sequence in the minigene sequence of anti-translation: HLA I class epi-position, HLA II class epi-position, antibody epitope, ubiquitination signal sequence and/or endoplasmic reticulum target signal.In addition, can present through the HLA that the side joint sequence of adjoining CTL or HTL epi-position that adds synthetic (for example gathering L-Ala) or natural generation is improved CTL and HTL epi-position; The epi-position that these bigger peptides comprise belongs to the scope of the invention.
Can the minigene sequence be changed into DNA through the oligonucleotide of assembling coding minigene normal chain and minus strand.The available technology of knowing is synthetic under proper condition, phosphorylation, purifying and the overlapping oligonucleotide of annealing (a long 30-100 base).Available for example T4 dna ligase connects the end of oligonucleotide.Minigene with this synthetic coding epitope polypeptide is cloned into required expression vector then.
Preferably comprise in the carrier and be proficient in standard adjustment sequence well-known to those having ordinary skill in the art and in target cell, express guaranteeing.Need some carrier elements: have the downstream cloning site to insert the promotor of minigene; What effectively stop transcribing gathers the adenosine signal; The intestinal bacteria starting point that is used for duplicating; With intestinal bacteria selective marker but (for example penbritin or kalamycin resistance).Many promotors can be used for this purpose, for example human cytomegalic inclusion disease virus (hCMV) promotor.Referring to USP 5,580,859 and 5,589,466 for example to understand other suitable promoter sequence.
Can add other carrier modification to optimize the expression and the immunogenicity of minigene.Under the certain situation, need intron to express, can mix the intron of one or more synthetic or natural generation at the transcriptional domain of minigene to carry out efficient gene.The expression that the sequence that in mammalian cell, adds the mRNA critical sequences and duplicate also can improve minigene.
In case selected expression vector, can minigene be cloned into the polylinker district in promotor downstream.This plasmid is transformed into suitable coli strain and prepares DNA with standard technique.Orientation and dna sequence dna and year intravital all other elements through restriction enzyme digestion drawing and dna sequence analysis checking minigene.The bacterial cell that comprises correct plasmid can be preserved as master cell bank and working cardial cell storehouse.
In addition, as if immunostimulatory sequence (ISS or CpG) plays a role in the immunogenicity of dna vaccination.Enhances immunogenicity can comprise these sequences outside the minigene encoding sequence of carrier if desired.
In some embodiments, can use the two-cistron expression vector that to make minigene coding epi-position and second protein (can strengthen or reduce immunogenicity).During co expression advantageously the example of the enhancing immunity protein of replying or polypeptide comprise molecule (for example LeIF), the costimulatory molecules that cytokine (for example IL-2, IL-12, GM-CSF), the inducing cell factor produce; Perhaps replied conjugated protein (
Figure G05817239920061201D000731
Epimmune of pan-DR for HTL; San Diego, CA).Auxiliary (HTL) epi-position can be attached to the interior target-seeking signal of born of the same parents and independently express with the CTL epi-position; This can be different from HTL epi-position guiding the cellular compartment of CTL epi-position.Need, this can make the HTL epi-position more effectively get into HLA II classpath, thereby promotes that HTL induces.Induce on the contrary with HTL or CTL, it is useful for some disease to reduce immunne response through coexpression immunosuppression molecule (for example TGF-b) specificity.
Can through for example in intestinal bacteria fermentation culture and then purifying produce the DNA of therapeutic dose.According to the technology of knowing, the working cardial cell storehouse of equivalent is inoculated into growth medium, in shaking bottle or bio-reactor, grow to saturated.DNA available standards bioseparation technology purifying, QIAGEN for example, Inc. (Valencia, the solid phase anionite-exchange resin that California) provides.The available gel electrophoresis of words or other method that need are separated DNA and super coiled DNA in open loop form and the line style form.
Can prepare the DNA of purifying so that inject with various preparations.The simplest method is to rebuild freeze dried DNA with sterile phosphate buffered saline (PBS).The method of this being called " naked DNA " is used to intramuscular (IM) administration at present in clinical trial.For improving the immunotherapy effect of gene DNA vaccine as far as possible, can adopt the method for the DNA of other preparation purifying.Describe many methods, and also can use new technology.This reagent can adopt cation lipid, glycolipid and shortly melt liposome (see that for example following document is said: WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6 (7): 682 (1988); USP 5,279,833; WO 91/06309; And Felgner etc., Proc.Nat ' l Acad.Sci.USA 84:7413 (1987).In addition; Peptide and be referred to as protectiveness, cross reaction property, non-cohesion compound (PINC; Protective; Interactive, non-condensing compound) compound also can form mixture with the DNA of purifying, with change stability for example, intramuscular is dispersed or to the variablees such as transportation of certain organs or cell type.
Target cell sensitization can be used as expression and the functional examination that HLA I class is presented of the CTL epi-position of minigene coding.For example, DNA is introduced into and is suitable as the mammal cell line that standard CT L chromium discharges the target of test.The preparation that used transfection method is final with farsighted person.Electroporation can be used for " naked " DNA, and cation lipid can be used for direct in-vitro transfection.The plasmid of expressing green fluorescent protein (GFP) can be by cotransfection so that through fluorescence-activated cell sorting (FACS) enrichment cells transfected.These cells are used chromium-51 (51Cr) mark then and are used as the target cell of epitope specificity CTL system; Discharge detection cytolysis through 51Cr, it has shown that the generation and the HLA of the CTL epi-position of minigene coding present.The expression of available similar test evaluation HLA epi-position is so that assessment HLA is active.
Immunogenicity is to detect the second method of minigene DNA preparation function in the body.Express the proteic transgenic mice of suitable people HLA with DNA product immunization.Dosage and approach depend on formulations employed (for example, the DNA that joins with PBS is adopted the IM approach, lipid compound DNA is adopted intraperitoneal (i.p.) approach).Immunity is gathered in the crops splenocyte and when having the peptide of each test epi-position of coding, is stimulated a week again after 21 days.Have the target cell test of the 51Cr mark of peptide then in the available standards technology for detection CTL effector cell dissolving.The target cell dissolving that is had with the HLA sensitization of the corresponding peptide epitopes of minigene coding epi-position has confirmed to induce CTL to reply in the dna vaccination preparation body.Confirm the immunogenicity of HTL epi-position in transgenic mice with similar method.
Perhaps, available for example USP 5,204, the trajectory described in 253 is carried the method administration of nucleic acid.Adopt this when technology, give only to contain the particle of DNA.In a further embodiment, can make DNA be attached to particle for example on the gold grain.
The bacterium or the viral delivery system of also available this area resin are carried minigene, for example can the expression constructs that code book is invented epi-position be mixed virus vector such as cowpox.
The combination of X.C.2.CTL peptide and auxiliary peptide
The vaccine composition that contains peptide of the present invention can be modified, for example by similarization, so that desired characteristic to be provided, as serum half-life improve, colony's fraction of coverage broadens or immunogenicity strengthens.
For example, peptide is connected to contain at least one can induce the sequence of the epi-position that t helper cell replys to strengthen the active ability of inducing peptide CTL.Although ctl peptide can be directly connected to the auxiliary peptide of T, through spacer molecule CTL epi-position/HTL epi-position is combined usually.This spacer molecule contains less relatively neutral molecule usually, like uncharged basically amino acid or amino acid analog thing under physiological condition.Spacer molecule can be selected from usually, for example the neutral spacer molecule of Ala, Gly or other nonpolar amino acid or neutral pole acidic amino acid.Be appreciated that the optional spacer molecule that exists not necessarily will contain identical residue, therefore can be assorted-or with-oligomer.When having spacer molecule, spacer molecule has at least one or two residues usually, and 3-6 residue more generally arranged, sometimes even 10 or more residues arranged.Can with the ctl peptide epi-position can be directly or interval through ctl peptide amino or C-terminal be connected to the auxiliary peptide epitopes of T.The N-terminal of the auxiliary peptide of immunogenic peptide or T can be by acidylate.
The htl peptide epi-position also can be modified to change its biological nature.For example, thus they can be modified to comprise D-amino acid and strengthened them to resistance towards proteases and therefore prolong their serum half-life, perhaps they can combine to strengthen its biological activity with other molecule (like lipid, protein, carbohydrate etc.).For example, the auxiliary peptide of T can combine one or more palmitinic acid chain at amino or C-terminal.
X.C.3.CTL peptide and T cell cause the combination of (priming) agent
In some embodiments, can comprise at least a ability inducing B lymphocyte or the lymphocytic component of T in the pharmaceutical composition of the present invention.Identified that lipid can cause CTL in vivo.Be connected to immunogenic peptide on the e-that for example can palmitic acid residues be appended to lysine residue and the a-amino and then through one or more bases (like Gly, Gly-Gly-, Ser, Ser-Ser etc.) that are connected.The peptide of lipidization can be used as the micelle or the particle that mix liposome then and directly uses, or with using after emulsion (like the incomplete Freund's adjuvant) emulsification.In an embodiment preferred, especially effectively immunogenic composition comprises the e-that appends to Lys and the palmitinic acid on the a-amino, makes it be incorporated into the N-terminal of immunogenic peptide through joint (like Ser-Ser) then.
Another example of the lipid that initiation CTL replys is the intestinal bacteria lipoprotein; When being covalently bound to suitable peptide, can be used to cause virus-specific CTL like three palmityls-S-glyceryl cysteinyl seryl-Serine (P3CSS) and (see for example Deres etc.; Nature 342:561,1989).Peptide of the present invention can with for example P3CSS coupling, and lipopeptid used to individuality to cause the specific immune response to target antigen.In addition, because the epi-position initiation of puting together with P3CSS-also can induce neutralizing antibody, therefore can two kinds of such compsns be combined more effectively to cause body fluid and cell-mediated replying.
X.C.4. the vaccine composition that contains the DC of useful CTL and/or htl peptide pulse
An embodiment of vaccine composition according to the invention comprises exsomatizing and gives to have from the PBMC of blood samples of patients or therefrom isolating DC the mixture of the peptide of epi-position.Can use the medicine that helps gathering in the crops DC, like Progenipoietin TM(Pharmacia-Monsanto, St.Louis, MO) or GM-CSF/IL-4.Wash DC to remove unconjugated peptide with peptide pulsed D C and before being infused into the patient again.In this embodiment, vaccine comprises the DC of peptide pulse, the mixture that its surface exists pulse peptide epitopes and HlA molecule to form.
The mixture of the available peptide pulsed D C that exsomatizes has some peptides to stimulate the CTL to PSCA to reply in the mixture.Can randomly contain helper cell (HTL) peptide, like HLA II class peptide natural or the loose restriction of synthetical, to promote that CTL replys.Therefore, vaccine of the present invention is used to treat the cancer of expression or over-expresses PSCA.
X.D.) adoptive immunotherapy
Antigenicity PSCA-related peptides also is used to in-vitro inducing CTL and/or HTL replys.Gained CTL or HTL cell can be used to treat the intravital tumour of patient, said patient to other traditional treatment pattern is reactionless maybe will be reactionless to therapeutic vaccine peptide of the present invention or nucleic acid.Through in tissue culture medium, cultivating the patient's or the compatible CTL of heredity or HTL precursor cell and antigen presenting cell (APC) together like dendritic cell and suitable immunogenic peptide, in-vitro inducing is replied the CTL or the HTL of specific antigen.Cultivate reasonable time (about usually 7-28 days) afterwards, precursor cell is activated and increases during this becomes the effector cell, and it is inculcated back the patient, and they will destroy (CTL) here or help to destroy (HTL) their specificity target cell (for example tumour cell).The dendritic cell of transfection also can be used as antigen presenting cell.
X.E.) use vaccine from treatment or prevention purpose
Pharmaceutical composition of the present invention and vaccine composition can be used to treat and/or prevent expression usually or cross and express the cancer of PSCA.In treatment is used, can give a certain amount of peptide of patient and/or nucleic acid composition, said amount is enough to cause effectively replys to antigenic B cell, CTL and/or HTL, perhaps can cure or at least part stop or slow down symptom and/or complication.Be fit to realize that the amount of this purpose is called as " treatment effective dose ".To depend on this purposes significant quantity, for example, concrete compsn, the administering mode of being used, the stage of being treated disease and seriousness, patient's body weight and healthy state and attending doctor's judgement.
Pharmaceutical composition of the present invention, immunogenic peptide or their DNA of encoding give to have the individuality of the tumour of expressing PSCA.Said peptide or its coding DNA can give separately or give with the form of the fusion sequence of one or more peptide sequences.The patient can use said immunogenic peptide to treat separately, perhaps suitable words also can with other treat-ment (as operation) combination therapy.
In treatment is used, when administration starts from being diagnosed out the PSCA associated cancer first usually.Increasing dosage then alleviates basically and continues for some time again up to symptom at least.The vaccine composition (promptly including but not limited to the for example dendritic cell of peptide mixt, multi-epitope polypeptide, minigene or TAA specific CTL or pulse) that is transported to the patient can change according to disease stage or patient's healthy state.For example, in the patient who suffers from the tumour of expressing PSCA, compare other instance, the vaccine that contains the PSCA specific CTL is more effective for killing the intravital tumour cell of patient with advanced cancer.
Carry the peptide epitopes that is enough to the effective stimulus cytotoxic T cell amount of replying normally important through a kind of administering mode; The compsn that also can stimulate helper cell to reply according to embodiment of the present invention.
The dosage that is used for initial treatment immunity occurs with unitary dose usually, and its scope lower limit is about 1,5,50,500 or 1,000 μ g, and the upper limit is about 10,000,20,000,30,000 or 50,000 μ g.The dose value scope that is used for the people is generally the about 500 μ g of per 70 kg body weight patients to about 50,000 μ g.Can improve dosage in several weeks to several months afterwards, increase dosage at about 1.0mg and about 50, between the 000mg peptide, this depends on through measuring definite reaction and the situation of ratio work available from the CTL and the HTL of blood samples of patients.But successive administration shows that up to clinical symptom or laboratory test tumour is excluded or reduces, and continues for some time again.Dosage, route of administration and dosage regimen will be judged according to methods known in the art.
In certain embodiments, peptide of the present invention and compsn can be used for serious morbid state, i.e. life-threatening or possible life-threatening state.At this moment, based on the minimum of exterior materials and the relative non-toxic nature of the peptide in the preferred present composition, can and be necessary to give the excessive peptide combinations of the said relatively dosage of patient and treat.
Vaccine composition of the present invention also can be only as preventive.Initial prophylactic immunization dosage occurs with unitary dose usually, is limited to about 1,5,50,500 or 1000 μ g under its scope, on be limited to about 10,000,20,000,30,000 or 50,000 μ g.The dose value scope that is used for the people is generally the about 500 μ g of per 70 kg of patient to about 50,000 μ g.After the primary vaccination vaccine, strengthen dosage, strengthen dosage at about 1.0mg and about 50, between the 000mg peptide with about 4 thoughtful 6 months predetermined spaces.The immunogenicity of vaccine can be assessed available from the CTL of blood samples of patients sample and the activity specific of HTL through measuring.
The pharmaceutical composition of treatment usefulness can be through using in parenteral, external application, mouth, nose, the sheath or at local (for example as emulsifiable paste or externally-applied ointment).Preferably through the parenteral administration pharmaceutical composition, for example intravenously, subcutaneous, intradermal or intramuscular.Therefore, the invention provides the compsn that supplies the parenteral medication, wherein comprise to be dissolved in or to be suspended in and to accept carrier, the solution of the immunogenic peptide in the preferred aqueous carrier.
Can use various aqueous carriers, for example water, buffered water, 0.8% salt solution, 0.3% glycocoll, mucinase etc.These compsns can be sterilized with the conventional sterilising technology of knowing, perhaps can be by sterile filtration.Obtained aqueous solution can be packaged into the preparation of direct use, or by freeze-drying, freeze dried goods need be heavily molten with sterile solution before administration.
Said compsn can contain pharmaceutically acceptable auxiliary substance to satisfy the needs of physiological condition; Like pH-regulator and buffer reagent, tension regulator, wetting agent, sanitas etc., concrete example if any sodium acetate, lactic acid receive, sodium-chlor, Repone K, calcium chloride, Arlacel-20, Emulphor FM etc.
The concentration of peptide of the present invention is variable in the pharmaceutical prepn, promptly its part by weight can from less than about 0.1% (be generally or at least about 2%) to 20%-50% or higher, and will mainly wait according to selected specific administration pattern and select through liquid volume, viscosity.
Usually the compsn that contains people unitary dose unit in the pharmaceutical composition; Said pharmaceutical composition comprises the acceptable carrier of people's unitary dose; Be aqueous carrier in one embodiment, and known to those skilled in the artly be used for giving human volume/mass ratio and carry out administration (for example see " Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences) the 17th edition, A.Gennaro compiles this compsn to be proficient in; Mack Publishing Co.; Easton, Pennsylvania, 1985).For example, 70kg body weight patient's initial immunity peptide dosage can be from about 1 to about 50, and 000mg is generally 100-5,000mg.For example, as far as nucleic acid, the expression vector of the naked nucleic acid form that initial immunity can give through IM (or SC or ID) at a plurality of positions in order to the amount of 0.5-5mg carries out.The also available particle gun of nucleic acid (0.1-1000mg) gives.3-4 strengthens dosage after week.Reinforcer can be the recombinant fowlpox virus of using with the dosage of 5-107 to 5 * 109pfu.
Antagonist, treatment generally includes through the acceptable approach that gives, and like intravenous injection (IV), repeats to resist-the psca antibody goods, and its dosage is generally the about 0.1-10mg of every kg body weight.Usually, the dosage of 10-500mg MAb is effectively and can well tolerable weekly.In addition, a kind of acceptable is anti--dosage of PSCAMAb goods is, every kg body weight patient is through the about 4mg initial load of IV dosage, then the dosage through the about 2mg/kg of IV weekly.Be proficient in those skilled in the art will appreciate that in concrete case, have many factors can influence optimal dosage.These factors comprise; For example, the antigenic circulation of the immunogenicity of the binding affinity of the transformation period of compsn, Ab, material, PSCA expression degree, the PSCA in the patient get rid of degree, required steady state concentration level, therapeutic frequency, unite chemotherapeutics or the influence of other reagent and the healthy state of particular patient of use with treat-ment of the present invention.Nonrestrictive preferred people's unitary dose is; 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 certain embodiments, dosage range is every kg body weight 2-5mg, for example, gives 1-3mg/kg then weekly; For example, be 0.5mg, 1,2,3,4,5,6,7,8,9,10mg/kg body weight then, give weekly, by a definite date 2,3 or 4 weeks; For example, be the 0.5-10mg/kg body weight then, give weekly, by a definite date 2,3 or 4 weeks; 400mg/m2 body surface area weekly 225,250,275,300,325,350,375; 1-600mg/m2 body surface area weekly; 225-400mg/m2 body surface area weekly; These dosage can give weekly, and by a definite date 2,3,4,5,6,7,8,9,19,11,12 or week more how.
In one embodiment, people's unit dosage of polynucleotide comprises suitable dosage ranges or the significant quantity of any result of treatment is provided.Understand like one of ordinary skill in the art, result of treatment depends on many factors, comprising the sequence of polynucleotide, the molecular weight and the route of administration of polynucleotide.Dosage is selected according to various parameters known in the art (the for example seriousness of symptom, medical history etc.) by doctor or other health care professionals usually.Usually; For the polynucleotide of about 20 bases; Dosage range can be selected from, for example, and the independent lower limit of selecting; According to appointment 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; To the independent upper limit of selecting greater than lower limit, according to appointment 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 can be following any: 0.1-100mg/kg, 0.1-50mg/kg, 0.1-25mg/kg, 0.1-10mg/kg, 1-500mg/kg, 100-400mg/kg, 200-300mg/kg, 1-100mg/kg, 100-200mg/kg, 300-400mg/kg, 400-500mg/kg, 500-1000mg/kg, 500-5000mg/kg or 500-10,000mg/kg.Usually, compare Nucleotide more directly is applied to diseased tissue, the administered parenterally approach needs the polynucleotide of higher dosage, and dosage also need increase when polynucleotide length increases.
In one embodiment, people's unit dosage of T cell comprises suitable dosage ranges or the significant quantity of any result of treatment is provided.Understand like one of ordinary skill in the art, result of treatment depends on many factors.Dosage is selected according to various various parameters known in the art (the for example seriousness of symptom, medical history etc.) by doctor or other health care professionals usually.Dosage can be about 104-106 cell, about 106-108 cell, about 108-1011 cell or about 108-5 * 1010 cells.Dosage can also be extremely about 1010 cells/m2 of about 106 cells/m2, or about 106 cells/m2 is to about 108 cells/m2.
Protein of the present invention and/or proteinic coding nucleic acid also can give through liposome, and liposome has following effect: 1) make protein target particular organization such as Lymphoid tissue; 2) selectivity target-seeking disease cell; Or 3) transformation period of prolongation peptide combinations.Liposome comprises emulsion, foam, micelle, insoluble individual layer, liquid crystal, phosphatide dispersion-s, multilayer or the like.In these goods, the part that the peptide that is transferred is used as liposome is mixed separately or is mixed with the molecule (as combining the antigenic monoclonal antibody of CD45) of the general acceptor of bind lymphocytes or other therapeutic or immunogenic components.Therefore, the liposome of required peptide of the present invention is housed or the liposome decorated with this peptide can be directed to the lymphocyte position, liposome will be carried said peptide combinations here then.The available standards carrier forms lipid and is formed for liposome of the present invention, wherein generally comprises the phosphatide and the steroid (like SUV) of electroneutral and negative charge.Usually will consider following factor when selecting lipid, for example the size of liposome, acid labile and liposome are in stability in blood.Many methods that prepare liposome are arranged, the method described in the for example following document: Szoka etc., Ann.Rev.Biophys.Bioeng.9:467 (1980) and USP 4,235,871,4,501,728,4,837,028 and 5,019,369.
For making the cell-targeting immunity system, the part that is impregnated in liposome for example can comprise specific antibody or its fragment of required immune cell surface determinant.The liposome suspension that contains peptide can give through approach such as intravenously, parts, and its dosage can and be treated the factors such as stage of hitting disease according to administering mode, the peptide that is transferred and changed.
Can use conventional non-toxic solid carrier for solids compsn, comprising, for example, pharmaceutical grade N.F,USP MANNITOL, lactose, starch, Magnesium Stearate, soluble saccharin, talcum.Mierocrystalline cellulose, glucose, sucrose, magnesiumcarbonate etc.Be oral administration; Can form pharmaceutically acceptable non-toxic composite through mixing any normally used vehicle (like top those carriers of listing); Usually contain the 10-95% activeconstituents in this compsn; Be one or more peptides of the present invention, and preferred concentration is 25%-75%.
For the aerosol administration, immunogenic peptide preferably provides with form and tensio-active agent and the propelling agent of segmentation together.The part by weight of peptide is generally about 0.01%-20%, preferably about 1%-10%.Certainly, tensio-active agent must be nontoxic, and preferably is dissolved in propelling agent.Representational this reagent is the lipid acid that contains 6-22 carbon atom (like caproic acid, sad, LAURIC ACID 99 MIN, palmitinic acid, Triple Pressed Stearic Acid, linolic acid, linolenic acid, oil base Triple Pressed Stearic Acid (olesteric acid) and oleic acid) and the ester or the partial ester of aliphatic polyhydroxy-alcohol or its cyclic anhydride.Can use the blended ester, for example blended or natural glyceryl ester.About 0.1%-20% of surfactant comprise composition weight, preferably about 0.25-5%.With conventional propellant balanced combination thing.The words that need also can contain carrier, can contain Yelkin TTS when for example carrying in the nose.
XI.) diagnosis of PSCA and prognosis embodiment
As described herein; Diagnosis, prediction and therapeutic test that PSCA polynucleotide, polypeptide, reactive cytotoxic T cell (CTL), reactive helper cell (HTL) and anti--polypeptide antibody is used to know, this is used for detecting and the dysregulated cellular growth diseases associated, like cancer; Especially the cancer listed of Table I (referring to; For example, its particular organization's expression pattern and the over-expresses in some cancer thereof, as it is said to be entitled as the embodiment of " healthy tissues and patient's sample in PSCA expression analysis ").
Hence one can see that, and PSCA is a kind of prostate gland related antigen PSA, and the generation that prostate cancer was identified and monitored to medical worker with this former phenotypic marker is existing for many years (for example sees Merrill etc., J.Urol.163 (2): 503-5120 (2000); Polascik etc., J.Urol.Aug; 162 (2): 293-306 (1999) and Fortier etc., J.Nat.Cancer Inst.91 (19): 1635-1640 (1999)).Also can use many other diagnostic flags, (see for example Tulchinsky etc., Int J Mol Med1999-7-4 (1): 99-102 and Minimoto etc., Cancer Detect Prev 2000 comprising p53 and K-ras; 24 (1): 1-12).Therefore; Open PSCA polynucleotide and polypeptide (and being used for identifying that there be the PSCA polynucleotide probes and anti--psca antibody of situation in these molecules) and their characteristic make those skilled in the art can these molecules be used for similar method, for example many related indication diagnostic tests of cancer that relate to.
Use the typical embodiments of the diagnostic method of PSCA polynucleotide, polypeptide, reaction-ive T cell and antibody to be similar to the for example diagnostic test of PSA polynucleotide, polypeptide, reaction-ive T cell and antibody of the well use of foundation.For example; (for example Northern analyzes as probe just as in the method for monitoring PSA over-expresses or prostate cancer transfer, using the PSA polynucleotide; See for example Sharief etc.; Biochem.Mol.Biol.Int.33 (3): 567-74 (1994)) and primer (pcr analysis for example; For example see Okegawa etc., J.Urol.163 (4): 1189-1190 (2000)) with observe PSA mRNA exist and/or level the same, the cancer metastasis of this gene is expressed in the cross expression or prostate cancer and other of monitoring PSCA with PSCA polynucleotide described here that can use the same method.Perhaps; (for example see Stephan etc. just as cross expression at monitoring PSA albumen; Urology 55 (4): 560-3 (2000)) or prostatic cell shift and (for example to see Alanen etc.; Pathol.Res.Pract.192 (3): 233-7 (1996)) produce with the PSA polypeptide in the method that the specific antibody of PSA is used for observing the proteic existence of PSA and/or level is the same, available PSCA polypeptide described here produces the transfer of crossing expression and/or prostatic cell and expressing other cancer cell of this gene that is used for monitoring PSCA.
Specifically; Relate to cancer cells and move to other zone of health (like lymphoglandula) owing to shift, so can adopt the detection of biological sample whether to exist the test of the cell of expressing PSCA polynucleotide and/or polypeptide that the evidence of transfer is provided from a kind of organ (like lung or prostate gland etc.) of source.For example; When containing the cell of expressing PSCA in the biological sample of finding from the tissue (lymphoglandula) that does not contain the cell of expressing PSCA usually; For example observe psca expression separating in the heterograft LAPC4 of lymphoglandula and bone transfer and LAPC9, this discovery is indicating to be shifted.
Perhaps; Available PSCA polynucleotide and/or polypeptide provide the evidence of cancer, for example when finding not express PSCA usually or expressing cell expressing PSCA or psca expression in the biological sample of PSCA when raising (seeing the psca expression in psca expression and the patient's sample shown in the corresponding figure etc. in the listed cancer of Table I for example) with different levels.In this mensuration; The technician also hopes through whether there being the second tissue limitations property mark except that PSCA in the detection of biological sample; Like PSA, PSCA etc., produce about the adminicle that shifts (for example seeing Alanen etc., Pathol.Res.Pract.192 (3): 233-237 (1996)).
Use and exist the PSCA polypeptide can explain that the state of some cell in this tissue is changed in the section of immunohistochemical method appraisement organization.As known in the art, the ability that antibody navigates to the cancer cells polypeptide expressed is the method for a kind of existence that diagnoses the illness, disease stage of living in, development and/or tumor invasion property.Compare corresponding non-malignant tumors tissue, this antibody also can detect the change that this polypeptide distributes in the cancer cells.
PSCA polypeptide and immunogenic composition also can be used for understanding the localized change of morbid state Central Asia cellular proteins.Cell changes over morbid state from standard state can make cellular form change and usually relevant with the variation of subcellular unit location/distribution.For example, the cell membrane protein of in normal cell, expressing with polarization mode can change in disease, and this causes protein to be distributed in whole cell surface with nonpolar mode.
Confirmed the phenomenon that cellular proteins location, the morbid state Central Asia changes through immunohistochemical method with MUC1 and Her2 protein expression.The top that normal epithelial cell has typical MUC1 distributes, and in addition locatees on the nuclear of gp in addition, and presents nonpolar dyeing pattern (Diaz etc., The Breast Journal, 7 usually in the malignant neoplastic disease focus; 40-45 (2001); Zhang etc., Clinical CancerResearch, 4; 2669-2676 (1998): Cao etc., The Journal of Histochemistry andCytochemistry, 45:1547-1557 (1997)).In addition, normal breast epithelium be Her2 albumen negative or only show that the substrate outside distributes, and the malignant T cell can be in whole cell surface expression protein (DePotter etc., International Journal of Cancer, 44; 969-974 (1989): McCormick etc., 117; 935-943 (2002)).Perhaps, in morbid state, proteinic distribution can become and is distributed to the kytoplasm from only being positioned the surface.MUC1 can be observed such example (Diaz etc., The BreastJournal, 7:40-45 (2001)).
Variation through the detected intracellular protein location/distribution of immunohistochemical method also provides important information for the feasibility of some treat-ment.This last point can be through in healthy tissues, being arranged in born of the same parents but is explained in the proteinic situation that malignant cell appears at cell surface; The cell surface location makes cell can successfully be used for diagnosis and the treat-ment based on antibody.When the protein positioning change took place PSCA, PSCA albumen was very useful with the immunne response relevant with it.Therefore, confirm that the ability whether 24P4C12 ubcellular albumen location changes makes PSCA albumen very useful with the immunne response relevant with it.Use PSCA compsn makes that being proficient in those skilled in the art can make important diagnostic and treatment decision.
When the PSCA polypeptide occurred under normal circumstances not producing in the tissue of PSCA, the immunohistochemistry reagent that is specific to PSCA also was useful for the transfer that detects the tumour of expressing PSCA.
Therefore, the antibody that PSCA polypeptide and immunne response thereof produce can be used for many important aspects, as is proficient in diagnosis known to those skilled in the art, prognosis, prevents and/or treats purpose.
Just as those skilled in the art are used to monitor the method for PSA with PSA polynucleotide passage and polynucleotide variant, PSCA polynucleotide passage and polynucleotide variant also can use in a similar manner.Specifically, the typical PSA polynucleotide that are used to monitor the PSA method are probe or the primers that are made up of PSA cDNA sequence fragment.For instance, being used for the primer of pcr amplification PSA polynucleotide must comprise incomplete PSA sequence in the polymerase chain reaction, to play a role.In this PCR reaction; Those skilled in the art produce many different polynucleotide passages usually; These fragments can be used as primer with the different piece of the interested polynucleotide that increase or make that amplified reaction is optimum (sees for example Caetano-Anolles; G.Biotechniques25 (3): 472-476,478-480 (1998); Robertson etc., Methods Mol.Biol.98:121-154 (1998)).Use this segmental another example to be provided at and be entitled as among the embodiment of " expression analysis of PSCA in healthy tissues and the patient's sample ", wherein the PSCA polynucleotide passage is used as probe to show the expression of PSCA RNA in cancer cells.In addition; The variant polynucleotide sequence is used as primer and the probe of corresponding mRNA usually and (for example sees Sawai etc. in PCR and Northern analyze; " the molecular biology fresh approach " that Fetal Diagn.Ther.1996 11-12 11 (6): 407-13 and Frederick M.Ausubel etc. compile, 1995)).Polynucleotide passage and variant here are useful, and they can combine target polynucleotide sequence (PSCA polynucleotide for example shown in Figure 1 or its variant) under highly rigorous condition.
In addition, contain the method that can be used to monitor PSA by the PSA polypeptide of the epi-position of antibody or T cell recognition, wherein said antibody or T cell-specific combine this epi-position.PSCA polypeptide fragment and polypeptide analog or variant also can use in a similar manner.This is common with polypeptide fragment or polypeptide variants generation antibody (like anti-PSA antibody or T cell) in many systems of this area; " molecular biology fresh approach " that Frederick M.Ausubel for example etc. compiles (seen by the fusion rotein system that uses like the technician; 1995, the 2 volumes Unit the 16th).In the literary composition, the function of each epi-position provides the reactive structure of antibody or T cell.Usually, those skilled in the art create many different polypeptides fragments, and these fragments can be used to produce the immunne response (seeing for example USP 5,840,501 and USP 5,939,533) that is specific to the polypeptide of interest different piece.For example, can preferably use the polypeptide that contains the biological motif of a kind of PSCA described here or have the subsequence of motif, this subsequence is proficient in those skilled in the art and identifies easily based on sequence known in the art.Polypeptide fragment, variant or analogue are normally useful here, as long as they contain the epi-position that can produce target peptide sequence (PSCA polypeptide for example shown in Figure 3) specific antibody or T cell.
As shown here, PSCA polynucleotide and polypeptide (and being used for identifying the PSCA polynucleotide probes and anti--psca antibody or T cell that has these molecules) have special nature, and this specific character makes them can be used for diagnosing cancer, the cancer listed like Table I.Measurement exists the psca gene product to be used to identify the patient carrying out preventative detection or further monitoring with the diagnostic method of the appearance of estimating disease condition described here (like prostate cancer) or generation, just as the PSA that has succeedd such.In addition; These materials have satisfied this area in some cases to having the micromolecular needs of or complementary characteristic similar with PSA; Said situation for example; Only (for example see Alanen etc. according to can not the clarify a diagnosis transfer in prostate gland source of the test of PSA; Pathol.Res.Pract.192 (3): 233-237 (1996)), therefore need to confirm the transfer in prostate gland source with PSCA polynucleotide and polypeptide materials such as (and being used for identifying the PSCA polynucleotide probes and anti--psca antibody that has these molecules).
At last; Except they purposes in diagnostic detection; PSCA polynucleotide described here have many other purposes, and for example they can be used to identify the interior and carcinogenic relevant chromosome abnormalty of psca gene chromosomal region (see below and be entitled as the embodiment of " karyomit(e) of PSCA is drawn ").In addition, except being used for diagnostic test, PSCA GAP-associated protein GAP described here and polynucleotide have many other purposes,, for example their tissues of can be used for the unknown source of forensic analysis (are seen for example Takahama K Forensic Sci Int 1996-6-28; 80 (1-2): 63-9).
In addition, to can be used to treat with the PSCA over-expresses be the pathological condition of characteristic for PSCA GAP-associated protein GAP of the present invention or polynucleotide.For example, the amino acid of Fig. 1 or nucleotide sequence, or its fragment can be used to produce to the antigenic immunne response of PSCA.Available function of regulating this molecule with antibody or other molecule of PSCA reaction, thus the treatment benefit can be provided.
XII.) inhibition of PSCA protein function
The present invention includes various inhibition PSCA combine its binding partners or with other protein be mutually related method and composition and the method that suppresses the PSCA function.
XII.A.) inhibition of with intracellular antibody PSCA being carried out
In a kind of method, the coding specificity combines the recombinant vectors of the single-chain antibody of PSCA to be introduced the psca expression cell through gene transmission technology.Therefore, the strand of coding is anti--psca antibody expresses in born of the same parents, ins conjunction with PSCA albumen, and so suppresses its function.The method of single-chain antibody is known in the engineered this born of the same parents.This intracellular antibody is also referred to as " intracellular antibody ", the selectively targeted intracellular specific compartment of ability, and the restraining effect basic weight that makes treatment is at this compartment.This technology successfully is used for this area (referring to for example Richardson and Marasco, 1995, TIBTECH the 13rd volume).Intracellular antibody in fact can eliminate the high abundance cell surface receptor expression (see for example Richardson etc., 1995, Proc.Natl.Acad.Sci.USA 92:3137-3141; Beerli etc., 1994, J.Biol.Chem.289:23931-23936; Deshane etc., 1994, Gene Ther.1:332-337).
Single-chain antibody comprises through the heavy chain of flexible joint polypeptide connection and the variable region of light chain, and as single expression of polypeptides.Randomly, single-chain antibody is used as with constant region of light chain bonded single chain variable fragment and expresses.The transportation signal is encoded the recombinant nucleotide vector of this single-chain antibody so that the required intracellular region of the accurate target of this intracellular antibody chamber through engineered one-tenth in the born of the same parents that will know.For example, the intracellular antibody of target endoplasmic reticulum (ER) adds leading peptide and the terminal ER stick signal peptide (like the KDEL amino acid motif) of optional C-through transformation.Engineeredly make it contain nuclear localization signal thereby make intracellular antibody in nucleus, have activity.Lipid part is combined with intracellular antibody intracellular receptor is limited in the kytoplasm side of plasma membrane.Intracellular antibody also plays a role in cytosol through target.For example, make the factor chelating in intracytoplasmic intracellular antibody and the cytosol, can prevent that like this them from getting into their n cell objective.
In one embodiment, intracellular antibody is used to catch endonuclear PSCA, thereby prevents that it is in endonuclear activity.In this PSCA intracellular antibody, add nuclear target signal to realize required target-seeking.Can design this PSCA intracellular antibody comes specificity to combine specific PSCA structural domain.In another embodiment, specificity combines the proteic kytoplasm intracellular antibody of PSCA to be used to prevent that PSCA from contacting with nucleus, thereby can prevent that them from bringing into play any biological activity (for example preventing PSCA and other factor formation transcription complex) in nucleus.
Express in specific cells for directing this intracellular receptor concretely, intracellular receptor is transcribed under the regulation and control of suitable tumor-specific promoters and/or enhanser.For making intracellular receptor specific expressed in prostate gland, can use for example PSA promotor and/or promotor/enhanser (seeing the USP of for example delivering on July 6th, 1,999 5,919,652).
XII.B.) with recombinant protein PSCA is suppressed
In the another kind method, recombinant molecule combines PSCA and therefore suppresses the function of PSCA.For example, these recombinant molecules can prevent or suppress its binding partners of PSCA contact/combination or related with other protein.For example, this recombinant molecule can contain the reactive part of PSCA specific antibody molecule.In one embodiment, the PSCA of PSCA binding partners combine the territory through engineered in dimeric fusion protein, so this fusion rotein contains two Fc part bonded PSCA ligand binding domains with human IgG (like the human IgG1).This IgG part can contain, for example, and CH2 and CH3 zone and hinge region, but do not contain the CH1 zone.This dimeric fusion protein can soluble form be suffered from the patient of the cancer relevant with psca expression, thereby this dimeric fusion protein specificity combines PSCA and blocks PSCA and the binding partners interaction.Also available known antibodies technology makes this dimeric fusion protein be combined into polyprotein.
The inhibition of XII.C.) PSCA being transcribed or translating
The present invention also comprises the method and composition that various inhibition psca genes are transcribed.Similarly, the present invention also provides inhibition PSCA mRNA to translate into method of protein and compsn.
In a kind of method, the method that the inhibition psca gene is transcribed comprises makes psca gene contact PSCA antisense polynucleotides.In the another kind method, the method that suppresses PSCA mRNA translation comprises makes PSCAmRNA contact antisense polynucleotides.In another method, suppress translation thereby cut PSCA courier with the PSCA specific ribozyme.This method based on antisense and ribozyme also can be to the regulation domain of psca gene, like PSCA promotor and/or enhancer element.The protein that similarly, can suppress the psca gene transcription factor is used to suppress PSCAmRNA and transcribes.Various polynucleotide and compsn useful in aforesaid method are described hereinbefore.The method of using antisense and ribozyme molecule to suppress to transcribe and translate is well known in the art.
Other factor of transcribing through disorderly PSCA transcriptional activity inhibition PSCA also can be used to treat the cancer of expressing PSCA.Similarly, the factor of disorderly PSCA processing also can be used to treat the cancer of expressing PSCA.The cancer treatment method that uses this type factor is also within the scope of the invention.
XII.D.) general process of treat-ment
Available transgenosis and gene therapy technology are come the tumour cell (polynucleotide and other PSCA that are antisense, ribozyme, coding intracellular antibody suppress molecule) of delivering therapeutic property polynucleotide molecule to synthetic PSCA.Many gene therapy methods known in the art.To the encode recombinant vectors, ribozyme of PSCA antisense polynucleotides, factor of can disorderly PSCA transcribing etc. of also available this gene therapy method is transported to the target tumour cell.
Above-mentioned treat-ment can be united with widely used operation, chemotherapy or radiotheraping method.Treat-ment of the present invention can reduce the dosage and/or the frequency of administration of chemotherapy (or other therapies), and this is favourable to the patient, especially can't the toxic patient of well tolerable chemotherapeutic for those.
The anti-tumor activity of particular composition (for example antisense, ribozyme, intracellular receptor) or these combinations of substances can be estimated with mensuration system in various external and the bodies.The external test of estimating therapeutic activity comprises that cell growth measurement, soft agar are measured and the active test of other indication tumor growth, combines mensuration can confirm to treat that compound will suppress bonded degree such as PSCA and binding partners combines test etc.
Can be in the body of suitable animal model inner evaluation PSCA therapeutic compsn effect.For example can use xenogenesis prostate cancer model, in this model, human prostata cancer explant or the xenotransplantation fabric texture that goes down to posterity are introduced into the low animal of immunological competence, like nude mice or SCID mouse (Klein etc., 1997, Nature Medicine 3:402-408).For example, PCT patented claim WO 98/16628 and USP 6,107,540 have been described the various xenograft models of human prostata cancer, and these models can be explained the development, micrometastasis of primary tumor and shift as the scleroblast of terminal illness characteristic.Mensuration capable of using suppresses its effects of prediction such as tumour formation, tumor regression or transfer.
Estimate and promote the in vivo tests of apoptosis also to can be used to estimate therapeutic compsn.In one embodiment, can detect the mouse that has tumor xenogeneic graft of handling with therapeutic compsn compares with understanding and untreatedly has the mouse that contrasts heterograft and whether have the apoptosis focus.The apoptosis focus of in the tumour of the mouse that process is treated, finding has been explained the therapeutic efficiency of compsn.
The therapeutic compsn that is used for aforesaid method can be formulated into pharmaceutical composition, contains the carrier that is fit to said carrying method in this pharmaceutical composition.Suitable carriers comprises when mixing with therapeutic compsn the anti-tumor function that keeps therapeutic compsn and any material that does not react with patient's immunogenicity usually.Its example includes but not limited to any standard pharmaceutical carrier, like sterile phosphate buffered saline solution, bacteriostatic water etc. (usually visible " Remington ' s Pharmaceutical Sciences " the 16th edition, A.Osal. compiles, 1980).
The treatment preparation can be dissolved and through any administration that can therapeutic compsn be transported to tumor locus.More effective route of administration includes but not limited to: in the intravenously, parenteral, intraperitoneal, intramuscular, knurl, in the intradermal, organ, normotopia etc.Contained this therapeutic compsn of preferred intravenous formulations can or be diluted in the SE or polyethylene bag that injection contains 0.9% aseptic sodium-chlor with antiseptic and inhibiting bacteria function water, aseptic non-anticorrosion water wiring solution-forming.The therapeutic protein goods can be preserved by freeze-drying and as sterilized powder, and preferred freeze dried under vacuum is being injected before with bacteriostatic water (containing for example phenylcarbinol sanitas) or sterilized water reprovision then.
Dosage with aforesaid method treatment cancer can change according to method is different with the target cancer with medication, and will depend on many other factorses known in the art usually.
XIII.) evaluation of PSCA regulator, signature analysis and purposes
Identify and use the method for regulator
In one embodiment, screen to identify regulator, said regulator is induced or is suppressed the particular expression pattern, suppresses or induce particular approach, preferably therefore produces relevant phenotype.In another embodiment, identified the gene of expressing for the particular case serious difference; Screen to identify and change the regulator that (improve or reduce) genes of individuals is expressed.In another embodiment, screen to identify the regulator of the biological function that changes the differentially expressed gene expression product.Moreover, identified the importance of gene in particular state, screen to identify combination and/or the bioactive reagent of regulatory gene product.
In addition, screening receives the inductive gene in to the candidate agent responsing reaction.Identify that regulator (suppresses the cancer expression pattern and produces the reagent of normal expression pattern; Maybe can regulate oncogene and cause the regulator of this gene at normal tissue expression), screen then to identify and in responsing reaction, receive the gene that specificity is regulated this reagent.Healthy tissues and can be presented at the gene of not expressing in healthy tissues or the cancerous tissue but in passing through the tissue of agent treated, expressing with the expression pattern of the cancerous tissue of agent treated relatively, perhaps vice versa.With described here being used for cancer gene or method of protein evaluation and using these reagent specific sequences.Specifically, these sequences and their encoded protein matter are used to the cell of mark or indentifying substance processing.In addition, produced the antibody of anti-reagent inducible protein matter and be used to cancerous tissue sample that new therapeutical agent target is treated.
Evaluation relevant and shaker test with regulator:
The test relevant with genetic expression
Protein of the present invention, nucleic acid and antibody are used to shaker test.Be used to screening assay with the protein of the protein of related to cancer, antibody, nucleic acid, modification and the cell that contains these sequences, for example estimate drug candidates to " gene expression pattern ", expression of polypeptides pattern or change the effect of biological function.In one embodiment, use expression pattern (preferably combining) to handle afterwards gene expression pattern (Dayis for example, GF etc., J Biol Screen 7:69 (2002) with candidate agent with the high flux screening technology; Zlokarnik etc., Science 279:84-8 (1998); Heid, Genome Res 6:986-94,1996).
The protein of cancer protein, antibody, nucleic acid, modification and contain cell natural or that modify cancer protein or gene and be used to screening assay.That is, the present invention includes the method that cancerous phenotype or cancer protein physiological function of the present invention are regulated in screening.But accomplish on this gene or accomplish through " gene expression pattern " or the biological function of estimating drug candidates.In one embodiment, use expression pattern (preferably combining), to see Zlokamik after handling, to monitor with candidate agent with the high flux screening technology, the same.
Various relate to gene of the present invention and proteinic tests have been carried out.Mensuration can be carried out at individual nucleic acid or protein level.That is, but after identifying the specific gene in cancer, raise the filler test compound express or combine the ability of cancer protein of the present invention to understand regulatory gene." adjusting " comprises raising here or reduces genetic expression.Preferred regulated quantity will depend on the initial change of genetic expression in healthy tissues and the cancerous tissue, and its variation is at least 10%, and will be preferred 50%, more preferably 100-300%, and be 300-1000% or higher in some embodiments.Therefore, in cancerous tissue, express 4 times of increases, often need to reduce about 4 times if healthy tissues is compared certain gene; Similarly, compare with healthy tissues in cancerous tissue and reduce by 10 times, often need test compounds that it is expressed and improve 10 times.Also can adopt the regulator that can worsen genetic expression in the cancer, for example, further analyze the rise expression of target gene.
Come the amount and the gene expression dose of gene expression with nucleic probe and quantitatively determined, but perhaps monitoring gene product itself, for example antibody and standard immunoassay test through using cancer protein.Also available protein science and stripping technique come quantitatively determined to express.
Express the compound of identifying the modification of gene expression through monitoring
In one embodiment, simultaneously many entities are carried out the genetic expression monitoring, promptly monitor expression map.This collection of illustrative plates generally includes one or more genes shown in Figure 1.In this embodiment, for example, the cancer nucleic acid probe is attached on the biochip with the cancer sequence in detection and the quantification specific cells.Perhaps can adopt PCR.Therefore can use microtiter plate, be dispersed with primer in its required hole.Can carry out PCR reaction then and each hole is analyzed.
Express monitoring to identify the compound of the expression of modifying one or more cancer correlated serieses (polynucleotide sequence listed) like Fig. 1.Usually before analyzing, in cell, add the test regulator.In addition, also screen to identify the adjusting cancer, regulate cancer protein of the present invention, to combine cancer protein of the present invention or disorderly cancer protein of the present invention and antibody or its binding partners bonded molecule.
In one embodiment, high-throughput screening method comprises provides the storehouse that comprises a large amount of potential treatment compounds (candidate compound).Screening this " combinatorial chemical library " has required chemically active library member (especially chemical species or subclass) to identify those in one or more mensuration then.The compound that identifies can be used as conventional " lead compound ", uses compound like screening, or as therapeutical agent.
In certain embodiments, screening potential regulator combinatorial library combines the ability of carcinoma polypeptide or regulates active.Usually; Be tested and appraised have some desired characteristic or activity (for example suppressing active) compound (being called " lead compound "), produce the variant of said lead compound and estimate the characteristic and the activity of these variant compounds, can produce new chemical entities with useful property.Usually, high flux screening (HTS) method is used to this analysis.
As stated, the genetic expression monitoring is used to detect candidate modulator (for example protein, nucleic acid or small molecules) usually.Add candidate agent and with cell cultures after for some time, the sample that contains target sequence to be analyzed for example is added on the biochip.
The available known technology of words that needs prepares target sequence.For example, handle sample so that lysis with methods such as known lysis buffer, electroporations, and purifying and/or amplification, PCR for example passed through.The for example available affinity tag that is covalently bound to Nucleotide carries out in-vitro transcription.Nucleic acid is used vitamin H-FITC or PE or usually with cy3 or cy5 mark.
Target sequence can be used fluorescent signal, chemiluminescence signal, chemical signal or radiated signal mark so that detect and probe specificity bonded target sequence.Mark also can be an enzyme, like SEAP or horseradish peroxidase, when suitable substrates is provided, can detect the product that these enzymes produce.Perhaps, mark is compound or the small molecules that is labeled, and like enzyme inhibitors, they combine with enzyme but not by enzyme catalysis or change.Mark also can be that for example epi-position mark or specificity combine the vitamin H of Streptavidin.With the vitamin H is example, labelled streptavidin as stated, thus detectable signal is provided for the bonded target sequence.Unconjugated labelled streptavidin was removed before analyzing usually.
Those skilled in the art will appreciate that these tests can be " the sandwich tests " that a plurality of probes were measured or used to direct cross, this method is generally described in 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 and 5,681,697.In this embodiment, prepare target nucleic acid usually as stated above, under the condition that can form hybridization complex it is being added on the biochip that contains many nucleic probes then.
The present invention has adopted multiple hybridization conditions, comprising above-mentioned height, the low rigorous condition of neutralization.Usually under the rigorous condition that only when target exists, could form the label probe hybridization complex, measure.Can control preciseness through changing the variable step parameter of thermodynamics, comprising but be not limited to temperature, methane amide concentration, salt concn, chaotropic salt concentration, pH, organic solvent concentration etc.Also available these parameters are controlled non-specific binding, and for example USP 5,681, and 697 is said.Therefore, under than higher rigorous condition, carry out some step to reduce comparatively ideal of non-specific binding.
The reaction that the realization that can in all sorts of ways is listed here.Reactive component can add simultaneously or with different order successively, and its preferred embodiment is following.In addition, can in reactant, add many other reagent.These reagent comprise salt, damping fluid, neutral protein matter (like BSA), stain remover etc., and they will help optimizing hybridization and detect, and/or reduce non-specific or the background interaction.The words that need also can be used the reagent that can improve determination efficiency, for example proteinase inhibitor, ribozyme suppressor factor, biocide etc., and this depends on the purity of sample preparation methods and target.Analysis detecting data is with changes of expression level between definite each expression of gene level and the various state, thus the formation gene expression atlas.
The mensuration relevant with biological activity
The invention provides and identify or screen the compound of regulating cancer related gene of the present invention or activity of proteins.This method is included in the detection compound that adds the preceding text definition in the cell that contains cancer protein of the present invention.Said cell contains the recombinant nucleic acid of code book invention cancer protein.In another embodiment, on many cells, detect the storehouse of candidate agent.
On the one hand; There is or do not exist physiological signal; Be exposed to perhaps under the situation of physiological signal and measure, said physiological signal for example has hormone, antibody, peptide, antigen, cytokine, growth factor, action potential, medicament (comprising chemotherapeutics), radiation, carcinogens or other cell (being cell-cells contacting).Among another embodiment, measure in the different steps of cell cycle.Identified in this way and regulated gene of the present invention or proteinic compound.Compound with pharmacologically active can strengthen or the activity of disorderly cancer protein of the present invention.In case identified, can be assessed similar structure so that the decisive constitutional features of authenticating compound.
In one embodiment, adjusting (for example suppressing) cancer cells splitted method is provided; This method comprises and gives a kind of cancer regulator.In another embodiment, adjusting (for example suppressing) method for cancer is provided; This method comprises and gives a kind of cancer regulator.Again in other embodiments, the method for handling cancer cells or suffering from the individuality of cancer is provided; This method comprises and gives a kind of cancer regulator.
In one embodiment, the method for regulating the state of the cell of expressing gene of the present invention is provided.State described here comprises the parameter that this area is accepted, like the growth of cell, propagation, viability, function, apoptosis, aging, location, enzymic activity, signal transduction etc.In one embodiment, the cancer suppressor factor is above-mentioned antibody.In another embodiment, the cancer suppressor factor is an antisense molecule.As described herein, the growth of various mensuration cells, propagation and the method that shifts be proficient in known to those skilled in the art.
Identify regulator through high flux screening
The test of identifying the suitable adjustable agent comprises high flux screening.Preferred test can detect enhancing or inhibition, the enhancing of expression of polypeptides or the inhibition or the enhancing of inhibition and polypeptide active that oncogene is transcribed.
In one embodiment, the regulator that the high flux screening method is identified is a protein, normally the proteinic fragment of the protein of natural generation or natural generation.Therefore, for example can use contain proteinic cell extract or protein sexual cell extract at random or directed digest.Made by this method and be used for the protein library that screens in the methods of the invention.In this embodiment, particularly preferably be bacterium, fungi, virus and mammalian proteins matter library, preferred mammal protein library, especially human protein library.Useful especially test compounds will be directed to target affiliated kinds of protein, for example enzyme and substrate, perhaps part and acceptor.
Using soft agar growth and colony to form identifies and the signature analysis regulator
Normal cell needs solid substrate to adhere to and to grow.After cell is transformed, just lose this phenotype and can leave the matrix growth.For example, cell transformed can be grown in the suspension culture of stirring or be suspended in the semisolid medium (for example semi-solid agar or soft agar).When with tumor suppressor gene transfection cell transformed, they can recover normal phenotype and need solid substrate again so that adhere to and grow.In detection, form the regulator of identifying the cancer sequence, when this sequence can suppress abnormal cell proliferation and conversion during at host cell inner expression with soft agar growth or colony.Regulator can reduce or eliminate the ability of host cell suspension growth in solid or semisolid medium (like agar).
The soft agar growth or the colony that suspend in measuring form technical description " Zooblast culture medium plinth TM " (Culture of Animal Cells a Manual of Basic Technique) (third edition, 1994) at Freshney.Also can be referring to the method part of (1996, the same) books such as Garkavtsev.
Evaluation contact inhibition and stand density restriction are to identify and the signature analysis regulator
Normal cell usually with tiling and the growth of organized pattern, contacts other cell up to them in cell culture.Their are understood contact inhibition and stop growing when other cell of cells contacting.Yet, cell transformed can contact inhibition and in the intralesional continued growth of disorder to high-density.Therefore, compare normal cell, cell transformed grows to higher saturation density.This can identify on form through in focus, forming unordered cell monolayer cell.During perhaps available saturation density ( 3H)-and the label index of thymus pyrimidine measures the stand density restriction, and similarly, MTT or Alamar are blue to be detected the multiplication capacity of showed cell and the ability of regulator influence propagation.See Freshney, (1994), the same.Cell transformed can be recovered normal phenotype when by the tumor suppressor gene transfection, receive contact inhibition and grow to than low density.
During this detects, during saturation density ( 3H)-label index of thymus pyrimidine is preferably to measure the stand density limiting method.With cancer correlated series transfection transformed host cells and make its under unrestricted culture condition saturation density growth 24 hours.Quantity through PM mixes confirm ( 3H)-per-cent of the cell of thymus pyrimidine mark.
Identify the regulator of cancer sequence with the growth that do not limit of contact, said sequence and cause abnormal cell proliferation and conversion.Regulator can reduce or eliminate the growth that do not limit of contact and make cell get back to normal phenotype.
Estimate growth factor or serum dependency to identify and the signature analysis regulator
Cell transformed is compared corresponding normal cell and is had lower serum dependency and (see for example Temin, J.Natl.Cancer Inst.37:167-175 (1966); Eagle etc., J.Exp.Med 131:836-879 (1970)); Freshney, the same.This part is because cell transformed can discharge various growth factors.Can the growth factor or the serum dependence degree of transformed host cells be compared with control cells.For example, in method, monitor the growth factor of cell or the serum dependency is regulated cancer correlated series of the present invention with evaluation and signature analysis compound.
Use the level of tumour-specific markers to identify and the signature analysis regulator
The amount of some factor (being called " tumour-specific markers " later on) that tumour cell discharges is higher than corresponding normal cell.For example; People's glioma discharges plasminogen activator (PA) with the level that is higher than the normal brain activity cell and (sees for example Gullino; " the potential disorder of vasculogenesis, tumor vessel formation and tumor growth " (Angiogenesis, Tumor Vascularization, and Potential Interference withTumor Growth); Draw from " biological response in the cancer " (Biologial Responses in Cancer) book 178-184 page or leaf (Mihich compiles, 1985)).Similarly, tumour cell is also to be higher than corresponding Normocellular horizontal release tumor angiogenesis factor (TAF).See for example Folkman, " vasculogenesis and cancer " (Angiogenesis and Cancer), Sem Cancer Biol. (1992)), and endothelioma discharges bFGF (Ensoli, B etc.).
The various technical descriptions of release of measuring these factors are in Freshney (1994), and are the same.Also can be referring to Unkless etc., J.Biol.Chem.249:4295-4305 (1974); Strickland & Beers, J.Biol.Chem.251:5694-5702 (1976); Whur etc., Br.J.Cancer 42:305 312 (1980); Gullino, " the potential disorder of vasculogenesis, tumor vessel formation and tumor growth " draws from " biological response in the cancer " book 178-184 page or leaf (Mihich compiles, 1985); Freshney, Anticancer Res.5:111-130 (1985).For example, can in method, monitor the compound of the level of tumour-specific markers with evaluation and signature analysis adjusting cancer correlated series of the present invention.
Through the invasion and attack of matrigel are identified and the signature analysis regulator
Can the degree or the extracellular matrix composition of invading matrigel be used to identify the test that can regulate the compound of cancer correlated series with signature analysis.Tumour cell is shown as positive correlation cancerating and invade between matrigel or other extracellular matrix composition.In this is measured usually with tumour generation cell as host cell.Tumor suppressor gene will reduce the invasive to host cell at these host cell inner expressions.Can use the technology of describing in the following document: Cancer Res.1999; 59:6010; Freshney (1994), the same.In brief, the available filter that scribbles matrigel or other extracellular matrix composition is measured the intrusion level to host cell.The distally of invading gel or passing filter is rated as invasiveness, and through cell count and miles of relative movement or through with 125I in advance labeled cell carry out Histological evaluation to carrying out radiocounting bottom the distally of filter or the plate then.See for example Freshney (1984), the same.
The evaluation tumor growth in vivo is to identify and the signature analysis regulator
In genetically modified organism or immunosuppression biology, detect the effect of cancer correlated series cell growth.Genetically modified organism is with the acceptable method preparation in various this areas.For example can make and knock out genetically modified organism, Mammals such as mouse for example, oncogene wherein is destroyed or inserts therein oncogene.Thereby inserting marker gene or other heterologous gene through homologous recombination at the genomic endogenous cancer gene locus of mouse produces and knocks out transgenic mice.Also the oncogene of available sudden change replaces endogenous oncogene, or makes endogenous oncogene mutation (for example through this area carcinogens) make this mouse.
For making transgenic chimaeric animals (like mouse), can the DNA construction be introduced the nucleus of embryonic stem cell.Cell with hereditary defect of new structure is injected into the host mouse embryo, and this embryo is implanted female receptor again.The some of them developing embryo becomes gomphosis mouse, and some sexual cell of these mouse are derived from mutational cell line.Therefore, can obtain to contain the new mouse species (seeing for example Capecchi etc., Science 244:1288 (1989)) of the hereditary defect of introducing through raising this gomphosis mouse.Available following method obtains gomphosis mouse: the USP 6,365,797 that on April 2nd, 2002 delivered; The USP 6,107,540 that on August 22nd, 2000 delivered; Hogan etc.; " mice embryonic operation laboratory handbook (Manipulating theMouse Embryo:A laboratory Manual); " teratocarcinoma and the embryonic stem cell actual development " that Cold Spring Harbor Laboratory (1988) and Robertson write, IRL Press, Washington; D.C., (1987).
Perhaps can use various immunosuppression or immunodeficient host animal.For example; Athymia " naked " mouse that obtains through genetic method (for example sees Giovanella etc.; J.Natl.Cancer Inst.52:921 (1974)), SCID mouse, thymusectomy mouse or irradiation mouse (for example see Bradley etc., Br.J.Cancer 38:263 (1978); Selby etc., Br.J.Cancer 41:52 (1980)) can be used as the host.To produce the invasive tumour at high proportion, the normal cell in similar source then can be not like this to be injected into homogenic host's transplantable tumour cell (usually about 106 cells).The cell of subcutaneous or normotopia injection expressing tumor correlated series in the host of having developed the invasive tumour.Then mouse is divided into control group and treatment experimental group (for example using modulators for treatment).After the suitable time, in preferred 4-8 week, measure tumor growth (for example through volume or through its two overall dimensions, or weight) and compare with contrast.The tumour that significantly reduces on the statistics (adopting for example Si Shi T check) is considered to growth and is suppressed.
The vitro detection of evaluation and signature analysis regulator
Can have the active compound of adjusting external the detection with evaluation.For example, make cancer polypeptide at first contact the potential regulator, cultivate the suitable time then, for example 0.5-48 hour.In one embodiment, through measuring protein or mRNA level in external definite cancer polypeptide level.Protein level adopts immunoassay (for example Western trace, ELISA etc.) to measure with selective binding cancer polypeptide or its segmental antibody.For measuring mRNA, preferably use augmentation detection (for example using PCR, LCR) or hybridization to detect (for example Northern hybridization, RNA enzyme protection, Dot blot).With directly or the detection reagent of montage mark detect the level of protein or mRNA, said detection reagent is the antibody of fluorescent mark or radiolabeled nucleic acid, radio-labeling or enzyme labelling etc. for example as stated.
Perhaps, the available action property cancer protein promotor that is connected in reporter genes such as luciferase, green fluorescent protein, CAT or P-gal is come the design report genic system.The reporter gene construction usually by transfection in cell.After handling with the potential regulator, be proficient in standard known to those skilled in the art count measurement report genetic transcription, translation or live vol (Davis GF, the same; Gonzalez, J. & Negulescu, P.Curr.Opin.Biotechnol.1998:9:624).
As stated, each gene and gene product are carried out in-vitro screening.In other words, the regulator of expressing this gene or its gene product is screened identifying after the specific gene of the important differential expression of particular state.
In one embodiment, the regulator of specific gene is expressed in screening.Usually only estimate one or several expression of gene.In another embodiment, screening is designed at first seek the differentially expressed proteinic compound of combination.Estimate the ability of these according a difference property of compound expression activities then.In addition, in case identified initial candidate compound, can further screen its variant with evaluation structure activity relationship better.
The combination of evaluation and signature analysis regulator detects
, combination of the present invention uses purifying or isolating gene product of the present invention in detecting usually.For example, produce the proteinic antibody of the present invention, carry out immunoassay to confirm this proteinic amount and/or location.Perhaps, the cell that contains cancer protein can be used for this mensuration.
Therefore, said method comprises makes cancer protein of the present invention combine candidate compound (like part), and confirms combining of this compound and cancer protein of the present invention.Embodiment preferred end user's cancer protein; Also can set up and use the animal model of human diseases.Simultaneously, be proficient in those skilled in the art and also can use other similar mammalian proteins matter.In addition, some embodiments have been used variant or deutero-cancer protein.
Usually, cancer protein of the present invention or part non-dispersive ground combine insoluble upholder.For example, said upholder can contain independently sample region of acceptance (microtiter plate, array etc.).Insoluble upholder can be processed with any compsn, and it can combine said compsn, is easy to from soluble substance, separate, and is perhaps compatible with whole screening method.The surface of this upholder can be solid or porous, and has the shape of any handled easily.
The example of suitable insoluble upholder comprises microtiter plate, array, film and pearl.They are usually by glass, plastics (like PS), polysaccharide, nylon, nitrocellulose or Teflon TMDeng processing.Microtiter plate and array are especially convenient, because available small volume of reagent and sample carry out big flow measurement simultaneously.To the concrete combining method of compsn and upholder regulation not, if it with reagent of the present invention with method is compatible, the activity of maintenance compsn and can not spreading.Preferred combining method comprises use antibody, when this antibody does not cause the space to check to ligand-binding site point or activation sequences during to upholder protein bound; Directly be attached to " viscosity " or ion upholder; Chemically crosslinked; At surperficial synthetic protein or reagent etc.After combining with upholder, protein or part/wedding agent remove excessive unconjugated material through washing.Cultivate with sealing sample region of acceptance with bovine serum albumin (BSA), casein or other irrelevant protein or other chemical molecular then.
In case cancer protein of the present invention combines with upholder, just can add detection compound and measure.Perhaps make candidate's wedding agent combine upholder to add cancer protein of the present invention then.Wedding agent comprises specific antibody, the non-natural wedding agent that passes through the screen chemical libraries evaluation, peptide analogs etc.
Interested especially is to identify the human cell is had hypotoxic reagent.Can use many detection methods for this purpose; Comprising proliferation assay, cAMP analysis, the protein-protein bound vitro detection of mark, electrophoretic mobility shift assay, the immunoassay of protein bound, functional analysis (phosphorylation analysis etc.), or the like.
Available many methods are measured combining of detection compound (part, wedding agent, regulator etc.) and cancer protein of the present invention.Detection compound can be labeled; And can directly confirm keying action, for example all or part of cancer protein of the present invention is added to solid support, the candidate compound (for example fluorescein-labelled) that adds mark, flush away excess reagent and confirms whether there is mark on the solid support.The words that need can be used different sealings and washing step.
In certain embodiments, have only a kind of component to be labeled, for example mark protein of the present invention or part.Perhaps use a plurality of components of different markers substance markers, for example also use the fluorophore tagged compound with the I125 labelled protein.Contiguous reagent (proximity reagent), for example quencher or energy transfer agent also are useful.
The competitive combination to identify and the signature analysis regulator
In one embodiment, through confirming combining of " detection compound " with the competitive binding experiment of " competition thing ".The competition thing is the bound fraction of binding target molecule (cancer protein for example of the present invention).The competition thing comprises the compound of antibody, peptide, binding partners, part etc. and so on.Under some situation, the competition thing replaces the competitive combination of detection compound.In one embodiment, detection compound is labeled.Can in protein, add detection compound or compete thing or the two, and place and enough carry out the bonded time.Cultivate making under the strongest active temperature (usually between 4-40 ℃).The cultivation time is optimised usually, for example to help rapid high flux screening; Common 0-1 hour just enough.Excessive reagent is removed or flush away usually.Add second component then, add or do not add the component of indication bonded mark afterwards.
In one embodiment, add the competition thing earlier, and then add detection compound.The competition thing is replaced the explanation detection compound and is combined cancer protein, the activity that therefore can combine and might regulate cancer protein.In this embodiment, arbitrary component can be labeled.Therefore, for example,, there is the compound displacement to be detected of description of symbols competition thing after detecting in the washings of compound if the competition thing is labeled.Perhaps, if detection compound is labeled, then exist description of symbols to be replaced on the upholder.
In another embodiment, at first add detection compound, cultivate and washing, add the competition thing then.The competition thing does not combine to explain that this detection compound combines the avidity of cancer protein to be higher than the competition thing.Therefore, if detection compound is labeled, thereby then exist mark competition thing to fail to combine the explanation detection compound can combine cancer protein of the present invention possibly regulate cancer protein on the upholder.
Therefore, competitive combined techniques comprises that otherness screening can regulate the active reagent of cancer protein of the present invention to identify.In this embodiment, said method comprises that the competition thing that makes in cancer protein and first sample combines.Second sample contains detection compound, cancer protein and competition thing.Confirm the combination of two sample competition things, combine the change of situation or different explanation existence can combine cancer protein and might regulate its active reagent between two samples.In other words, if the combination of competition thing is different from first sample in second sample, then this reagent can combine cancer protein.
Perhaps, screen the drug candidates of identifying the cancer protein that combines natural cancer protein but can not combine to modify with otherness.For example, confirm the structure of cancer protein and use it for rational medicinal design, thus the synthetic proteinic reagent of modifying with interactional reagent in this site and common debond site.In addition, the ability that also strengthens or reduce this protein active through screening of medicaments has been identified the active drug candidates of the natural cancer protein of influence.
Also can use positive control and negative control in the analysis.Control sample and specimen should be carried out at least three times to obtain significant result on the statistics.All samples is cultivated the time that is enough to make reagent and protein bound.Washing sample is with the material of removing non-specific binding and confirm bonded and the amount of the reagent of mark normally after cultivating.For example, when using radio-labeling, sample can be in the scintillometer inside counting to confirm the amount of bonded compound.
Screening also can be used other reagent in analyzing.These reagent comprise salt, neutral protein matter (for example BSA), stain remover etc., use them will help protein-protein best combination and/or reduce non-specific or background interacts.Also can use the reagent that can improve determination efficiency, for example proteinase inhibitor, ribozyme suppressor factor, biocide etc.The mixture that adds each component with a definite sequence is to provide the combination that needs.
Use polynucleotide to reduce or suppress protein of the present invention
Of WO 91/04753, can the polynucleotide regulator of cancer be introduced the cell that contains target nucleotide sequences through forming with the conjugate of ligand binding molecules.Suitable ligand binding molecules includes but not limited to: other part of cell surface receptor, growth factor, other cytokine or combination cell surface receptor.The combination of preferred ligand binding molecules can not influence ligand binding molecules basically and combine its corresponding molecule or acceptor, perhaps stops justice or antisense oligonucleotide or its combining form to get into the ability of cell.Perhaps, the polynucleotide regulator of cancer can be introduced into the cell that contains target nucleic acid sequence, and is for example through forming polynucleotide-lipid complex body, of WO 90/10448.Except that the treatment method, in above-mentioned screening method, also can use antisense molecule or knock out and knock in model.
Inhibition and GEM 132
In certain embodiments; Through using antisense polynucleotides or inhibition small nuclear RNA (snRNA); Promptly with mRNA nucleic acid sequence encoding (cancer protein for example of the present invention), mRNA or its subsequence is complementary and can be preferably and the nucleic acid of their specific hybrids, downward modulation or suppress the activity of cancer GAP-associated protein GAP fully.Antisense polynucleotides combines to have reduced mRNA translation and/or stability with mRNA.
In the present invention, antisense polynucleotides can comprise the Nucleotide of natural generation or the synthetic kind that is formed by subunit or its close homologue of natural generation.Antisense polynucleotides also can contain sugar moieties or the interior connection of sugar that changes.Its example has thiophosphatephosphorothioate and other to contain the kind of sulphur, known they all can be used for this area.As long as analogue can promptly be included within the present invention with the effective hybridization of Nucleotide of the present invention.See for example Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA.
This antisense polynucleotides can be synthetic easily with recombination method, perhaps can be external synthetic.Some manufacturers sell this synthesis device, comprising Applied Biosystems.The method for preparing other oligonucleotide such as thiophosphatephosphorothioate and alkyl derivative also be proficient in well-known to those having ordinary skill in the art.
Antisense molecule used herein comprises antisense or positive MODN.For example, available positive MODN is blocked with combining of antisense strand and is transcribed.Antisense comprises the single-chain nucleic acid sequence (RNA or DNA) that can combine cancer molecule said target mrna (justice) or DNA (antisense) sequence with positive MODN.Antisense of the present invention or positive MODN comprise and containing usually at least about 12 Nucleotide, preferably contain the fragment of 12-30 the Nucleotide of having an appointment.The ability description of specifying proteinic cDNA sequence to produce antisense or positive MODN from coding exists, for example, and (BioTechniques 6:958 (1988)) such as Stein Cohen (Cancer Res.48:2659 1988) and van der Krol.
Ribozyme
Remove antisense polynucleotides, available ribozyme comes target-seeking and suppresses transcribing of cancer related nucleotide sequences.Ribozyme is to cut off other RNA RNA molecule molecule by catalytic.Different types of ribozyme had been described; Comprising I group ribozyme, hammerhead ribozyme, hairpin ribozyme, ribonuclease P and axehead ribozyme (can be referring to for example Castanotto etc., Adv.in Pharmacology 25:289-317 (1994) have roughly understand) with characteristic to different ribozymes.
The general characteristic of hair clip ribozyme is described in, for example, and Hampel etc., Nucl.Acids Res. 18:299-304 (1990); European Patent Publication No 0360257; USP 5,254,678.Its preparation method is to be proficient in well-known to those having ordinary skill in the artly (to see for example WO 94/26877; Ojwang etc., Proc.Natl.Acad.Sci.USA 90:6340-6344 (1993); Yamada etc., Human Gene Therapy1:39-45 (1994); Leavitt etc., Proc.Natl.Acad Sci.USA 92:699-703 (1995); Leavitt etc., Human Gene Therapy 5:1151-120 (1994); And Yamada etc., Virology205:121-126 (1994)).
In phenotypic screen, use regulator
In one embodiment, the cancer cell population detection compound that has cancer correlated expression characteristic." give " or " contact " here is meant regulator is added in the cell, regulator is through taking in and in born of the same parents, playing a role or play a role at cell surface to the mode of action of this cell.In some embodiments, the nucleic acid of coded protein appearance reagent (being peptide) is added into the virus formulation thing, for example adenovirus or retrovirus construction, and add cell, can realize the expression of peptide reagent like this, for example PCT US97/01019.Also available regulatable gene therapy system.In case give cell modulator, the words washed cell that needs and preferably under physiological condition with cell culture for some time.Harvested cell and generate new gene expression atlas then.The cancerous tissue phenotype that this reagent place of screening regulates (induce or suppress) changes.This reagent of variation explanation of at least one (preferably having a plurality of) gene expression atlas has effect to the cancer activity.Similarly, the activity of regulator has also been indicated in the change of biological function or signal path.Can filter out the new drug that changes this phenotype through defining this cancerous phenotype characteristic.Need not know the medicine target in this way, and need not express on the platform and show, and need not change the transcript level of target protein in traditional genes matter.The inhibit feature of regulator will be used as surrogate markers.
As stated, gene or gene product are screened.In other words, after the gene that identifies the concrete differential expression important, the regulator of expressing this gene or its gene product itself is screened for particular state.
Use regulator to influence peptide of the present invention
Activity or cancerous phenotype with various experimental measurement cancer polypeptides.For example through detecting the effect of above-mentioned parameter measuring and adjusting agent to cancer polypeptide.Assess the influence of detection compound with the active physiological change of influence to polypeptide of the present invention.After confirming the function result, can assess various effects with complete cell or animal; For example; In the cases of cancer relevant with solid tumor, these effects comprise that tumor growth, metastases, neovascularization, hormone discharge, the variation of transcribing variations (for example through the Northern trace), cellular metabolism of known and unknown genetic marker such as cell is grown or the change of pH and born of the same parents in the variation of second messenger such as cGNIP.
Identify the method for qualitative cancer correlated series
The expression of range gene sequence is relevant with cancer.Therefore can confirm disease based on two mutants or variant cancer gene.In one embodiment, the invention provides the method for identifying the cell that contains variant cancer gene, for example confirm to exist in the cell sequence of (all or part of) at least a endogenous cancer gene.This can realize through multiple sequencing technologies.The present invention includes the genotypic method of individual cancers of identifying, for example confirm all or part of sequence of at least a gene of the present invention in the individuality.This carries out at least a individual tissue usually, the listed tissue of Table I for example, and can comprise the different samples of estimating many tissues or homologue.This method can comprise that the sequence of sequenced genes and known cancer gene (being wild type gene) compare to confirm to exist family member, homologue, two mutants or variant.Can all or part of sequence of this gene and the sequence of known cancer gene be compared to determine whether to exist any difference then.This can carry out through multiple known homology program, like BLAST, Bestfit etc.As described here, there are difference and ill or ill being associated of possibility between patient's cancer gene order and the known cancer gene order.
In an embodiment preferred, the cancer gene is used as probe to confirm the copy number of cancer gene in the genome.The cancer gene is used as probe to confirm the chromosomal localization of cancer gene.Information such as chromosomal localization can be used for diagnosis or prediction, when especially in cancer gene locus, identifying chromosome abnormalty such as transposition.
XIV.) RNAi and the treatment of adopting siRNA (siRNA)
The invention still further relates to the siRNA oligonucleotide, specifically is to comprise PSCA coding region or 5 " at least one segmental double-stranded RNA in UTR district or any the PSCA sequence is had specific complementary strand or antisense oligonucleotide.In one embodiment, this class oligonucleotide is used to illustrate the function of PSCA, or is used to screen or assess the regulator of PSCA function or expression.In another embodiment, through the genetic expression of use siRNA transfection reduction PSCA, and make the multiplication capacity of this antigenic conversion cancer cells of endogenous expression significantly reduce; Through the mensuration to for example relevant with the multiplication capacity that reduces cell viability metabolism reading, the cell of handling with specificity PSCAsiRNA shows that survival rate reduces.Therefore, PSCA siRNA compsn comprises siRNA (double-stranded RNA), and it is corresponding to PSCA proteic nucleic acid ORF sequence or its subsequence; The length of these subsequences is generally 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30; 31,32,33,34,35 or greater than 35 continuous RNA Nucleotide, and contain complementary and non-complementary sequence with at least a portion of mRNA encoding sequence.In a preferred implementation, the length of said subsequence is 19-25 Nucleotide, and most preferably length is 21-23 Nucleotide.
It is a kind of novel method that makes gene silencing in vitro and in vivo that RNA disturbs, and therefore little double-stranded RNA (siRNAs) is of great value therapeutical agent.SiRNA makes the reticent ability of special gene activity be used in the disease animal model now, and also has been used for the mankind.For example, confirmed to contain anti-concrete target siRNA siRNA solution through fluid administration give mouse be treatment effectively.
The pioneering research of Song etc. shows can be with one type of complete natural nucleic acid---and siRNA (siRNA) is as therapeutical agent; Even need not it is carried out further chemically modified (Song; E.; Deng, " RNAinterference targeting Fas protects mice from fulminant hepatitis " Nat.Med.9 (3): 347-51 (2003)).This research provides the first individual internal evidence certificate that siRNA is infused and can palliate a disease into animal.In this case, authors have given injected in mice and have been designed for reticent FAS albumen (a kind of necrocytosis acceptor, it can inducing hepatocyte and other necrocytosis when overactivity in inflammatory reaction)) siRNA.Next day, give animal Fas specific antibody.Control mice is at a few days internal cause acute hepatic failure and death, keeps not taking place serious disease and survival and surpass 80% the mouse with the siRNA treatment.Their hepatocellular about 80%-90% have mixed exposed siRNA oligonucleotide.In addition, before the back forfeiture of 3 weeks was renderd a service, this RNA molecule continued to play a role and reaches 10 days.
When being used for the human treatment, send siRNA through inducing the active effective system of long-acting RNAi.Main warning (caveat) in the clinical application is siRNA is delivered in the suitable cell.As if liver cell especially be easy to accept the RNA of external source.At present, the target that is positioned at liver is very attractive, and this is because this organ extremely is easy to by nucleic acid molecule and virus vector institute target.Yet other organ also is preferred with organizing target.
Use contains the siRNA preparation of the compound that promotes the permeate through cell membranes transportation in treatment, to improve the administration of siRNA.Another embodiment is that nucleicacidase is had resistance, has the synthetic siRNA through chemically modified that serum stability makes thus that also the time length of RNAi effect increases.
Therefore, the siRNA technology is a kind of through sending to the therapy of the individuality of suffering from cancer (for example in the table 1 listed cancer) with the treatment human malignancies to the siRNA molecule of PSCA.This siRNA of giving can reduce the cancer cells of expressing PSCA growth, a kind of antitumor therapy, sickness rate and/or mortality ratio that reduction is relevant with malignant tumour are provided.
When in external or body, measuring, the effect that the gene product of this pattern knocks out is very significant.When adopting in vitro method to detect the reduction of PSCA protein expression, be easy to confirm its in vitro effects through the cancer patients or the tissue that siRNA are applied to culturing cell (as previously mentioned) or equivalent.
XV.) manufacturing of test kit/product
For being used for laboratory described here, prognosis, prevention, diagnosis and therepic use, the present invention includes test kit.This test kit can comprise carrier, packing or divided to hold the container of one or more contents such as bottle, test tube etc., comprises an independent component that is used for this method separately during each container is interior, and comprises working instructions.For example, can comprise in the container by or can be by the probe of detectable label.This probe can be antibody or the polynucleotide that are specific to protein of the present invention or gene or courier respectively.When said method utilizes nucleic acid hybridization to detect target nucleic acid, also can contain in the container of test kit and be used for the Nucleotide of amplifying target nucleic acid sequence.Contain acceptor in the container of test kit, as combining the protein of vitamin H, for example avidin or Streptavidin, they are attached to reporter molecules such as enzyme labelling, fluorescent mark or labelled with radioisotope; This reporter molecules can use with for example nucleic acid or antibody.Said test kit is at all or part of aminoacid sequence that can contain Fig. 1 or its analogue, or the nucleic acid molecule of this aminoacid sequence of encoding.
Test kit of the present invention comprises said vesse and one or more other container relevant with it usually, contains in these containers from commercial and user's position and the material of needs comprises damping fluid, thinner, filler, syringe needle, needle tubing; The bottle of carrier, packing, sign composition and/or operation instruction and/or label on the test tube and working instructions.
Label can appear on the container or with container and occur; To instruct how said composition is used for specific treatment or non-therepic use; For example prognosis, prevention, diagnosis or laboratory purposes, and how can to instruct in vivo and external use are like described here those.Explain or out of Memory also can appear on specification sheets or the label that said specification sheets or label provide or are provided on the test kit with test kit.Label can appear on the container or with container and provide.When the letter, numeral or the further feature that constitute label be molded into or embed container originally on one's body the markers label can appear on the container; Sign and to provide with container when label appears at box or carrier internal clock that this container is housed.Label can indicate how compsn is used for diagnosis, treatment, prevention or prediction symptom, and the said symptom for example tumour of the listed tissue of Table I forms.
Term " test kit " and " manufacturing article " interchangeable use.
In another embodiment of the invention; Make in the article and comprise compsn; Like aminoacid sequence, small molecules, nucleotide sequence and/or antibody, for example be used for diagnosis, prognosis, prevent and/or treat tissue the article that (those tissues of listing like Table I) tumour forms.Make in the article and contain at least one container and a at least label usually.Suitable containers comprises, for example, and bottle, bottle, syringe and test tube.Said container can be made of a variety of materials, like glass, metal or plastics.Can contain aminoacid sequence, small molecules, nucleotide sequence, cell mass and/or antibody in the container.In one embodiment, contain in the container and be used for the reagent that detects the polynucleotide of cell mRNA expression map and be used for this purpose.In another embodiment, contain antibody, its binding fragment or binding proteins specific in the container,, or be used for related experiment chamber, prognosis, diagnosis, prevention and therapeutic purpose with the protein expression of PSCA in estimating cell and organizing; The guidance that also has this purposes and/or the specification sheets that on this container or therewith occur, and can contain the reagent and other compsn or instrument that is useful on these purposes.In another embodiment, contain in the container and be used for the material of trigger cell or HI and relevant guidance and/or specification sheets.In another embodiment, contain the material that is useful on adoptive immunotherapy in the container, as like cytotoxic T cell (CTL) or helper cell (HTL), and relevant guidance and/or specification sheets; Also can contain the reagent and other compsn or instrument that are useful on this purpose.
Also can contain the compsn that effectively to treat, diagnose, predict or prevent certain symptom in the container and can contain sterile port (for example, said container can be intravenous solution bag or bottle, the stopper that has available hypodermic needle to pierce through on it).Promoting agent in the compsn can be specificity to combine PSCA and to regulate the antibody of PSCA function.
Said manufacturing article also can contain second container, wherein contain pharmaceutically acceptable damping fluid, like PBS, Ringer's solution and/or glucose solution.It also can contain other from commercial with user's position and the material of needs comprises the guidance of other damping fluid, thinner, filler, whisking appliance, syringe needle, needle tubing and/or working instructions and/or usage.
Embodiment
Several embodiment through following do further description and explanation to various aspects of the present invention, and these embodiment do not mean that limitation of the scope of the invention.
Embodiment 1
The expression analysis of PSCA variant in healthy tissues and patient's sample
V.1 PSCA is called as PSCA in this article, and former research has confirmed that PSCA expresses as antigen in prostate cancer.The expression of PSCA is all arranged in surpassing 80% former prostate cancer and most of prostate gland metastatic carcinoma.PSCA also is expressed in carcinoma of gallbladder, ovarian cancer and the carcinoma of the pancreas; Such tumour is all listed in the Table I.Find that through immunohistochemical analysis cell surface at most of urethra transitional cell carcinomas, 60% former pancreas adenocarcinoma all has the overexpression of PSCA.The article of the many patent documentations (PCT/US98/04664, PCT/US/28883, PCT/US00/19967) and the peer review (Saffran etc., Proc NatlAcad Sci U S A.2001-2-27; 98 (5): 2658-2663; Amara etc., Cancer Res.2001-6-15; 61 (12): 4660-65; Reiter etc., Proc Natl Acad Sci USA.1998-2-17; 95 (4): 1735-40; Argani etc., Cancer Res.2001-6-1; 61 (11): the data of all having reported relevant psca expression 4320-24).
The present invention has studied the specific expressed situation of different PSCA variants in healthy tissues and cancer patients's sample.Institute's designed primer can distinguish PSCA v.1/v.2/v.4, PSCA v.3 and the difference of PSCA between v.5; PSCA PCR product length v.1/v.2/v.4 is 425bp; PSCA PCR product length v.3 is 300bp, and PSCA PCR product length v.5 is 910bp (Fig. 1 I (a)).
From normal gall-bladder, brain, heart, kidney, liver, lung, prostate gland, spleen, Skelettmuskel, testis, pancreas, colon, stomach-tissue and one group of prostate cancer, carcinoma of gallbladder, kidney, colorectal carcinoma, lung cancer, ovarian cancer, mammary cancer, metastatic carcinoma and carcinoma of the pancreas tissue, prepare cDNA article one chain (Fig. 1 I (b)).It is contrast that the primer of use Actin muscle carries out the resulting product of pcr amplification.Utilize the specific primer of different variants to carry out sxemiquantitative PCR, 30 circulations are carried out in amplification.
The result shows that v.5 PSCA mainly is expressed in mammary cancer, metastatic carcinoma and the carcinoma of the pancreas, also has low expression level in colorectal carcinoma and lung cancer.In prostate cancer, carcinoma of gallbladder, kidney, colorectal carcinoma, lung cancer, ovarian cancer, mammary cancer, metastatic carcinoma and carcinoma of the pancreas, detected PSCA PCR product v.1/v.2/v.4.In healthy tissues; Only in prostate gland, stomach-tissue, detected PSCA PCR product v.1/v.2/v.4; V.1/v.2/v.4, v.5 the PSCA that in kidney and lung tissue, has yet detected low expression level and does not detect PSCA in all healthy tissuess.In all samples that is detected, all do not detect PSCA PCR product v.3.
Design can distinguish PSCA v.4 with PSCA primer (Fig. 1 J (a)) v.5.PSCA PCR product length v.4 is 460bp, and PSCA PCR product length v.5 is 945bp.
In normal gall-bladder, brain, heart, kidney, liver, lung, prostate gland, spleen, Skelettmuskel, testis, pancreas, colon, stomach-tissue and one group of prostate cancer, carcinoma of gallbladder and multiple heterograft mixture (prostate cancer, kidney and carcinoma of gallbladder heterograft) tissue, prepare cDNA article one chain (Fig. 1 J (b)).It is contrast that the primer of use Actin muscle carries out the resulting product of pcr amplification.Utilize the specific primer of different variants to carry out sxemiquantitative PCR, 30 circulations are carried out in amplification.
The result is presented in prostate cancer, carcinoma of gallbladder, multiple heterograft mixture, normal kidney and the prostata tissue has PSCA expression v.4.Only in normal prostate tissue and Proteins in Carcinoma of Gallbladder, detected the expression of PSCAv.5.
The expression of PSCA variant in healthy tissues is limited, and in cancer patients's sample, can detect the expression of PSCA variant, and this explanation PSCA variant can be used as therapeutic, prognostic, experimental, the preventative and diagnosis target position of human tumor.
Embodiment 2
The shearing variant of PSCA
Term used herein " variant " comprises transcript variant and SNP (SNP).The transcript variant is the variant of the ripe mRNA of same gene, owing to alternative transcription or alternative splicing form.But the alternative transcription thing is meant from the different transcript of same gene transcription initiation site.Splice variant is meant the mRNA variant that same transcript forms through different montages.In eukaryote, after many exon genes transcribed out from genomic dna, the RNA precursor that is produced need pass through montage could form functional mRNA, and this mRNA has only an exon, is used to translate into aminoacid sequence.Accordingly, a given gene has 0 to a plurality of different alternative transcription things, and each transcript all has 0 to a plurality of splice variants.Each transcript variant all is made up of its unique exon, comprises the different coding and/or non-coding (5 ' or the 3 ' end) part in transcript precursor source.Transcript variant identical, the similar or different protein of can encoding, this protein can have identical or similar function, also different functions can be arranged.Mutein possibly expressed in identical time, identical tissue, also possibly in identical time, different tissue, express, and perhaps in different time, identical tissue, expresses, and perhaps in different time, different tissue, expresses.Transcribe variant encoded protein matter and possibly have similar or different ubcellulars or location, extracellular (like type in secretor type and the born of the same parents).
Many methods of this area all can be used for identifying the transcript variant.For example, alternative transcription thing and splice variant can or use total length transcript and est sequence to identify through full-length clone.At first the somebody source EST of institute is divided into different bunches, these bunches have direct or indirect homogeny each other.The second, will be divided into different Asia bunch with the EST in the cluster, be combined into a consensus sequence.The original series of gene and consensus sequence or other full length sequence are relatively.Each consensus sequence all is a potential splice variant of this gene.It is known in the art that several conclusive evidence methods are arranged, and identifies variant as waiting through RNA trace, full-length clone or use probe library.Even the variant that is identified is not a full-length clone, this variant fragment also can be used as research tool, for example is used for antigen prepd through technology well known in the art, perhaps is used for further cloning the total length splice variant.
In addition, this area has some computer programs now, and these programs can be identified the transcript variant according to genome sequence.Transcript variant qualification program based on genome sequence comprises FgenesH (A.Salamov and V.Solovyev, " Ab initio gene finding in Drosophila genomic DNA, " GenomeResearch.2000-4; 10 (4): 516-22); Grail (URLcompbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (URLgenes.mit.edu/GENSCAN.html).Southan is seen in the discussion of relevant splice variant Identification Tools, C., A genomic perspective on human proteases, FEBS Lett.2001-6-8; 498 (2-3): 214-8; De Souza, S.J. etc., Identification of human chromosome 22transcribed sequences with ORF expressed sequence tags, Proc.NatlAcad Sci U S is A.2000-11-7; 97 (23): 12690-3.
The existing multiple technologies in this area can be used for further confirming the parameter of transcript variant; Checking and 5 ' the RACE checking that like the checking of full-length clone, protein group, with PCR is the basis waits (sees that protein group verifies: Brennan; S.O. etc.; Albumin banks peninsula:a new termination variantcharacterized by electrospray mass spectrometry, Biochem Biophys Acta.1999-8-17; 1433 (1-2): 321-6; Ferranti P etc., Differential splicing ofpre-messenger RNA produces multiple forms of mature caprinealpha (s1)-casein, Eur J Biochem.1997-10-1; 249 (1): 1-7.With PCR is the checking on basis: Wellmann S etc.; Specific reverse transcription-PCR quantificationof vascular endothelial growth factor (VEGF) splice variants byLightCycler technology, Clin Chem.2001-4; 47 (4): 654-60; Jia, H.P. etc., Discovery of new human beta-defensins using a genomics-based approach, Gene.2001-1-24; 263 (1-2): 211-8.With PCR is the checking and 5 ' RACE checking: the Brigle on basis; K.E. etc.; Organization of the murine reduced folate carrier geneand identification of variant splice forms, Biochem Biophys Acta.1997-8-7; 1353 (2): 191-8).
As known in the art, the genome district of tumour is vicissitudinous.When the genetic mapping in the genome district of a specific tumors changed, corresponding variation also can take place in the alternative transcription thing or the splice variant of this gene.A special expression pattern (seeing table 1) that is exactly the PSCA relevant described herein with tumour.The alternative transcription thing and the splice variant that also comprise PSCA in the tumour are like one or more tissues in these tissues and some other tissue-derived tumour.Therefore these variants also can be regarded as tumour mark of correlation/antigen.
Utilize total length psca gene and est sequence to identify four other transcript variants, be named as PSCA respectively v.2, v.3, v.4 and v.5.Primary transcript PSCA exon border v.1 is presented in the Table VI.The sequence of PSCA and PSCA variant is shown among Fig. 1.
Embodiment 3
The SNP of PSCA
SNP (SNP) is meant that nucleotides sequence is listed in the variation of the single base pair on the specific site.All exist four possible nucleotide bases right genomic on arbitrarily to locating point: A/T, C/G, G/C and T/A.Such as this paper description, allelotrope is meant in a given gene a series of multi-form, the difference between the isoallele is not its dna sequence dna, and can influence its product (RNA and/or protein).
The SNP of the last generation of cDNA is called as cSNP.This cSNP can cause the variation of the protein generation aminoacid sequence of genes encoding, thereby proteinic function also possibly change.Some SNP can cause heredopathia; Some SNP in addition are relevant to the quantitative change of the reaction of environmental factors such as food and medicine with phenotype quantitative change and different people.Therefore; SNP and/or allelotrope make up many purposes that have of (being called as haplotype); Analysis (P.Nowotny like genetic correlation between the evaluation of the confirming of diagnosis, drug reaction and the dosage of heredopathia, disease related gene and the individuality; J.M.Kwon and A.M.Goate, " SNPanalysis to dissect human traits, " Curr.Opin.Neurobiol.2001-10; 11 (5): 637-641; M.Pirmohamed and B.K.Park, " Genetic susceptibility toadverse drug reactions, " Trends Pharmacol.Sci.2001-6; 22 (6): 298-305; J.H.Riley, C.J.Allan, E.Lai and A.Roses, " The use of single nucleotidepolymorphisms in the isolation of common disease genes ", Pharmacogenomics.2000-2; 1 (1): 39-47; R.Judson, J.C.Stephens and A.Windemuth, " The predictive power of haplotypes in clinical response, " Pharmacogenomics.2000-2; 1 (1): 15-26).
SNP can use the existing several different methods in this area to identify (P.Bean, " The promisingvoyage of SNP target discovery, " the Am.Clin.Lab.2001 10-11 month; 20 (9): 18-20; K.M.Weiss, " In search of human variation, " Genome Res.1998-7; 8 (7): 691-697; M.M.She, " Enabling large-scale pharmacogeneticstudies by high-throughput mutation detection and genotypingtechnologies, " Clin.Chem.2001-2; 47 (2): 164-172).For example, detect through gel method such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) and find that the dna fragmentation with polymorphum can identify its SNP through checking order.The sequence of DNA sample that in addition also can be through direct mensuration Different Individual or the sequence through more different DNA samples disclose SNP.Utilize the sequence data that accumulates fast in public database and the private database access; People also can utilize computer program relatively to find SNP (Z.Gu through sequence; L.Hillier and P.Y.Kwok; " Single nucleotidepolymorphism hunting in cyberspace, " Hum.Mutat.1998; 12 (4): 221-225).Many methods all can be used for verifying SNP; Confirm the genotype and the haplotype of Different Individual; Comprising directly checking order and high-throughput chip method (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-throughput SNP genotyping with theMasscode system, " Mol.Diagn.2000-12; 5 (4): 329-340).
Described method has been found out v.2 13 SNP of transcript of PSCA above the utilization.We have used variant 2 rather than variant 1, and this is because the indeterminate base of variant 2 is less than variant 1.Accordingly, PSCA SNP v.2 is determined and is positioned on site 57 (t/c), 367 (c/t), 424 (a/c), 495 (c/g), 499 (c/t), 563 (c/t), 567 (g/a), 627 (g/a), 634 (t/g), 835 (g/a), 847 (g/a), 878 (g/a) and 978 (c/g).Allelic transcript of selectivity or protein are named as variant PSCAv.6 to v.18, shown in Figure 1B and Fig. 1 G.
V.6 the Nucleotide gone up changes and has changed initiator codon v.1, so its translation just begins up to running into next ATG (the last AUG of being of mRNA), and the protein of generation lacks 9 amino acid than albumen v.1.V.7 and the Nucleotide of v.8 going up change that to be reflected on the protein level be reticent.
12 among these 13 SNP also are present on the variant 4.12 SNP that PSCA is v.4 relevant are named as PSCA respectively and v.19 arrive v.30.Variant 19 to 27 coding different amino acid are shown in Fig. 1 H.
Embodiment 4
Preparation reorganization PSCA in prokaryotic system
For the PSCA and the PSCA variant of express recombinant in prokaryotic cell prokaryocyte, with total length PSCA and PSCA variant cDNA sequence or its fragment cloning in any expression vector well known in the art.Express following one or more fragments of PSCA variant: full length sequence shown in Figure 1, perhaps contain the fragment of 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 more a plurality of continuous amino acids of PSCA, PSCA variant or its analogue.
A. in-vitro transcription with the translation construction:
PCRII: in order to prepare PSCA justice and the antisense RNA probes that is used for the RNA in situ detection, preparation coding total length PSCA cDNA or its segmental pCRII construction (Invitrogen, Carlsbad CA).Contain Sp6 and T7 promotor at the pCRII carrier, lay respectively at and insert segmental both sides, these two promoters driven PSCARNA transcribe, and the RNA that transcribes out can be used as probe and is used for RNA in situ hybridization test.These probes are used in analyzes PSCA in cell and in-house expression on the rna level.(Madison synthesizes PSCA albumen in WI) for Promega, Corp. to represent the PSCA rna transcription thing of psca gene cDNA amino acid coding region to can be used for external translating system such as TnTTM Coupled ReticulolysateSystem.
B. bacterium construction:
The pGEX construction: for preparation fusion in bacterium has the proteic reorganization PSCA of glutathione s-transferase (GST) albumen; Total length or part PSCA cDNA albumen coded sequence are cloned into (the Amersham Pharmacia Biotech of pGEX plasmid family of GST fusion vector; Piscataway, NJ) in.These constructions can controllably be expressed aminoterminal fusion GST, carboxyl terminal is 6 * Histidine epi-position (reorganization PSCA albumen of 6 * His).The existence of GST and 6 * His mark mainly is for suitable affinity matrix purification of Recombinant fusion rotein from the inductive bacterium, and anti-GST antibody and anti-His antibody can be discerned this fusion rotein.Through on 3 ' end clone primer of open reading frame (ORF), adding that 6 Histidine codons just can make albumen add last 6 * His mark.Through introducing protease cracking site, as on pGEX-6P-1, introducing the PreScissionTM recognition site, can the cracking from the PSCA GAP-associated protein GAP of GST mark be got off.Penbritin drug resistant gene and pBR322 replication orgin be used for the pGEX plasmid in intestinal bacteria screening and duplicate.
The pMAL construction: for the reorganization PSCA albumen that MBP (MBP) is arranged is merged in preparation in bacterium; Total length or part PSCA cDNA albumen coded sequence are cloned into pMAL-c2X and pMAL-p2X carrier (New England Biolabs; Beverly makes it the gene fusion with MBP in MA).These constructions can controllably be expressed aminoterminal fusion MBP, carboxyl terminal is the reorganization PSCA albumen of 6 * Histidine epi-position mark.The existence of MBP and 6 * His mark mainly is for suitable affinity matrix purification of Recombinant fusion rotein from the inductive bacterium, and anti-MBP antibody and anti-His antibody can be discerned this fusion rotein.Through on 3 ' end clone primer, adding that 6 Histidine codons just can make albumen add last 6 * His mark.Adding factor Xa recognition site is to get off for the pMAL mark is gone up cracking from PSCA.PMAL-c2X and pMAL-p2X carrier are respectively applied for the kytoplasm and the periplasmic expression of recombinant protein.Periplasmic expression helps albumen and utilizes disulfide linkage to fold.
The pET construction: in order in bacterial cell, to express PSCA, with total length or part PSCA cDNA albumen coded sequence be cloned into pET family carrier (Novagen, Madison, WI) in.These carriers can controllably express to merge in bacterium has protein and epi-position mark that increases solubility or the reorganization PSCA albumen that does not contain fusion rotein; The protein that increases solubility comprises NusA and sulphur hydrogen reduction albumen (Trx), and the epi-position mark comprises 6 * His and S-Tag TM, these marks can be used for the purifying and the detection of recombinant protein.For example, the PSCA albumen that utilizes pET NusA emerging system 43.1 construction of expression vector to make to give expression to is NusA on aminoterminal merges.
C. yeast construction:
The pESC construction: in order in yeast saccharomyces cerevisiae, to express PSCA to prepare recombinant protein and to carry out functional study; Total length or part PSCA cDNA albumen coded sequence are cloned in the carrier of pESC family; These carriers contain a kind of in following four kinds of selection markers respectively: HIS3, TRP1, LEU2 and URA3 (Stratagene; La Jolla, CA).These carriers make us in same primary yeast cell, from the same plasmid of 2 different genes or cloned sequence, controllably express different albumen, and these two genes or cloned sequence contain FlagTM and Myc epi-position mark respectively.This system can be used for confirming the protein-protein interaction of PSCA.In addition, in yeast, express and to produce the posttranslational modification similar, like glycosylation and phosphorylation with eukaryotic cell expression.
The pESP construction: in order in grain brewer yeast (Saccharomyces pombe), to express PSCA, total length or part PSCA cDNA albumen coded sequence are cloned in the carrier of pESP family.These carriers controllably high level expression merge the PSCA protein sequence that GST is arranged at aminoterminal or carboxyl terminal, and GST helps the purifying of recombinant protein.The existence of FlagTM epi-position makes us detect recombinant protein by enough anti-FlagTM antibody.
Embodiment 5
Production reorganization PSCA in higher eucaryote
A. Mammals construction:
For express recombinant PSCA in eukaryotic cell, can PSCA cDNA sequence or its variant of total length or partial-length be cloned into any one in the multiple expression vector known in the art.In these constructions, express the zone below PSCA one or more: PSCA v.1, the amino acid/11-123 in PSCA variant or their analogue or 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 more a plurality of continuous amino acid wherein.
Can this construction be transfected into any in the multiple mammalian cell (for example 293T cell).Available anti-PSCA polyclonal serum as herein described carries out probe mark to the lysate of the 293T cell of transfection.
The pcDNA4/HisMax construction: in order in mammalian cell, to express PSCA, with the PSCAORF of PSCA or its part be cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA).Come the expression of kinesin matter with cytomegalovirus (CMV) promotor and SP16 transcriptional enhancer.This recombinant protein has 6 Histidines (6 * His) epi-positions and XpressTM that merge with aminoterminal.Said pcDNA4/HisMax carrier also comprises Trobest (BGH) polyadenylation signal and transcription termination sequence improving the stability of mRNA, and is used for SV40 starting point that episome duplicates and the simple carrier that can obtain to save in the clone of expressing large T antigen.Rely on the screening of damp wet star (Zeocin) resistant gene to express said proteinic mammalian cell, and rely on Ampicillin Trihydrate resistant gene and the screening of ColE1 starting point and keep the plasmid in the intestinal bacteria.
The pcDNA3.1/MycHis construction: in order in mammalian cell, to express PSCA, with the PSCA ORF of PSCA or its part with total Kozak translation initiation site be cloned into pcDNA3.1/MycHis VersionA (Invitrogen, Carlsbad, CA).With cytomegalovirus (CMV) promoters driven protein expression.This recombinant protein has 6 Histidines (6 * His) epi-positions and the myc epi-position that merges with carboxyl terminal.Said pcDNA3.1/MycHis carrier also comprises Trobest (BGH) polyadenylation signal and transcription termination sequence improving the stability of mRNA, and is used for SV40 starting point that episome duplicates and the single carrier that can obtain to save in the clone of expressing large T antigen.Can adopt the neomycin resistance gene screening to express said proteinic mammalian cell, and rely on Ampicillin Trihydrate resistant gene and the screening of ColE1 starting point and keep the plasmid in the intestinal bacteria.
The pcDNA3.1/CT-GFP-TOPO construction: in order in mammalian cell, to express PSCA and can to use fluorescence to detect recombinant protein, with PSCA ORF or its part with total Kozak translation initiation site be cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA).With cytomegalovirus (CMV) promoters driven protein expression.This recombinant protein has the green fluorescent protein (GFP) that merges with carboxyl terminal, makes that non-intruding, the interior detection of body and RESEARCH ON CELL-BIOLOGY are more easy.Said pcDNA3.1CT-GFP-TOPO carrier also comprises Trobest (BGH) polyadenylation signal and transcription termination sequence improving the stability of mRNA, and is used for SV40 starting point that episome duplicates and the single carrier that can obtain to save in the clone of expressing large T antigen.Rely on the neomycin resistance gene screening to express said proteinic mammalian cell, and rely on Ampicillin Trihydrate resistant gene and the screening of ColE1 starting point and keep the plasmid in the intestinal bacteria.In pcDNA3.1/NT-GFP-TOPO, be prepared in aminoterminal and have other construction that GFP merges to cross over the proteic total length of PSCA.
PAPtag: with PSCA ORF or its part be cloned into pAPtag-5 (GenHunter Corp.Nashville, TN).This construction has produced the SEAP fusions of PSCA protein carboxyl terminal, has then merged IgG κ signal sequence at aminoterminal.Also made up SEAP and IgG κ signal sequence aminoterminal is blended in the N-terminal construction of PSCA albumen.Optimize gained reorganization PSCA albumen so that it is secreted in the medium of transfection mammalian cell, and can be used for differentiating the protein (for example part or acceptor) with the PSCA protein-interacting.With CMV promoters driven protein expression, this recombinant protein also is included in 6 * His epi-position and the myc that carboxyl terminal merges, so that detection and purifying are easier.Rely on the damp wet star resistant gene screening in the carrier to express said proteinic mammalian cell, and rely on the plasmid in the Ampicillin Trihydrate resistant gene screening intestinal bacteria.
Ptag5: PSCA ORF or its part are cloned into pTag-5.This carrier is similar to pAPtag, when there not being SEAP to merge.The PSCA albumen that is produced by this construction has N-terminal IgG κ signal sequence and the 6 * His epi-position tag and the myc that are positioned at carboxyl terminal, thereby feasible detection and affinity purification are more easy.Optimize gained reorganization PSCA albumen so that it is secreted in the medium of transfection mammalian cell, and differentiate the protein (for example part or acceptor) with the PSCA protein-interacting as immunogen or part.With CMV promoters driven protein expression.Rely on the damp wet star resistant gene screening in the carrier to express said proteinic mammalian cell, and rely on the plasmid in the Ampicillin Trihydrate resistant gene screening intestinal bacteria.
PsecFc: PSCA ORF or its part are cloned into psecFc.The psecFc carrier is through with immunoglobulin G while 1 (IgG) Fc (hinge area, CH 2District, CH 3The district) (Invitrogen California) assembles to be cloned into pSecTag2.This construction has produced the IgG1 Fc fusions of PSCA protein carboxyl terminal, has then merged the IgGK signal sequence at the N end.Also used the PSCA fusions that has adopted mouse IgG1 Fc district.Optimize gained reorganization PSCA albumen so that it is secreted in the medium of transfection mammalian cell, and can be used as the protein (for example part or acceptor) of immunogen or discriminating and PSCA protein-interacting.With CMV promoters driven protein expression.Rely on the hygromycin gene screening in the carrier to express said proteinic mammalian cell, and rely on the plasmid in the Ampicillin Trihydrate resistant gene screening intestinal bacteria.
Shown in Figure 8 is proteic expression of PSCA.psecFc and purifying in the 293T cell.
PSR α construction: in order to produce the mammal cell line of constitutive expression PSCA, PSCAORF or its part of PSCA is cloned into pSR α construction.Through pSR α construction being transfected into 293T-10Al packing strain or pSR α and helper plasmid (packaging sequence that contains disappearance) cotransfection being gone into 293 cells, produce both sexes respectively and have a liking for same retrovirus.Use the multiple mammal cell line of this retroviral infection, make clone gene PSCA be integrated into host cell system.Repeat the expression of (LTR) kinesin matter with long terminal gene.Rely on the neomycin resistance gene screening in the carrier to express said proteinic mammalian cell, and rely on Ampicillin Trihydrate resistant gene and ColE1 initial screening and the plasmid that keeps in the intestinal bacteria.Thereafter can with this reverse transcription carrier be used for infecting and for example adopt PC3, NIH 3T3, TsuPr1,293 or the rat-1 cell produce various clones.
Shown in Figure 6 for adopting the psca expression of PSCA.pSR α construction in recombined small-mouse, rat and human cell line.Retrovirus vector to have people PSCA cDNA and neomycin resistance gene or only to contain neomycin resistance gene infects specified mouse, rat and human cell line.Obtain stable recombinant cell lines through G418 drug screening.To measure psca expression with the painted FACS of the anti-PSCA MAb of 1G8 (5 μ g/ml).Be depicted as the FACS distribution plan of each clone, shown that the fluorescence drift only takes place, and has indicated the psca expression of cell surface in PSCA cells transfected system.These clones can be used as immunogen, MAb screening reagent and be used for functional analysis in the Mab exploitation.
Through making epitope tag (FLAG for example TMTag) be blended in other pSR α construction of carboxyl terminal preparation of PSCA sequence, detect to use anti-Flag antibody.For example, with FLAG TMSequence 5 ' gat tac aag gat gac gac gat aag 3 ' (SEQ ID NO:76) is added to clone's primer of ORF 3 ' end.Prepare other pSR α construction to produce the proteic aminoterminal of total length PSCA and carboxyl terminal GFP and myc/6X His fusion rotein.
Other virus vector: preparation is used for virus-mediated other construction of sending and expressing of PSCA.In viral delivery systems (for example adenovirus virus and bleb amplicon vector), obtain to cause the high virus titer of PSCA high level expression.Through pcr amplification PSCA encoding sequence or its fragment, and its subclone gone into AdEasy shuttle vectors (Stratagene).Recombinate and viral the packing according to manufacturers instruction to produce adenovirus carrier.Perhaps, PSCA encoding sequence or its fragment cloning are gone into HSV-1 carrier (Imgenex) to produce herpesvirus vector.Then this virus vector is used to infect various clones, for example PC3, NIH 3T3,293 or the rat-1 cell.
Regulating and expressing system: in order to control the expression of PSCA in mammalian cell; PSCA encoding sequence or its part are cloned into mammiferous regulating and expressing system; For example T-Rex system (Invitrogen), GeneSwitch system (Invitrogen) and closely regulate the ecdysone system (tightly-regulatedEcdysone System, Stratagene).These systems are used to study time and the concentration dependent effect of reorganization PSCA.Then with these carriers be used for controlling PSCA various clones (for example PC3, NIH 3T3,293 or the rat-1 cell) expression.
B. baculovirus expression system
In order in baculovirus expression system, to produce reorganization PSCA albumen, PSCA ORF or its part are cloned into baculovirus conversion carrier pBlueBac 4.5 (Invitrogen), this carrier provides His-tag at the N end.Particularly, pBlueBac-PSCA and helper plasmid pBac-N-Blue (Invitrogen) cotransfection are gone into SF9 (fall army worm (Spodoptera frugiperda)) insecT cell to produce recombinant baculovirus (seeing the specification sheets of Invitrogen for details).From cell conditioned medium liquid, collect baculovirus then, carry out purifying through the plaque analysis.
Then, with the baculovirus infection HighFive insecT cell (Invitrogen) of purifying to produce reorganization PSCA albumen.Can use anti-PSCA or anti-His-tag antibody to detect reorganization PSCA albumen.But purifying PSCA albumen, and use it for various with the cell be the basis analysis or with its as immunogen to produce specific polyclone of PSCA and monoclonal antibody.
C. the expression vector that is used for PSCA forward homologue (ortholog)
With the forward homologue of PSCA mouse and monkey be cloned into pcDNA3.1/MycHis VersionA (Invitrogen, Carlsbad, CA).Adopt cytomegalovirus (CMV) promoters driven protein expression.This recombinant protein has 6X His epi-position and the myc epi-position that is blended in carboxyl terminal.These carriers make that PSCA forward homologue is able to express, to test the cross reactivity of anti-people PSCA monoclonal antibody.
Also mouse and the monkey forward homologue with PSCA is cloned into pSR α construction.This pSR α construction has produced the mammal cell line of constitutive expression PSCA forward homologue.Adopt cytomegalovirus (CMV) promoters driven protein expression.This recombinant protein has 6X His epi-position and the myc epi-position that is blended in carboxyl terminal.These carriers make PSCA forward homologue be able to express with the cross reactivity of testing anti-people PSCA monoclonal antibody and the functionally active that is used to study PSCA forward homologue.Through pSR α construction being transfected into 293T-10A1 packing strain or pSR α and helper plasmid (packaging sequence that contains disappearance) cotransfection being gone into 293 cells, produce both sexes respectively and have a liking for same retrovirus.Use the various mammal cell lines of this retroviral infection, make this clone gene, promptly PSCA forward homologue is integrated into host cell system.
Shown in Figure 7 for being transfected into mouse and the expression of ape and monkey PSCA.pcDNA3.1/MycHis behind the 293T cell.With mouse PSCA.pcDNA3.1/MycHis or ape and monkey PSCA.pcDNA3.1/MycHis or pcDNA3.1/MycHis vehicle Control transfection 293T cell.After 40 hours, adopt anti-PSCA monoclonal antibody with the flow cytometry collecting cell and analyze.
Embodiment 6
Antigenicity collection of illustrative plates and secondary structure
Can find the amino acid collection of illustrative plates of PSCA variant 1,3 and 4 through the ProtScale website (expasy.ch/cgi-bin/protscale.pl) on the visit World Wide Web ExPasy molecular biology server.
These collection of illustrative plates: wetting ability (hydrophilicity, Hopp T.P., Woods K.R., 1981.Proc.Natl.Acad.Sci.U.S.A.78:3824-3828); Hydrotherapy property (hydropathicity, Kyte J., Doolittle R.F., 1982.J.Mol.Biol.157:105-132); But contact residues per-cent (Janin J., 1979Nature 277:491-492: average flexibility (Bhaskaran R. and PonnuswamyP.K., 1988.Int.J.Pept.Protein Res.32:242-255); β-corner (Deleage, G., Roux be Engineering 1:289-294 B.1987Protein); And other collection of illustrative plates well known in the art, as listed on the ProtScale website, all can be used for identifying the antigenic region on each PSCA misfolded proteins.ProtScale parameter below the above-mentioned amino acid collection of illustrative plates of each of PSCA variant is all available is described: 1) window size is 9; 2) compare with window kind system, window edge accounts for 100% weight; And 3) amino acid collection of illustrative plates value through after the stdn between 0 to 1.
But wetting ability, hydrotherapy property and contact residues per-cent be used for confirming hydrophilic amino acid (be wetting ability with can contact amino acid per-cent greater than 0.5, wetting ability is less than 0.5 amino acid) distributed areas.This zone may be exposed in the aqueous environments, is positioned at proteinic surface, therefore can be used as immune recognition site, like the recognition site of antibody.
The amino acid that average flexibility and βZhuan Jiao have determined not comprise in secondary structure such as βZhe Die and the α spiral (be βZhuan Jiao and on average flexibility greater than 0.5 amino acid) distributed areas.This zone also possibly be exposed to protein surface, therefore also can be used for immunity identification, like the identification of antibody.
The antigen sequence of the PSCA misfolded proteins of indicating in the above-mentioned collection of illustrative plates can be used for preparing immunogen (no matter being peptide or its coding nucleic acid) to produce therapeutic and diagnostic anti-psca antibody.This immunogen can comprise any 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50 or more a plurality of continuous amino acid of the listed PSCA misfolded proteins of Fig. 1; Or these amino acid whose corresponding nucleics of encoding; Also can extrapolate its amino acid collection of illustrative plates, because the sequence that variant contained is the same with variant shown in Figure 1.Special needs to be pointed out is; Peptide based immunogens of the present invention can comprise 5 amino acid whose peptide zones containing Fig. 1 at least; Wherein contained amino acid number can increase progressively by arbitrary integer, the amino acid position that is wherein comprised in the wetting ability collection of illustrative plates intermediate value of Fig. 5 greater than 0.5; At least contain 5 amino acid whose peptide zones of Fig. 1, wherein contained amino acid number can increase progressively by arbitrary integer, and the value of the amino acid position that is wherein comprised in the wetting ability collection of illustrative plates is less than 0.5; At least contain 5 amino acid whose peptide zones of Fig. 1, wherein contained amino acid number can increase progressively by arbitrary integer, but the value of the amino acid position that is wherein comprised in contact residues per-cent collection of illustrative plates is greater than 0.5; At least contain 5 amino acid whose peptide zones of Fig. 1, wherein contained amino acid number can increase progressively by arbitrary integer, and the value of the amino acid position that is wherein comprised in the flexibility collection of illustrative plates is greater than 0.5; And 5 amino acid whose peptide zones containing Fig. 1 at least, wherein contained amino acid number can increase progressively by arbitrary integer, and the value of the amino acid position that is wherein comprised in the βZhuan Jiao collection of illustrative plates is greater than 0.5.Peptide based immunogens of the present invention also comprises the nucleic acid of the above-mentioned peptide section of encoding.
All immunogens of the present invention, no matter peptide or nucleic acid, embodied that can the human unitary dose perhaps is included in the compsn, this compsn comprises the pharmaceutical excipient with the Human Physiology environmentally compatible.
PSCA misfolded proteins 1,3,4 and 6 secondary structure; Promptly predict the position whether α spiral, extended chain and random coil exist and exist; HNN-Hierarchical Neural Network method capable of using is inferred (NPS:Network Protein SequenceAnalysis TIBS in March, 2000 according to proteinic one-level aminoacid sequence; The 25th volume, No 3 [291]: 147-150; Combet C.; BlanchetC.; Geourjon C. and Del é age G.; Http:// pbil.ibcp.fr/cgi-bin/npsa_automat.pl? Page=npsa_nn.html), this method can find (expasy.ch/tools/ on the World Wide Web) through visit ExPasy molecular biology server.Form by 30.89% α spiral, 21.95% extended chain and 47.15% random coil through analyze finding PSCA variant 1.PSCA variant 3 is made up of 14.89% α spiral, 28.51% extended chain and 76.60% random coil.PSCA variant 4 is made up of 9.52% α spiral, 8.99% extended chain and 81.48% random coil.PSCA variant 6 is made up of 24.56% α spiral, 21.93% extended chain and 53.51% random coil.
Whether exist the various film prediction algorithms of striding of striding on available World Wide Web, the film district ExPasy molecular biology server to analyze (.expasy.ch/tools/) on the PSCA misfolded proteins.
Embodiment 7
The generation of PSCA polyclonal antibody
Polyclonal antibody can if desired, can add adjuvant in the preparation through preparing to Mammals injection one or many immunological reagent.In general, immunological reagent and/or adjuvant come immune Mammals through the subcutaneous injection or the peritoneal injection of multidigit point.Except the PSCA protein variant with total length is come the immunity; Can also design immunogen with computer program, for example find antigenic sequence and can be by the sequence of immune host's immune system recognition (seeing that title is the embodiment of " antigenicity characteristic and secondary structure ") according to the amino acid sequence analysis result.Can know this fragment possess hydrophilic property, flexibility, βZhuan Jiao configuration through prediction, be positioned at proteinic surface.
For example, recombinant bacteria fusion rotein or the polypeptide that contains wetting ability, flexibility, the βZhuan Jiao zone of PSCA protein variant can be used as antigen and is used in NZw body preparation polyclonal antibody or the title described monoclonal antibody of embodiment for " generation of PSCA monoclonal antibody ".For example, in PSCA variant 1, this type zone includes, without being limited to amino acid 28-56 and amino acid 66-94.In variant 3, this type zone includes, without being limited to amino acid 6-18, amino acid 27-39, amino acid/11 03-133 and amino acid/11 77-189.In variant 6, this type zone includes, without being limited to amino acid/11 9-35 and amino acid 57-85.Known in by the mammalian body of immunity, to have immunogenic protein be useful connecting one on the immune factor.This para-immunity originality albumen includes, without being limited to keyhole
Figure G05817239920061201D001161
hemocyanin (KLH), serum albumin, bovine thyroglobulin and soybean trypsin inhibitor.In one embodiment, the peptide of the amino acid/11 03-133 of coding PSCA variant 4 connects a KLH and is used for immunizing rabbit.In addition, immunological reagent can also comprise total length or partial sequence, its stand-in or the fusion rotein of PSCA misfolded proteins.For example, can utilize recombinant DNA technology that the aminoacid sequence of PSCA variant is merged with any one fusion rotein companion well known in the art mutually, as contain the fusion rotein of glutathione s-transferase (GST) and HIS mark.In one embodiment, utilize recombinant technology that the amino acid/11 8-98 and the GST of PSCA variant 1 are merged, in the pGEX expression vector, express, be used for immunizing rabbit and mouse behind the purifying, can prepare polyclonal antibody and monoclonal antibody respectively.This fusion rotein can come out with suitable affinity matrix purifying in the derivative bacterium.
Can be used for the constant region that other recombinant bacteria fusion roteins of the present invention comprise maltose binding protein, LacZ, sulphur hydrogen reduction albumen, NusA or Tegeline and (see that " the molecular biology fresh approach " the 2nd that " in prokaryotic system, preparing PSCA " part and Frederick M.Ausubu etc. compile roll up Unit the 16th, 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 can use Mammals expressed proteins antigen.These antigens can be expressed (seeing " preparation reorganization PSCA in eukaryotic system " part) with mammalian expression vector such as Tag5 and Fc-fusion vector, utilize this carrier to express and can keep the posttranslational modification process, like the glycosylation of native protein.In one embodiment, the cDNA of PSCA variant 1, N end leading peptide and C end GPI anchorin are cloned in the Tag5 mammalian secretory carrier, and at the 293T cell inner expression.Utilize metal-chelate to close chromatographic technique purification of recombinant proteins from the 293T cell tissue culture supernatant of stably express recombinant vectors.The Tag5 PSCA albumen that is purified into just can be used as immunogen.
In immunologic process, be useful with the immunoreation that strengthens host animal with adjuvant mixing or emulsification antigen.The example of adjuvant includes, without being limited to complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl lipid A, a kind of synthetic trehalose two diphtherinic acid salt (trehalose dicorynomycolate).
In a typical immunization method, rabbit reaches 200mg through subcutaneous injection earlier, and the fusion rotein or the peptide that are generally 100-200mg carry out immunity, and fusion rotein is connected with KLH, mixes with complete Freund's adjuvant (CFA).Whenever subcutaneous injection 200mg biweekly then is generally 100-200mg full freund's adjuvant (IFA) the blended immunogen of toing many or too much for use.The 7-10 days blood sample collections in each immunity back detect, and measure sero-fast titre through ELISA.
For reactivity and the specificity of measuring immune serum; Like rabbit anteserum with PSCA variant 3 or 4 proteic gst fusion protein immunity; Total length PSCA variant cDNA separately is cloned in the pCDNA 3.1myc-his expression vector (Invitrogen sees embodiment " preparation reorganization PSCA in eukaryotic system ").After arriving the construction transfection in the 293T cell; Through western blotting technique with resistance body serum and anti-His antibody (Santa Cruz Biotechnologies; Santa Cruz is CA) with the atopy of cell lysate hybridization with the mutain of definite and sex change.In addition, utilize the immunoprecipitation method of fluorescent microscope, flow cytometry and anti-293T and other reorganization PSCA variant express cell to analyze immune serum to confirm the specific recognition activity of native protein.Also can utilize the cell of endogenous expression PSCA to carry out Western blot, immunoprecipitation, fluorescent microscope and flow cytometry and measure proteic reactivity and specificity.
The tame rabbit anti-serum of purifying PSCA variant fusion rotein such as GST and MBP fusion protein immunization with remove can with the antibody of fusion partner serial response, utilize that only contain fusion partner or except that fusion partner, also contain the affinity column of uncorrelated fusion rotein can the purifying antiserum(antisera).For example, at first (chromatography column Calif.) comes purifying to the antiserum(antisera) in GST-PSCA variant 1 fusion rotein source for BioRad, Hercules through containing the covalently bound AffiGel matrix of GST albumen.Come purifying through the chromatography column that contains the covalently bound Affigel matrix of MBP-PSCA fusion rotein then.Come the purifying antiserum(antisera) to separate IgG component wherein through G albumen affinity column again.The serum of the serum in other Hi s-labelled antigens and peptide immunizing rabbit source and removal fusion partner carries out affinity purification through the chromatography column that contains original protein immunogen or free peptide.
Embodiment 8
The generation of PSCA monoclonal antibody (MAb)
In one embodiment; The therapeutic polyclonal antibody of PSCA and PSCA variant (" Mab ") comprises those antibody that can react with epi-position or the specific epi-position of consensus sequence to each protein-specific; The biological function of said antibody capable combination, internalization (internalize), prevention or adjusting PSCA or PSCA variant, for example those can removal and aglucon and the interactional sequence of binding partners.The immunogen that is used to prepare this MAb comprise those codings comprise extracellular region or the immunogen of complete PSCA protein sequence, prediction comprise functional motif the zone, or comprise certain the regional immunogen that has antigenic PSCA protein variant through the Computer Analysis prediction of aminoacid sequence.Immunogen comprises peptide, recombinant bacteria albumen (the for example PSCA pET carrier proteins (Fig. 6) of GST-PSCA fusion rotein (Fig. 8) and His mark), by the purifying His labelled protein (Fig. 7) of mammalian cell expression and the IgG FC fusion rotein of people and mouse.In addition, but through retrovirus transduction and after genetic engineering modified the cell of high level expression PSCA variant 1, for example RAT1-PSCA, 293T-PSCA, 3T3-PSCA or 300.19-PSCA can be used for immune mouse.
In order to prepare the monoclonal antibody of PSCA, usually at first through in mouse insole (FP), using and suitable immunological adjuvant blended 5-50 μ g protein immunogen or 10 6-10 7Individual psca expression cell carries out immunity.The example that is used for the suitable immunization method of FP is to carry out FP injection first with TiterMax (Sigma), uses the alum glue that contains (Qiagen) then.Behind initial injection, weekly mouse is carried out 2 immunity until execution, obtain the B cell from lymphoglandula and be used for merging.
In immunologic process, blood sample collection is to monitor immunoreactive titre and specificity.In most of the cases; In case find to have obtained enough reactivities and specificity through ELISA, Western blot, immunoprecipitation, fluorescent microscope or Flow cytometry; Just can electricity consumption cytogamy method (BTX ECM2000) carries out the preparation of cytogamy and hybridoma.
In one embodiment, the invention provides the monoclonal antibody of called after Ha1-1.16, Ha1-5.99, Ha1-4.117, Ha1-4.120, Ha1-4.121 and Ha1-4.37.Differentiate said antibody and demonstration and cell surface or immobilized PSCA reaction and combine.
Adopt allos mouse (Xenomice) technology
Figure G05817239920061201D001191
to produce Mab to PSCA; Locus in this technology on murine heavy chain and the κ light chain has been inactivated and main people's heavy chain and κ light chain Tegeline seat inserts: Ha1-1.16, and it is to produce after can producing the allos mouse 13 times of people γ 1 with the PSCA-GST immunity; Ha1-5.99, it can produce with Rat1-PSCA cellular immunization and produce for 2 times with the PSCA-tag5 injection behind the allos mouse 6 times of people γ 2 again; Ha1-4.117, HA1-4.37, Ha1-4.120 and Ha1-4.121, they are to produce for 4 times with the PSCA-tag5 injection after can producing the allos mouse 6 times of people γ 1 with Rat1-PSCA cellular immunization again.Said anti-PSCA Mab, Ha1-1.16, Ha1-5.99, Ha1-4.117, Ha1-4.120 and Ha1-4.121 are combined in the endogenous cell surface PSCA that expresses in the prostate cancer heterograft cell.
The antibody of called after Ha1-5.99, Ha1-4.117, Ha1-4.120, Ha1-4.37, Ha1-1.16 and Ha1-4.121 is delivered to (through Federal Express) American type culture collection (ATCC) on May 4th, 2004; P.O.Box 1549; Manassas; VA 20108, and given preserving number PTA-6703, PTA-6699, PTA-6700, PTA-6702, PTA-6698, PTA-6701 respectively.
After from each hybridoma, isolating mRNA; Measure the dna encoding sequence of anti-PSCA MAb Ha1-1.16, Ha1-5.99, Ha1-4.117, Ha1-4.120, Ha1-4.121 and Ha1-4.37 with Trizol reagent (Life Technologies, Gibco BRL).The total RNA of purifying, and carry out quantitatively.Use Gibco BRL Superscript Preamplification system to produce the cDNA of first chain by oligomerization (dT) 1218 primers.Personnel selection immunoglobulin variable heavy chain primer and the variable light chain primer amplification first chain cDNA of human normal immunoglobulin.The PCR product cloning is gone into pCRScript carrier (Stratagene, La Jolla).A plurality of clones are checked order, measure heavy chain and variable region of light chain.The nucleic acid of heavy chain and variable region of light chain and aminoacid sequence are listed among Fig. 2 and Fig. 3.Psca antibody and kind are that the comparison of V-D-J sequence is shown among Fig. 4 A-Fig. 4 M.
Embodiment 9
The screening of psca antibody and discriminating
Employing comprises that the avidity test group of the PSCA that expresses on ELISA, FACS, epi-position grouping and the pair cell surface is incompatible to adopting title to screen for the antibody that step produced described in the embodiment of " generation of PSCA monoclonal antibody (MAbs) " and differentiating.
A. through FACS screening people MAb.
Through FACS former hybridoma to the MAb of PSCA screened.Experimental design is following: the hybridoma supernatant (pure) or the antibody purified (carrying out serial dilution) in 50 μ l/ holes are added in the 96 hole FACS plates, mix with the cell of expressing PSCA (endogenous or reorganization, 50,000 cells/well).This mixture was hatched under 4 ℃ 2 hours.After hatching end,, detect antibody with 100 μ l and (resist-hIgG-PE) under 4 ℃, hatched 45 minutes with FACS damping fluid washed cell.After hatching end,, use formaldehyde fixed, analyze with FACScan with FACS damping fluid washed cell.Adopt CellQuest Pro software to carry out data analysis.What solid histogram was represented is the data available from control cells, and what hollow histogram was represented is the data (Fig. 9) available from the PSCA positive cell.
The positive hybridoma that will from original screening thing, identify is transferred in 24 orifice plates, collects supernatant and is used for the conclusive evidence screening.The conclusive evidence screening comprises: B300.19-PSCA/300.19-neo, Rat1-PSCA/Rat1-neo, PC3-PSCA/neo, SW780 (bladder cancer cell lines), LAPC9AI (prostate cancer cell line), HPAC (pancreatic cancer cell system) are carried out FACs analyze, and adopt Tag5-PSCA, GST-PSCA, GST-PSCA N end, Med.C end and pET-PSCA to carry out elisa assay.
B.PSCA people MAb relative affinity is analyzed
Test hybridoma supernatant is to confirm the relative binding affinity of their pair cells surface PSCA.In FACS damping fluid (FB) with the hybridoma supernatant from μ g/ml serial dilution to inferior sub-ng/ml; Adopt the LAPC9AI cell in the FACS binding analysis, to assess.High affinity antibody provides high MFI value.Adopt CellQuestPro software to obtain the MFI value of each point, and use it for and adopt the avidity of Graphpad Prism software to calculate (Table VII and Table VIII): S type dosage-response (variable slope) formula.The result of relative affinity analysis is shown among Figure 10.
C. epi-position is divided into groups
Through of the combining form of assessment psca antibody, psca antibody is divided into groups according to epi-position to the LAPC9AI cell.In brief, make a spot of each antibody biotinylation; Then with each biotinylated antibody 4 ℃ with the abiotic elementization antibody of excessive (100x) in the presence of hatched 1 hour.Usually, if excessive antibodies and biotinylated antibodies in identical epi-position, can be competed between them.After hatching end, washed cell was hatched it 45 minutes with streptavidin-PE under 4 ℃.Behind the unconjugated streptavidin-PE of flush away, adopt the FACS pair cell to analyze.Carry out data analysis (Table VII) with MFI mensuration.Shown in Table X I, represent that with the outstanding cell of yellow the MFI in these cells is the background contrast to each biotinylated antibody from body competition (self-competition, 100% competition).The cell that no color indicates representes that two kinds of antibody compete (low MFI), two kinds of different epi-positions of high MFI (outstanding with blueness) two kinds of antibodies of expression each other.Antibody with identical combination pattern is incorporated into same epi-position in antibody.6 epi-position groups in the antibody have been tested.Table X I shows that PSCA 4.121 is incorporated into its unique epi-position.
Embodiment 10
The characterized of psca antibody and expression
A. with the cross reactivity of monkey PSCA and mouse PSCA
Screening MAb, and they and the response capacity that mouse and ape and monkey are originated carried out characterized.This characteristic is used for understanding when using mouse with the ape and monkey animal model on the MAb and cell and organizes combining of PSCA.Clone macaque (Cynomolgus) and mouse psca gene are expressed and instantaneous infection 293-T cell in retrovirus.Adopt following experimental design will _ _ _ _ hatch with various antibody.Test antibody is hatched with the 293-T cell of expressing macaque or mouse PSCA, or hatch as negative control with the 293T cell of expressing the neo gene.Adopt anti-hIgG-PE to detect the two anti-specific recognition of measuring.The typical histogram of describing the specificity cross reactivity is shown among Figure 11.Being shown in summary in the Table X shows in all anti-people's psca antibodies except a kind of antibody all and monkey PSCA generation cross reaction, only a kind of anti-people's psca antibody and mouse PSCA generation cross reaction.
B. measure through the avidity of FACS
Anti-people's psca antibody of test 7 group (7) is gone up the binding affinity of PSCA to SW780 cell (a kind of human bladder cancer cell's strain of expressing high-level PSCA).With the antibody purification of 23 series dilution in 1: 2 and SW780 cell (50,000 every holes of cell) with the final concentration of 167nM-0.01pM incubated overnight together under 4 ℃.After hatching end, washed cell, and it is detected antibody with anti-hIgG-PE under 4 ℃, hatched 45 minutes.Behind the unconjugated detection antibody of flush away; Use the facs analysis cell: adopt CellQuest Pro software to obtain the MFI value of each point, and use it for and adopt the avidity of Graphpad Prism software to calculate: S type dosage-response (variable slope) formula (Table VII and Table VIII).The result of the relative affinity analysis of 7 kinds of (7) antibody is shown among the Table I X.
Embodiment 11
The internalization of psca antibody
Use the PC3-PSCA cell to carry out the internalization research of Ha1-4.121.In brief, with Ha1-4.121 with cell under 4 ℃, hatch 90 ℃ so that said antibodies in said cell surface.Then cell is divided into two groups, under 37 ℃, continues to hatch, perhaps under 4 ℃, hatch as contrast (no internalization) to carry out the antibody internalization.37 ℃/4 ℃ are carried out pickling is incorporated into cell surface with removal PSCA 4.121 after hatching.The infiltration of carrying out subsequently can detect the PSCA of antibodies in internalization.After detecting two anti-hatching, observe with the facs analysis cell or under fluorescent microscope.About 30% Ha1-4.121 is by internalization (Figure 12) after hatching 2 hours under 37 ℃.
Embodiment 12
Antibody-mediated Secondary cases is killed and wounded (secondary killing)
The antibody of anti-PSCA mediation saporin dependency in the cell of expressing PSCA is killed and wounded.B300.19-is expressed the cell (750 cells/well) of PSCA and go into 96 orifice plates inoculation in the 1st day.In each hole, add isopyknic 2 * concentration that contains in the fate subsequently and specify anti-and 2 times of excessive and saporin toxin (AdvancedTargeting Systems; San Diego, CA) anti-people of link coupled (Hum-Zap) or the anti-goat (substratum of polyclonal antibody of goat-Zap).Cell was hatched under 37 ℃ 5 days.Incubation period, add MTS (Promega) after finishing in each hole, continued to hatch 4 hours.Measure the OD value at 450nM place.Result among Figure 13 (A) shows that psca antibody HA1-4.121 and HA1-4.117 mediate saporin dependent cell toxic action in the B300.19-PSCA cell, but not specific human IgG1 control antibodies does not then have effect.Result among Figure 13 (B) show add with the saporin link coupled fail to see others' Fc two anti-can not mediated cell toxic action (Figure 13 (A) and Figure 13 (B)).These results show that medicine or toxic protein can optionally be delivered to through using suitable anti-PSCA MAb in the cell of expressing PSCA.
Embodiment 13
The CDCC of antibody mediated immunity mediation
Psca antibody is assessed to confirm the ability of their mediation immune dependent sexual cell toxic actions.With RHB damping fluid (RPMI 1640, Gibco Life Technologies, 20mM HEPES) dilution psca antibody (0-50 μ g/ml).At the cell of RHB damping fluid washing expression B300.19-PSCA, with 10 6The density of individual cell/ml suspends cell again.In typical the analysis, (Cedarlane, Ontario Can) add in flat tissue culture 96 orifice plates with 50 μ l cell suspensions together with the psca antibody of 50 μ l, the dilution rabbit complement serum of 50 μ l.With mixture at 37 ℃, 5%CO 2Hatch 2 hours in the incubator to promote the cytolysis of complement-mediated.(Biosource Intl.Camarillo CA), and continued to hatch 4-5 hour under 37 ℃ in each hole, to add the Alamar Blue of 50 μ l then.Use 96 hole photofluorometers to read fluorescence, excite, the emission of 590nm place at the 530nm place.The result shows to have Ig1 (HA1-4.121) or IgG2 isotype (HA1-5.99.1) but be not that the psca antibody of IgG4 isotype (HA1-6.46) can mediate complement-dependent dissolving (Figure 14).
ADCC (antibody-dependent cytotoxicity effect) is that a kind of immune-mediated dissolving to the cell that combines in the antibody of specificity cell surface antigen with target is attacked.In this application, it is PSCA.The Fc part of immunocyte identification antibody excites the solvability that causes necrocytosis to attack through the Fc γ acceptor that is incorporated into white corpuscle, monocyte and NK cell.The ability of this reaction of psca antibody mediation can be passed through external use 51Chromium, europium or fluorescence molecule marked tumor cell are hatched them in the presence of people PSCA MAb and PMBC.Can recently measure the SL of tumour cell through the dissolving percentage of measuring the target tumour cell.The conventional end eventually point of being measured comprises: adopt suitable detection method to measure radioactivity, europium or the optical dye that from dead cell, discharges.Perhaps, can measure intracellular enzyme (serum lactic dehydrogenase for example, release LDH).
Embodiment 14
F (Ab ') 2 Segmental generation
The F of generation MAb (Ab ') 2Fragment is externally to keep MAb molecule divalence antigen binding site with the interior therapeutic model but lack the effect of the MAb molecule in immunological effect Fc district in order to be used for studying.With the 20mM sodium-acetate buffer pH4.5 of the MAbHa1-4.121 of 20mg with or not with immobilized stomach en-(Pierce.Rockford IL) hatches together the specified time.Remove complete MAb and Fc fragment through the albumin A chromatography through digesting.Shown in Figure 15 for complete without the unreduced MAb of digestion, at the appointed time take out do not reduce equal portions through the material of digestion and finally digest F (ab ') 2The PAGE coomassie dyeing gel of the sample that is reduced of product.This reagent can be used for treating the animal that lotus has the tumour of expressing PSCA.Can distinguish the inherent biological activity and by the activity of immune dependent property mechanism mediation with the observed anti-tumor activity of this antibody fragment.
Embodiment 15
Adopt people's antibody expression of recombinant DNA method
For the anti-PSCA MAb of express recombinant in transfectional cell, divide the upper reaches that are clipped to people's heavy chain IgG1 and light chain Ig κ constant region with anti-PSCA variable heavy chain and sequence of light chain clone.Complete anti-PSCA people's heavy chain and light chain box are cloned into the downstream of the CMV promotor/enhanser in the cloning vector.Comprise the polyadenylation site in the downstream of this MAb encoding sequence.The construction of the anti-PSCA MAb of the expression of this reorganization is transfected into 293T, Cos and Chinese hamster ovary celI.To the cell surface PSCA among oozy HA1-4.121 antibody from reorganization 293-T cell and the figure Pia-3A combine assess, and compare (Figure 16) with same antibody by original hybridoma generation.
Embodiment 16
The combination experiment of HLA I quasi-molecule and II quasi-molecule
The combination experiment of HLA I quasi-molecule and II quasi-molecule utilizes the HLA molecule of purifying to carry out, and used method is that the document public reported (is seen PCT patented claim WO 94/20127 and WO 94/03205; Sidney etc., Current Protocols in Immunology 18.3.1 (1998); Sidney etc., J.Immunol.154:247 (1995); Sette etc., Mol.Immunol.31:813 (1994)).In brief, the MHC molecule of purifying (5-500 nM) and various unlabelled peptide supressors and 1-10nM 125The peptide probes of I mark combines, and hatches the back altogether and through gel-filtration the MHC-peptide complex is separated with free peptide, measures peptide bonded part.In general, in trial test, measure the concentration of the titre of every kind of MHC prepared product under the situation that need exist with the needed HLA molecule of gross activity of definite 10-20% of combination at the radiolabeled peptides of fixed amount.Inhibition is subsequently carried out with the HLA that directly combines to test with regard to these concentration.
Because the concentration of affinity tag is less than the concentration of HLA, IC under these conditions 50More than or equal to the concentration of HLA, so measured IC 50Value should be approximately equal to real K DValue.The concentration of peptide supressor generally at 120 μ g/ml between the 1.2ng/ml, should carry out 2 to 4 times fully independently the test.In order to compare the resulting data of different tests, need to calculate the relative association index of every kind of peptide, the IC of the positive contrast of method of calculation 50IC divided by each detection of peptides 50(generally being the unmarked version of radiolabeled probe's peptide).For the comparison in making up DB and making an experiment, need compile relative associated value.These values can convert IC again to 50The nM value, the IC of the positive contrast of conversion method 50The nM value is divided by the relative associated value of purpose peptide.This data compiling method is accurate and stable when being used for that comparison makes an experiment resulting data in the resulting data of different time detection of peptides or with the purifying MHC of different batches.
Above-mentioned binding analysis test also can be used for analyzing the peptide (seeing Table IV) that contains HLA hyper-base preface and/or motif.
Embodiment 17
The structure of " minigene " Polyepitope DNA plasmid
Present embodiment has been discussed the structure of minigene expression plasmid.Certainly, the minigene plasmid can comprise B cell, CTL and/or HTL epi-position or the epitope mimic thing of various combinations described herein.
The minigene expression plasmid generally all comprises a plurality of CTL and htl peptide epi-position.In the present embodiment, contain HLA-A2 ,-A3 ,-peptide epitopes of B7 hyper-base preface is connected with epi-position that contains DR hyper-base preface and/or DR3 epi-position with the peptide epitopes that contains HLA-A1 and-A24 motif.The peptide epitopes that contains HLA I quasi-molecule hyper-base preface or motif in the selected PSCA of being source has just comprised a plurality of hyper-base preface/motifs like this, thereby has guaranteed to have colony's coverage of a broad in the plasmid.Equally, the HLA II class epi-position of selecting PSCA to originate also can provide colony's coverage of broad, promptly in the minigene construction, not only has the epi-position that contains HLA DR-1 ,-4 ,-7 hyper-base prefaces but also have the epi-position that contains HLA DR-3 motif.Then CTL and the HTL epi-position selected are inserted in the minigene, it is expressed in expression vector.
This construction also possibly comprise the HTL epi-position is directed to the sequence on the endoplasmic reticulum.For example; Ii albumen can be fused on the described one or more HTL epi-positions in this area, and wherein the proteic CLIP sequence of Ii is deleted, and replacement is with HLA II class epitope sequences; HLA II class epi-position just may be directed on the endoplasmic reticulum like this, and epi-position combines with HLA II quasi-molecule herein.
Present embodiment has been described the construction process of the expression plasmid that contains minigene.Other expression vector also can be used for the minigene compsn, and these carriers also are well known to those skilled in the art.
The minigene DNA plasmid of present embodiment comprises a total Kozak and a total mouse κ Ig light chain signal sequence, is CTL and/or the HTL epi-position of selecting according to principle described herein subsequently.Open reading frame of these sequence codifieds utilizes on pcDNA 3.1Myc-His expression vector can merge Myc and His antibody epitope mark.
On average comprise about 70 Nucleotide, wherein have the overlapping oligonucleotide of 15 overlapping oligonucleotides can use the method for chemosynthesis synthetic, through the HPLC purifying.Oligonucleotide encode selected peptide epitopes and suitable oligonucleotide, Kozak sequence and the signal sequence of being connected.Utilize PCR to extend the eclipsed oligonucleotide and just can be assembled into final multi-epitope minigene through three group reactions.Utilize Perkin/Elmer 9600PCR appearance under following condition, to carry out 30 circulations: 95 ℃ 15 seconds, renaturation temperature (than low 5 ℃ of the right minimum Tm calculated value of each primer) 30 seconds, and 72 ℃ 1 minute.
For example prepare minigene according to following process.Two kinds of oligonucleotide are used in first round PCR reaction, every kind of 5mg, renaturation and extension: in an example, use 8 kinds of oligonucleotide; I.e. 4 pairs of primers, oligonucleotide 1+2,3+4,5+6 and 7+8 are blended in the 100ml reaction solution, wherein contain the Pfu polymerase buffer (1 *=10mMKCL; 10mM (NH4) 2SO4,20mM Tris-Cl, pH8.75; 2mM MgSO4,0.1%TritonX-100,100mg/ml BSA), each 0.25mM of dNTP and 2.5U Pfu polysaccharase.Through the dimerisation products of gel-purified total length, comprise two kinds of reaction solution mixing, renaturation of product 1+2 and 3+4 and product 5+6 and 7+8 and extend 10 circulations.Two kinds of reaction solutions are respectively got a meromict then, carry out 5 round-robin renaturation and extension, add the both sides primer with the amplification full length product.The gel-purified full length product is cloned among the pCR-blunt (Invitrogen), through each clone of order-checking screening.
Embodiment 18
The degree of plasmid construction thing and induction of immunity originality thereof
A plasmid construction thing, like above-mentioned embodiment constructed plasmid, the ability of its induction of immunity originality can be through at external use epi-position express nucleic acid construction transduction or transfection APC, and then measures the ability that APC offers epi-position and confirm.This test can be confirmed " antigenicity ", but end user APC.This test determination be the ability that epi-position is offered by APC, if an epi-position can just can be confirmed this ability through the density of quantitatively determined cell surface epi-position-HLA I quasi-molecule mixture so by the T cell recognition.Quantitatively determined can be confirmed (to see Sijts etc., J.Immunol.156:683-692,1996 from the peptide quantity that APC goes up wash-out through direct mensuration; Demotz etc.; Nature 342:682-684,1989), perhaps confirm the quantity of peptide-HLA I quasi-molecule mixture through measuring the amount of dissolving release or lymphokine release; Dissolving discharges or lymphokine release is the target cell inductive by ill or transfection; Just can confirm that then the dissolving release or the lymphokine that reach par discharge needed peptide concentration (seeing Kageyama etc., J.Immunol.154:567-576,1995).
In addition, immunogenicity also can be through injection mouse in the body, and external test CTL and HTL are active then confirms; CTL is active and the HTL activity tests with determination of cytotoxic activity respectively and proliferation test is confirmed; Like Alexander etc., Immunity 1:751-761,1994 are described in detail.
For example, for the ability that definite DNA minigene construction that contains a ultra motif peptide of HLA-A2 is at least induced CTL in vivo, the naked cDNA immunity of intramuscular injection 100mg HLA-A2.1/Kb transgenic mice.In order to compare the cDNA immunity inductive CTL of institute level, control animals is carried out immunity with peptide combinations, and wherein peptide combinations contains a plurality of epi-positions, and these epi-positions are positioned on the polypeptied chain, and are the same as minigene encoded polypeptides chain.
Separating Morr. cell in the immune animal body stimulates twice respectively with two kinds of compsns (peptide epitopes of minigene coding and the peptide epitopes of multi-epitope peptide coding), then the cytotoxic activity through 51Cr release test mensuration peptide specific.The result can show the ctl response intensity of anti-A2 restricted epitope, thereby minigene vaccine and polyepitope vaccines immunogenicity in vivo have been described.
Therefore, the result of this test has proved that minigene can excite the immunoreation to HLA-A2 hyper-base preface peptide epitopes, and the multi-epitope peptide vaccine also is like this.Utilize other HLA-A3 also can estimate HLA-A3 and HLA-B7 motif or the hyper-base preface epi-position inducibility to CTL with the HLA-B7 transgene mouse model through similar test, the result finds that minigene also can inspire the immunoreation to these epi-positions.
For the ability that the minigene of estimating coding II class epi-position is induced HTL in vivo, intramuscular injection 100mg DNA immunity DR transgenic mice or the restricted mouse of I-Ab-, the latter be used to estimate can with those epi-positions of suitable mouse MHC molecule generation cross reaction.For the comparison dna immunity inductive HTL of institute level, establish one group of control animal, with the immunity of complete Freund's adjuvant emulsive peptide combinations.Separating Morr. cell in the immune animal body is purified into CD4+T cell wherein, and promptly HTL stimulates respectively with two kinds of compsns (peptide of minigene coding).Through 3The H thymus pyrimidine mixes proliferation test and measures htl response (seeing Alexander etc., Immunity1:751-761,1994).The result shows the htl response that has produced certain intensity, thereby has explained that minigene has immunogenicity in vivo.
The DNA minigene that makes up according to the description of the foregoing description also can be used as vaccine and unites and strengthen the factor through just exempting from-strengthen strategy evaluation.Strengthen the factor and comprise that recombinant protein (sees Barnett etc., Aids Res.and Human Retroviruses 14, supplementary issue 3:S299-S309; 1998) or vaccinia virus recombinant; As the minigene or the DNA that express the complete target protein of coding (see Hanke etc., Vaccine 16:439-445,1998; Sedegah etc., Proc.Natl.Acad.Sci USA 95:7648-53,1998; Hanke and McMichael, Immunol.Letters 66:177-181,1999 and Robinson etc., Nature Med.5:526-34,1999).
For example, being used for the first efficient of exempting from-strengthen the DNA minigene of strategy at first estimates with transgenic mice.In the present embodiment, the A2.1/Kb transgenic mice is immune with the DNA minigene that IM adds 100mg coding immunogenic peptide, and immunogenic peptide wherein comprises a peptide that contains HLA-A2 hyper-base preface at least.After incubation period (3-9 week), mouse carries out booster immunization through the peritoneal injection vaccinia virus recombinant, and ID is every mouse 107pfu, the identical sequence of vaccinia virus expressible dna minigene coding.The contrast mouse is with 100mgDNA or do not contain the vaccinia virus recombinant immunity of minigene, perhaps with the dna immunization of coding minigene, but without the vaccinia virus booster immunization.After incubation period through 2 weeks, get the splenocyte of mouse again, pass through the cytotoxic activity of its peptide specific of ELISPOT analysis of experiments immediately.In addition, in the A2 restricted peptides epi-position stimulation of external use minigene or vaccinia virus recombinant coding, measure the cytotoxic activity of peptide specific then through α, β and/or γ IFN ELISA.
It is stronger than independent DNA that the result finds just to exempt from-strengthen the used immunoreation to the ultra motif peptide of HLA-A2 that minigene excited of strategy.Utilize HLA-A11 or HLA-B7 transgene mouse model can similarly analyze to estimate the ability that HLA-A3 or HLA-B7 motif or hyper-base preface epi-position are induced CTL.In human body, implement the method description of the embodiment of face " utilization is just exempted from-strengthened strategy and induces ctl response " as follows of elementary strengthened scheme.
Embodiment 19
The polyepitope vaccines compsn in many antigens source
PSCA peptide epitopes of the present invention can make up with the epi-position in other tumor associated target antigens sources, and the vaccine composition of formation can be used for preventing or treating the tumour of expressing PSCA and this tumor associated target antigens.For example; A kind of vaccine composition provided by the invention exists with the likeness in form of single chain polypeptide; This polypeptied chain contains a plurality of epi-positions in PSCA and taa source; Wherein express the target tumour of PSCA and also express this taa usually, perhaps this vaccine composition is the mixture of one or more discontinuous epi-positions.In addition, the form injection of all right minigene construction of vaccine or BMDC, BMDC wherein is peptide epitopes on external being loaded.
Embodiment 20
Utilize the peptide immune response evaluated
Peptide of the present invention can be used for analyzing immunoreation to judge whether to exist specific antibody, CTL or the HTL to PSCA.The available Ogg of this analysis etc., Science 279:2103-2106, the method for 1998 descriptions is carried out.In the present embodiment, peptide of the present invention is used to diagnose as reagent rather than immunogen or the purpose of judging prognosis.
In the present embodiment, the human leucocyte antigen (HLA) tetramer mixture (" tetramer ") of hypersensitivity is used to analyze as representational example and is in the probability various disease stage or produce PSCA HLA-A*0201-specific CTL with the HLA A*0201 positive individuals after the PSCA peptide immunity that contains the A*0201 motif.Method according to document (Musey etc., N.Engl.J.Med.337:1267,1997) is described is synthesized tetramer mixture.In brief, HLA heavy chain of synthesizing and purifying (being A*0201 in the present embodiment) and b2-microglobulin in prokaryotic expression system.Stride film-kytoplasm tail and modify heavy chain through deletion in sequence that contains BirA enzyme catalysis biotinylation site of COOH end interpolation.Make heavy chain, b2-microglobulin and peptide folding again through dilution.Utilize the fast protein liquid chromatography technology to isolate the refolding product of 45-kD, vitamin H (Sigma, St.Louis, Missouri), utilize BirA to make this product biotinylation under the condition that exists of 5 '-Triphosaden and mg ion.Ratio with 1: 4 mol ratio adds streptavidin-phycoerythrin connector, and tetramer product is concentrated to 1mg/ml.Resulting product is called as tetramer-phycoerythrin.
For the blood sample to the patient is analyzed, about 1 * 10 6PBMC centrifugal 5 minutes at 300g is resuspended in the cold phosphate buffered saline buffer of 50ml then.Utilize tetramer-phycoerythrin, anti-CD8-Tricolor and anti-CD38 to carry out trichromatic analysis.PBMC and tetramer and antibody were hatched on ice 30 to 60 minutes altogether, used formaldehyde fixed after the washed twice.The door that sets is greater than 99.98% of control sample.The contrast of tetramer comprises A*0201 negative individuals and the positive non-ill donor of A*0201.Measure the shared percentage ratio of the painted cell of tetramer through flow cytometry then.The result can demonstrate the cell count that comprises the epi-position Restricted CTL in the PBMC sample; Thereby can confirm the immunoreation intensity to the PSCA epi-position at an easy rate, therefore can judge the aversion response that excited behind PSCA or the vaccine or the situation of therapeutic reaction of being exposed to.
Embodiment 21
Induce ctl response through just exempting to strengthen strategy
Be used for confirming that with the foregoing description " degree of plasmid construction thing and induction of immunity originality thereof " is described dna vaccination is similar first the exempting from of the intravital curative effect of transgenic mice-strengthen also can be used for vaccine evaluation in the intravital curative effect of people under the described hereinafter principle of strategy.This vaccine immunity scheme comprises uses the naked DNA initial immunity, and then with recombinant virus or the recombinant protein/polypeptide or the peptide mixt booster immunization of adjuvant blended coding vaccine.
For example, initial immunity can be used expression vector, as embodiment " structure of minigene Polyepitope DNA plasmid " and in constructed expression vector, to a plurality of sites, dosage is 0.5-5mg with the form intramuscular injection (or subcutaneous injection or ID) of naked nucleic acid.Also can inject nucleic acid (0.1-1000mg) with particle gun.After the incubation period in week, carry out booster immunization through 3-4 again one time.Booster immunization can be used 5 * 107 to 5 * 109pfu recombinant Borrel virus.Other recombinant virus also can be used for booster immunization like MVA, canary pox virus, adenovirus or adeno-associated virus, perhaps multi-epitope albumen or peptide mixt.For the curative effect of vaccine evaluation, gather patient's blood sample before immunity, behind the initial immunity and behind the booster immunization.From the fresh blood sample of heparinization, separate PMNC through Ficoll-Hypaque density gradient centrifugation, the frozen pipe refrigerated storage of packing into after resuspended with frozen storing liquid.The CTL of analytic sample and HTL are active.
Test-results shows that the immunoreation intensity that is produced is enough to reach the purpose that produces anti-PSCA therapeutic or protective immunity.
Embodiment 22
The complementary polynucleotide
To be used to detect, reduce or suppress the expression of natural PS CA with PSCA encoding sequence complementary sequence (Fig. 1 or Fig. 3) or its any part.Though this paper has described the purposes of the oligonucleotide that contains 15 to 30 base pairs approximately, basic similarly method also can be used for littler or bigger sequence fragment.Utilize the oligonucleotide of the encoding sequence appropriate design of OLIGO 4.06 softwares (National Biosciences) and PSCA.In order to suppress to transcribe,, be used to stop combining of promotor and encoding sequence according to 5 ' unique sequences Design complementary oligonucleotide.In order to suppress translation, the complementary oligonucleotide that is designed can stop rrna to be attached on the PSCA encoding transcription thing.
Embodiment 23
Utilize the specific antibody purification of PSCA PSCA natural or reorganization
Utilize the specific antibody of PSCA through the PSCA immunoaffinity chromatography purifying natural or reorganization.To resist psca antibody to be covalently bound on activatory chromatographic resin such as the CNBr activatory SEPHAROSE (AmershamPharmacia Biotech) and prepare immune affinity chromatographic column.In conjunction with later specification sheets sealing and washing resin according to manufacturer.
The medium that contains PSCA is through immune affinity chromatographic column, then at the condition that allows the PSCA preferentially adsorbed high ionic strength buffers liquid of stain remover (as contain) washing chromatography column down.With breaking antibody/PSCA bonded condition (like pH2 to the damping fluid of pH3, or the chaotropic agent of high density, like urea or thiocyanate ion) elution chromatography post, collect GCR.P again.
Embodiment 24
Evaluation can with the interactional molecule of PSCA
PSCA or its bioactive fragment are with 121 1 Bolton-Hunter reagent marks (seeing (1973) Biochem.J.133:529 such as Bolton).The candidate molecules of in the hole of above-mentioned porous culture plate, arranging and the PSCA of mark are hatched altogether, and the institute of detecting the PSCA mixture that contains mark after the washing is porose.The resultant data of the PSCA of different concns are used to calculate PSCA and candidate molecules bonded quantity, avidity and interaction.
Embodiment 25
Measure PSCA in vivo to the promoter action of tumor growth
Confirm that through evaluation expression or the cells whose development and the growing state that lack PSCA PSCA albumen is in vivo to the effect of tumor growth.For example, in the one side subcutaneous injection 1 * 10 of SCID mouse 6Contain 3T3 cell, the prostate cancer cell strain (like the PC3 cell) of tkNeo empty carrier or PSCA.Have at least two kinds of strategies available: the constitutive expression of PSCA is carried out in (1) under the adjusting of promotor such as constitutive promoter; The promotor of this composing type can be from the genome of virus; Like polyomavirus, bird pox virus (UK2 that on July 5th, 1989 published, 211,504), adenovirus (like adenovirus 2), bovine papilloma virus, Avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40); Or come from mammiferous allogeneic promoter; Like actin promoter or immunoglobulin promoter, as long as these promotors are compatible with derivable carrier system, and regulatable expression is carried out in (2) under the control that can induce carrier system; Like ecdysone, tsiklomitsin etc., as long as this promotor is compatible with host cell systems.If tumour can touch; So just use the caliper measurements tumor size; Judge the whether at faster speed growth of psca expression cell after after a while, and its aggressive characteristic of the plastidogenetic tumour of psca expression whether change (like transfer ability raising, vascularization, the susceptibility of chemotherapeutics is reduced).
In addition, implant 1 * 10 can for the mouse original position 5Identical cell is judging whether PSCA is influential to intraprostatic local growth, and whether PSCA influences transfer (Miki T etc., Oncol Res.2001 in transcellular ability, particularly lymphoglandula and the bone; 12:209; Fu X etc., Int.J Cancer.1991,49:938).The PSCA bone tumor forms and the influence of growth can be estimated through injection prostate tumor cells in shin bone.
This test also can be used for analyzing the retarding effect of PSCA to candidate therapeutic property compsn, like PSCA endosome, PSCA antisense molecule and ribozyme.
Embodiment 26
Restraining effect in the tumour body of PSCA mediated monoclonal antibody
PSCA significantly expresses in cell surface at tumor tissues, and restricted expression in healthy tissues, this just makes PSCA become the desired target of Antybody therapy.PSCA equally also is the target position of T cellular immunization treatment.Therefore; Recombinant cell lines such as PC3-PSCA and 3T3-PSCA (see Kaighn, M.E. etc., Invest Urol; 1979.17 (1): 16-23) with the human prostate heteroplastic transplantation model; For example (Saffran etc., PNAS 1999,10:1073-1078) can be used for estimating the treatment effect of anti-PSCA monoclonal antibody in human prostata cancer xenotransplantation mouse model and human pancreas cancer xenotransplantation mouse model for LAPC 9AD.
Antibody can be studied in mouse original position prostate gland or carcinoma of the pancreas heteroplastic transplantation model the effect of tumor growth and metastatic carcinoma formation.Antibody can be not connect any material as present embodiment is discussed, also can be connected to as known in the art on the therapeutic carrier.Anti-PSCA MAb can suppress the formation of tumour in pancreas and prostate gland heteroplastic transplantation model.Anti-PSCA MAb can also suppress the growth of existing TIS, prolongs the survival time of tumor-bearing mice.The anti-PSCA MAb of these presentation of results can be used for treating in several kinds of local and advanced prostate cancer, carcinoma of the pancreas and the Table I those listed cancers and (sees Saffran, D. etc., PNAS10:1073-1078; Or access site pnas.org/cgi/doi/10.1073/pnas.051624698).
Inject anti-PSCA MAb and can cause the inhibition of existing TIS growth, stop metastases, survival time that so just can the significant prolongation tumor-bearing mice to position at a distance.These researchs show that PSCA is the desired target of immunotherapy, and PSCA MAb is effective when being used to treat local tumor and shifting prostate gland and pancreas tumor.The PSCA monoclonal antibody that the present embodiment explanation does not connect any material can suppress the human prostate tumor xenogeneic graft effectively in the intravital growth of SCID mouse; Accordingly, the combination of this type monoclonal antibody also is effective.
Utilize multiple PSCA MAb to suppress tumour
Material and method
The PSCA monoclonal antibody:
Can prepare the monoclonal antibody of anti-PSCA according to the described method of embodiment " preparation of PSCA monoclonal antibody (MAb) ".Through ELISA, Western blot, FACS and immunoprecipitation technical measurement antibody and PSCA bonded ability.The epi-position spectrum data of the anti-PSCA MAb that obtains through ELISA and Western blot shows that this antibody can discern the epi-position on the PSCA albumen.Utilize these antibody that prostate tumor tissue and cell are carried out immunohistochemical analysis.
Utilize Protein-G or albumen-A Sepharose chromatography column monoclonal antibody purification from ascites or hybridoma tissue culture supernatant, with PBS dialysis ,-20 ℃ of storages after the filtration sterilization.(Bio-Rad, Hercules CA) measure protein content to utilize Bradford assay.Preparation therapeutic monoclonal antibodies or comprise the mixture of different monoclonal antibodies is used to treat mouse subcutaneous or in-situ injection LAPC9 AD and HPAC tumor xenogeneic graft.
Clone and heterograft
Prostate cancer cell line PC3 and LNCaP clone and fibroblast NIH 3T3 (American type culture collection) are kept at respectively among the RPMI and DMEM that is supplemented with L-glutamine and 10%FBS.
Through Hubert etc., Proc Natl Acad Sci USA, 1996,96 (25): the retrovirus gene transformation described in 14523 produces PC3-PSCA and 3T3-PSCA cell mass.
The LAPC-9 heterograft is expressed the wild-type androgen receptor; Can produce PSA (PSA), it is implanted to (Craft, N. etc. in 6-8 male ICR-severe combined immunodeficiency (SCID) mouse (Taconic Farms) body in age in week through subcutaneous trocar; 1999,5:280).The single cell suspension for preparing the LAPC-9 tumour cell according to the method for descriptions such as Craft.
The xenotransplantation mouse model
At the right abdominal part hypodermic 1 * 10 of male SCID mouse 6Prepare subcutaneous (s.c.) tumour with matrigel (CollaborativeResearch) by the tumour cell of 1: 1 mixed.In order to detect the influence that antibody forms tumour, at the antibody of start injection on the same day of injection tumour cell.The mouse IgG (ICN) or the PBS of control mice injection purifying; Perhaps the irrelevant antigenic monoclonal antibody purification that human archeocyte is not expressed is discerned in injection.In preliminary experiment, find mouse IgG and PBS the no significant difference that influences to tumor growth.Utilize clamp to measure the size of tumour, tumor size equals length., the diameter of Subcutaneous tumor puts to death mouse when surpassing 1.5cm.
With carrying out in-situ injection after ketamine/xylazine anesthesia.When carrying out prostate gland orthotopic transplantation test, cut mouse web portion, expose prostate gland.Matrigel blended LAPC or PC3 tumour cell (2 * 10 6) be expelled in the prostatic utriculus, volume injected is 10 μ l.In order to monitor the growth of tumor situation, mouse is touched inspection, gather a mouse blood sample weekly to measure the expression level of PSA.
Anti-PSCA MAb can suppress to express the xenotransplantation growth of tumor of PSCA
Utilize the former bit model of HPAC and LAPC9 to estimate the influence that anti-PSCA MAb forms tumour.Compare with the Subcutaneous tumor model, the TIS transplantation model need be injected directly into tumour cell respectively in mice pancreatic or the prostate gland, causes the formation of local tumor; Tumour is to the transfer at position, distant place; The deterioration of mouse healthy state, and mouse dead subsequently (Saffran, D. etc.; PNAS, the same).These characteristics make former bit model more can represent the progression of human diseases, can make us judge the treatment effect of monoclonal antibody according to clinical index of correlation.
Correspondingly tumor cell injection is gone into the mouse prostate gland, after 2 days mouse is divided into 2 groups, use a) the anti-PSCA Ab of 250-1000 μ g; Or b) control antibodies is treated for 3 times weekly, continues 2-5 week.
The major advantage of TIS model is to study the incidence and development of metastases.Utilize the antibody of antineoplastic specificity cell surface protein such as the anti-CK20 antibody of prostate cancer to analyze formation (Lin S etc., the Cancer Detect Prev.2001 that can study metastatic tumor in the mouse body that has TIS through IHC; 25:202).
Another advantage of TIS model is to study neovascularization and vasculogenesis.Tumor growth partly depends on the formation of neovascularity.Originate though the vasoganglion of capillary vessel system and formation is the host, the xenotransplantation tumour also can be regulated initial sum structure (Davidoff etc., the ClinCancer Res.2001 of new vessel; 7:2870; Solesvik O etc., Eur.J Cancer Clin Oncol.1984,20:1295).Utilize method well known in the art can study the influence of antibody and small molecules, for example analyze tumor tissues and microenvironment on every side thereof through IHC to neovascularization.
The anti-PSCA MAb of injected in mice or the control antibodies of in 4 weeks, giving existing TIS.All load the tumors of higher load to guarantee in mouse lung, forming metastatic tumor for two groups of mouse.Put to death mouse then, analyze in its bladder, liver, bone and the lung whether have tumour cell through IHC.The result of these tests proves that anti-psca antibody has the activity that anti-efficiently prostate cancer forms and makes progress in the mouse heteroplastic transplantation model.Anti-psca antibody can suppress the formation of tumour, stops existing growth of tumor, the survival time of extended treatment mouse.And the transfer to the site, distant place has extremely strong retarding effect to anti-PSCA MAb to local tumor of prostate, even there is the tumors of higher load.Therefore, anti-PSCA MAb helps to improve main clinical indices (tumor growth), prolongs survival time, improves patient's healthy state.
PSCA Mab is to the influence of human prostata cancer growth in the mouse
Adopt aforesaid method, with LAPC-9AI tumour cell (2.0 * 10 6Individual cell) male SCID mouse is gone in subcutaneous injection.With mouse random packet (n=10 in each group), in the time of the 0th day, give HA1-4.120 or isotype Mab contrast initial treatment as indicated through intraperitoneal (i.p.).2 times animal is treated weekly, administration was 7 times altogether, until the 28th day that studies.Every as indicated 3-4 days with caliper measurement monitoring tumor growth.The result show the anti-PSCA monoclonal antibody of people Ha1-4.120 suppressed significantly subcutaneous implantation SCID mouse the human prostata cancer heterograft growth (p＜0.05) (Figure 18).
In another test, with LAPC-9AI tumour cell (2.0 * 10 6Individual cell) male SCID mouse is gone in subcutaneous injection.When gross tumor volume reaches 50mm 3The time, with mouse random packet (n=10 in each group), give specified HA1-5.99.1 or isotype Mab contrast initial treatment through intraperitoneal (i.p.) as indicated.2 times animal is treated weekly, administration was 5 times altogether, until the 14th day that studies.Every as indicated 3-4 days with caliper measurement monitoring tumor growth.The result show the anti-PSCA monoclonal antibody of total man Ha1-5.99 significantly suppressed subcutaneous implantation SCID mouse foundation androgen-dependent human prostata cancer heterograft growth (p＜0.05) (Figure 19).
In another test, with LAPC-9AD tumour cell (2.5 * 10 6Individual cell) male SCID mouse is gone in subcutaneous injection.When gross tumor volume reaches 40mm 3The time, with mouse random packet (n=10 in each group), peritoneal injection (i.p.) improves the HA1-4.121 or the isotype MAb contrast begin treatment of concentration as indicated.Weekly animal is carried out 2 treatments, administration was 7 times altogether, until the 21st day that studies.Detected growth of tumor with caliper in every as indicated 3-4 days.The result of this research shows that HA1-4.121 has suppressed the growth of people's androgen-dependent prostate cancer heterograft of the foundation in the subcutaneous implantation SCID mouse.The result on statistics for being significantly: the 14th, 17 and 21 days (p＜0.05 of 300 μ g dose groups; The Kruskal-Wallis check; Two tail α=0.05) and 700 μ g dose groups the 10th, 14,17 and 21 days (p＜0.05, Kruskal-Wallis check, two tail α=0.05) (Figure 20).
In another test, will be available from the LAPC-9AD tumour cell (2.0 * 10 of patient and androgen-dependent 6Individual cell) is injected into the prostatic episternites of male SCID mouse.Made tumor growth about 10 days, mouse is carried out random packet.Contrast begin treatment with 500mg people HA1-4.117, HA1-4.121 or isotype at implantation tumour after 10 days.Send antibody through intraperitoneal 2 times weekly, administration is 7 times altogether.4 days the time, put to death animal behind the last potion, take out primary tumo(u)r and also weigh.The result shows that the anti-PSCA monoclonal antibody of people Ha1-4.121 (p＜0.01) and Ha1-4.117 (p＜0.05) have significantly suppressed the growth (Figure 21) of the LAPC-9AD prostate cancer heterograft of Chang Weiyi implantation SCID mouse.
In another test, will be available from patient's androgen-dependent LAPC-9AD tumour cell (2.0 * 10 6Individual cell) is injected in the prostatic episternites of male SCID mouse.Made tumor growth about 9 days, mouse is carried out random packet.The animal that is randomized into survival group comprises 11 mouse of isotype MAb contrast and 12 mouse of HA1-4.121 treatment group.2 usefulness 1000 μ g Ha1-4.121 or 1000 μ g isotype MAb contrast intraperitoneal is treated animal weekly, and administration is 9 times altogether.The result shows that HA1-4.121 remarkable (logarithm hierarchical verification: p＜0.01) has prolonged the survival time of the SCID mouse that has people's androgen-dependent tumor of prostate.Treated the last time back 110 days, 2 mouse in the HA1-4.121 treatment group keep the state (Figure 22) of no palpable tumour.
The coupling of PSCA Mabs and TX is in the effect of mouse
In another test, with LAPC-9AI tumour cell (2 * 10 6Individual cell/animal) male SCID mouse is gone in subcutaneous injection.When gross tumor volume reaches 65mm 3The time, as indicated animal is weaved into 4 different groups (n=10 in each group) at random.In the time of the 0th day, begin weekly 2 dosage with 500 μ g and give Ha1-4.121 or isotype MAb contrast, administration is 6 times altogether.In the time of the 17th day, give last potion.Dosage with 5mg/kg in the time of the 0th, 3 and 7 day gives TX through intravenously.Detected growth of tumor with caliper in every 3-4 days.The result of this research shows with single the treatment with control antibodies and compared in the 28th day, and Ha1-4.121 reaches 45% (ANOVA/Tukey check: p＜0.05) as the medicament of single usefulness to the growth-inhibiting of non-androgen-dependent prostate cancer heterograft in the SCID mouse.Compare with giving the control antibodies treatment separately, give isotype MAb contrast and add that TX reaches 28% to the inhibition of tumor growth, does not have the significance on the statistics.Compare with independent control antibodies, unite and give HA1-4.121 and TX and have the enhanced effect and make the inhibition of tumor growth reach 69% (ANOVA/Tukey check: p＜0.01).When HA1-4.121 is added that with HA1-4.121 or isotype MAb contrast the TX group is compared with the combined group of TX, all demonstrated (the ANOVA/Tukey check: p＜0.05) (Figure 23) of the significant difference on the statistics.
The influence that people PSCA MAb grows to human pancreas cancer in mouse
In another test, with people HPAC pancreatic cancer cell (2 * 10 6Individual/mouse) subcutaneous injection go into immunodeficiency type ICR SCID mouse (Taconic Farm, Germantown, NY).With mouse random packet (n=10 animal/group), in using specified people PSCA monoclonal antibody begin treatment on the same day.Send antibody (500mg/ mouse) through intraperitoneal 2 times weekly, administration is 8 times altogether.The result shows that the anti-PSCA monoclonal antibody of people Ha1-4.121, Ha1-4.117 and Ha1-1.16 have significantly suppressed the growth of subcutaneous transplantation human pancreas cancer heterograft in the SCID mouse.Use the t check to carry out statistical analysis (two tails, α=0.05) (Figure 24).
In another test, with HPAC cell (3.0 * 10 6Individual cell) Chang Weiyi implants in the pancreas of SCID mouse.Be 3 groups (n=9 of every group) with the mouse random assignment as indicated.Transplanting the same day with HA1-4.121 (250 μ g or 1000 μ g) or isotype MAb contrast (1000 μ g) begin treatment.2 intraperitoneal give antibody weekly, and administration is 10 times altogether.Last potion was put to death animal after 13 days, and the taking-up primary tumo(u)r is also weighed.The result of this research shows that the HA1-4.121 of two kinds of dosage levels being tested has all significantly suppressed the normal position growth of human pancreas cancer heterograft in the SCID mouse.250 μ g and 1000 μ g AGS-PSCA have suppressed tumor growth respectively and have reached 66% and 70% (Kruskal-Wallis/Tukey check: be respectively p＜0.01 and p＜0.01) (Figure 25).
In necrotomy, in control antibodies treatment group, observe the MET of visible to lymphoglandula and far-end organ.In two HA1-4.121 treatment groups, all do not observe the visible MET.From all animals, take out lymphoglandula, lung and liver, the existence of dividing a word with a hyphen at the end of a line property of histology tumour.The personnel selection cytokeratin dyes to the section of taking from each animal lung and lymphoglandula, and microscopically is confirmed the quantity of MET.The histologic analysis result shows that lymphoglandula (LN) MET in the animal of treating with HA1-4.121 significantly reduces (p=0.0152 is recorded by the check of Fishers particularity).The incidence that shifts and invade also significantly reduces (p=0.0152 is recorded by the check of Fishers particularity) in the animal with two kinds of concentration HA1-4.121 treatments.Only the lung MET quantity with the HA1-4.121 of 1.0mg dosage treatment mouse significantly reduces (p=0.0498 is recorded by the check of Fishers particularity) (Figure 26).
The influence that people PSCA MAb grows to bladder cancer in mouse
In another test, with people SW780 transitional cell bladder carcinoma cell line (2 * 10 6Individual/mouse) subcutaneous injection go into immunodeficiency type ICR SCID mouse (Taconic Farm, Germantown, NY).With mouse random packet (n=10 animal/group), the people PSCA MAb begin treatment shown in use on the same day.2 intraperitoneal are sent antibody (250mg/ mouse) weekly, and administration is 7 times altogether.The result shows that HA1-4.117 (p=0.014), HA1-4.37 (p=0.0056), HA1-1.78 (p=0.001), Ha1-5.99 (p=0.0002) and HA1-4.5 (p=0.0008) have significantly suppressed the growth of subcutaneous implantation SW780 tumor of bladder in the SCID mouse.Carry out statistical analysis (two tails, α=0.05) (Figure 27) with the t check.
The result of these tests shows that PSCA Mab can be used for treatment and the diagnostic purpose that listed cancer in the Table I is treated and controlled.
Embodiment 27
Adopt anti-psca antibody the mankind are treated and to diagnose
Anti-PSCA monoclonal antibody can be safely and is used for diagnosis, prevention, prediction and/or the therapeutic purpose to the mankind effectively.Be presented at through Western blotting and immunohistochemical analysis cancerous tissue and tumor xenogeneic graft with anti-PSCA Mab and have strong extensive dyeing in the cancer, in healthy tissues, then have significantly reduced level or undetectable level.To in the cancer with metastatic disease in the detection of PSCA shown the purposes of this Mab as diagnosis and/or prognostic indicator.Therefore, anti-psca antibody can be used for diagnostic uses, and for example the biopsy samples to kidney carries out immunohistochemical analysis from suspected patient, to detect cancer.
Detect discovery through flow cytometry, anti-PSCA Mab combines with the cancer cells specificity.Therefore; Anti-psca antibody can be used in the whole body imaging diagnostic uses, for example put exempt from scintigraphy and radioimmunotherapy (referring to for example, Potamianos S. etc.; Anticancer Res 20 (2A): 925-948 (2000)), to detect the locality and the metastatic cancer of expressing PSCA.Come off or be discharged into the PSCA extracellular region in born of the same parents' external environment, for example alkaline phosphatase diester B10 (Meerson, N.R., Hepatology 27:563-568 (1998)) make to adopt available from the suspected patient serum and/or the diagnostic detection of carrying out PSCA of the anti-psca antibody in the urine sample become possibility.
Specificity combines the anti-psca antibody of PSCA to can be used for treating the cancer of expressing PSCA.Use this anti-psca antibody with non-binding molecular form and combining form, the multiple treatment or the molecule (for example prodrug, enzyme or ri) that forms images in antibody described in the said combining form and this area, known combine.In preclinical study; Non-binding and the anti-psca antibody of bonded have been tested to tumor prevention in the SCID mouse cancer xenograft models (for example cancer kidney model AGS-K3 and AGS-K6) and growth inhibiting effect (referring to for example, title be the embodiment of " the in-vivo tumour inhibition of PSCA mediated monoclonal antibody ").Bonded and uncombined anti-psca antibody all can be used as the treatment pattern in people's clinical trial, no matter be separately or described in following examples, combine with other therapies.
Embodiment 28
Human clinical's research of anti-psca antibody treatment in end user source and diagnosing human tumour in the body
Antibody of the present invention can be discerned the epi-position on the PSCA, is used to treat some tumour, like those listed in Table I tumours.Consider multiple factor, comprising the expression level of PSCA, those listed tumours are at present preferred indications in tumour such as the Table I.Interrelate with these indications, we have successfully implemented following three kinds of clinical protocol.
I.) assisting therapy: as the assisting therapy measure, anti-psca antibody combined chemotherapy medicine or antitumor drug and/or radiotherapy are treated the patient.The primary tumo(u)r target position, those tumours listed like Table I are handled with the treatment measure of standard, handle with anti-psca antibody more in addition in a line and the second line treatment measure of standard.The validity of setting in the regimen is to judge according to the ability of dwindling and reducing standard chemotherapy drug use dosage of tumor mass.The minimizing of standard chemotherapy drug use dosage also descends the toxicity relevant with dosage of chemotherapeutics, therefore makes us can adopt other treatment measure and/or extended treatment cycle.Anti-psca antibody can be used for uniting use with chemotherapeutics or antitumor drug in several auxiliary clinical studyes, like Zorubicin (advanced prostate cancer), cis-platinum (late period head and neck cancer and lung cancer), taxol (mammary cancer) and Dx (preclinical study).
II.) single therapy: use anti-psca antibody treatment tumour to be meant separately only to use antibody and without chemotherapeutic or antitumour drug to the patient.In one embodiment, the single therapy measure is used to treat the patient who suffers from late tumor and serious transfer.Patient's the state of an illness shows to a certain degree stable.Evidence antibody also has certain curative effect to the tumour patient of stubbornness.
III.) contrast medium: through with radionuclide (like iodine or yttrium (I 131, y 90) be connected on the anti-psca antibody and prepare radiolabeled antibody, this antibody can be used as diagnostic reagent and/or contrast medium.When being used for this purpose, the antibody of mark navigates in the MET of solid tumor and expression PSCA.When anti-psca antibody when the contrast medium, antibody can be used as the additives of solid tumor operative treatment, is used for before the art scanning and postoperative scanning to confirm whether tumour is eliminated clean and/or recurrence.In one embodiment, ( 111In)-and psca antibody is used for I phase human clinical trial as contrast medium, and screening suffers from patient's (analogous cases are seen J.Natl.Cancer Inst.83:97-104 (1991) such as Divgi) of the tumour of expressing PSCA.The patient carries out the anteposition and position, back gamma-rays radiography of standard subsequently.From the radiography result, can identify the position of primary lesion and MET.
Dosage and route of administration
As well-known to those skilled in the art, can through with clinical in other used similar medicines definite dosage of comparing.Therefore, the dosage scope of anti-psca antibody is 5 to 400mg/m 2,, can consider lower dosage according to the result of safety research.With known antibodies the affinity of its target position is compared, the relative affinity of anti-PSCA is those skilled in the art's used parameters when confirming similar dosage regimen.In addition, anti-psca antibody is a human antibody completely, compares with chimeric antibody, and its clearance rate is relatively low; Therefore required dosage was also wanted corresponding minimizing when the patient used this antibody, maybe be 50 to 300mg/m 2Between still effective.Dose unit is used mg/m 2, rather than conventional dose unit mg/kg, come administration according to surface-area, be convenient to confirm the required dosage of the people of the various bodily forms from infant to grownup like this.
There are three kinds of different medications to can be used for the administration of anti-psca antibody.Conventional intravenously administrable is the used standard medications of most of tumours.But; If tumour is positioned at peritoneal cavity, like the tumour in the tumour in entovarial tumour, the bile duct and other lacunas, peritoneal injection is considered to preferably; Can reach higher antibody dosage at tumor locus like this, also can make the clearance rate of antibody drop to minimum simultaneously.Some solid tumor contains blood vessel, and is just proper through regional perfusion's administration like this.Regional perfusion makes the antibody of high dosage focus on tumor locus, makes the short-term clearance rate of antibody drop to minimum simultaneously.
Clinical development plan (CDP)
General introduction: CDP utilizes and develops anti-psca antibody and assisting therapy, single therapy and as the relevant clinical application potential of contrast medium.Prove its security through clinical study earlier, and then prove the validity of its repeat administration.Clinical trial is an open label, and standard chemotherapy and standard care add anti-psca antibody and make comparisons.Should be noted that an inclusion criteria relevant with screening the experimenter is to express the level of PSCA according to its tumour that biopsy obtains.
The same with any albumen or the measure of antibody infusion of therapeutic, main relevant with following factors to the consideration of security: (i) cytokine release syndrome, like ypotension, generate heat, tremble, shiver with cold; (ii) be directed against the generation (like the antibody to therapeutic antibodies of patient's generation, or HAHA reacts) of the immunogenic response of medicine; And (iii) to expressing the Normocellular toxicity of PSCA.Carry out the detection of standard and follow up a case by regular visits to monitor these safety indexes.Test shows that anti-psca antibody is safe when being used for human body.
Embodiment 29
Human clinical trial: the monotherapy that adopts the anti-psca antibody of people
Anti-psca antibody is safe in above-mentioned assisting therapy clinical trial, and II phase human clinical trial has also proved conclusively validity and confirmed best dosage for single therapy.Find later on that through this clinical trial its security is the same with the result of above-mentioned assisting therapy clinical trial with clinical efficacy, just the patient does not use chemotherapeutics when accepting anti-psca antibody.
Embodiment 30
Human clinical trial: the diagnosing image that adopts the anti-psca antibody of people
As stated, it is safe carrying out assisting therapy according to above-mentioned safety standards with antibody, so we utilize anti-psca antibody to carry out clinical trial once more as diagnostic contrast agents.Those schemes that testing program and this area are described are similar basically, like Divgi etc., and J.Natl.Cancer Inst.83:97-104 (1991).The result finds that antibody is safely and effectively as diagnostic reagent.
Embodiment 31
Remove therapy with the anti-psca antibody of people and chemotherapy, radiotherapy and/or hormone
Human clinical trial for the assisting therapy measure
The I clinical trial phase of the anti-psca antibody in people source that at first carries out 6 intravenous injection dosage is to estimate the relevant security of itself and solid tumor treatment, and these solid tumor are meant the tumour that Table I is listed.In this research; The assisting therapy measure of removing as antitumor drug or chemotherapeutics or hormone with the anti-psca antibody of single dose is carried out the I clinical trial phase and is estimated its security, such as this paper description but be not limited only to cis-platinum, Dx, Zorubicin, taxol, leuprorelin, Zoladex, flutamide, Casodex, RU-23908 etc.Testing program comprises the anti-psca antibody of about 6 single doses, and 6 dosage raise gradually, from about 25mg/m 2To about 275mg/m 2, the arrangement of process of the test is following or can similar following arrangement:
The 0th day the 7th day the 14th day the 21st day the 28th day the 35th day
MAb dosage 25mg/m 275mg/m 2125mg/m 2175mg/m 2225mg/m 2275mg/m 2
Chemotherapeutic
+ + + ?+ ?+ ?+
(standard dose)
After carrying out Antybody therapy and chemotherapy, monitor the patient in 1 week closely at every turn.To note estimating above-mentioned safety indexes especially: (i) cytokine release syndrome, like ypotension, generate heat, tremble, shiver with cold; (ii) be directed against the generation (like the antibody to therapeutic antibodies of patient's generation, or HAHA reacts) of the immunogenic response of medicine; And (iii) to expressing the Normocellular toxicity of PSCA.Carry out the detection of standard and follow up a case by regular visits to monitor these safety indexes.Also want the clinical manifestation of evaluate patient, particularly the situation of dwindling through MRI or the observed knurl piece of other shadowgraph techniques.
The anti-psca antibody of evidence is safely and effectively, and the purpose of II clinical trial phase is further to prove conclusively its validity and confirms optimal therapeutic dosage.
Embodiment 32
RNA disturbs (RNAi)
RNA disturbs (RNAi) technology to be applied in the various cell analysis relevant with tumour.RNAi is a kind of PTGS mechanism that is excited by double-stranded RNA (dsRNA).The degraded of RNAi inductive specific mrna makes protein expression and follow-up gene function thereof change.In mammalian cell, these dsRNA that are called as short interfering rna (siRNA) have the correct structure of activation target in the RNAi approach of degraded (especially some mRNA).Referring to Elbashir S.M. etc. " Duplexes of 21-nucleotide RNAs MediateRNA interference in Cultured Mammalian Cells ", Nature 411 (6836): 494-8 (2001).Therefore, the RNAi technology can be successfully applied to mammalian cell with reticent target gene.
Uncontrolled cellular proliferation is a sign of cancerous tumor cell; Therefore, the assessment in cell survival/proliferation assay is correlated with to PSCA.Therefore, RNAi is used for the antigenic function of PSCA.In order to produce siRNA, can significantly reduce the algorithm of the oligonucleotide of PSCA protein expression level when having adopted prediction to have key molecule parameter (G: C content, fusing point are stable etc.) and transfered cell to PSCA.According to this embodiment, used the PSCAsiRNA compsn that comprises corresponding to the siRNA (double-stranded short interfering rna) of PSCA protein nucleic acid ORF sequence or its subsequence.Therefore; The length of the siRNA subsequence of using with this mode is generally 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 or more a plurality of continuous RNA Nucleotide.At least the part complementation and the incomplementarity of these siRNA sequences and mRNA encoding sequence.In preferred embodiment, the length of this subsequence is 19-25 Nucleotide, and most preferably length is 21-23 Nucleotide.In preferred embodiment, these siRNA have obtained the effect that PSCA antigen knocks out in expressing the proteic cell of PSCA, and have the effect that is described below.
In various kinds of cell system, test selected siRNA (PSCA.b oligomer) with survival/propagation MTS method of testing (mensuration cell metabolic activity).(that is, MTS) can detect survivaling cell exclusively, this is because viable cell has metabolic activity and can four nitrogen salts be reduced to the first membranization compound with color thus based on the colorimetric analysis of four nitrogen salts; Dead cell then can not.In addition, according to following test design, this PSCA.b oligomer has obtained the effect that PSCA antigen knocks out and has had the effect that is described below in expressing the proteic cell of PSCA.
Mammals siRNA transfection: carry out previous day of siRNA transfection, with 2 * 10 3The different clones of individual cells/well plant in the substratum (containing the antibiotic RPMI1640 of 10%FBS w/o) with 80 μ l (96 well plate format), are used for survival/MTS test.Parallel with PSCA specific siRNA oligomer, in each test, comprised following sequence as contrast: a) with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and annealing buffer (not containing siRNA) simulate cells transfected; B) (sequence of target is luciferase-4 specific siRNA: 5 '-AAGGGACGAAGACGAACACUUCTT-3 ') (SEQ ID NO:77); And c) (sequence of target is the Eg5 specific siRNA: 5 '-AACTGAAGACCTGAAGACAATAA-3 ') (SEQ ID NO:78).The final concentration that uses of SiRNA is 10nM and 1 μ g/ml Lipofectamine 2000.
Test design is following: at first (the serum-free transfection media is diluted to 0.1uM μ M (10 times concentrate) with siRNAs in Invitrogen), at room temperature hatches 5-10 minute at OPTIMEM.Lipofectamine2000 is diluted to 10 μ g/ml (10 times concentrate) is used for all transfections, at room temperature (RT) hatched 5-10 minute.10 times of concentrated Lipofectamine 2000 of an amount of dilution are mixed with 10 times of concentrated siRNA of dilution with 1: 1 ratio, at room temperature hatch 20-30 minute (5 times of spissated transfection solution).5 times of concentrated transfection solution of 20 μ l are added in each sample, after hatching 96 hours under 37 ℃, measure.
The MTS test: the MTS test is the colorimetric method of the viable cell number in a kind of definite propagation, CDCC or the chemical-sensitive property testing; This test is that [3-(4 with four nitrogen salt compounds; 5-dimethylthiazole-2-yl)-and 5-(3-carboxyl p-methoxy-phenyl)-2-(4-sulfo group phenyl)-2H-four nitrogen, inner salt; MTS (b)] and electron pair mixture (sulfoethyl (ethosulfate) azophenlyene; PES).Test as follows: small volume of solution reagent is directly added in the culture hole, hatched 1-4 hour, read the light absorption value at plate appearance record 490nm place then with 96 holes.The You Se Jia Za product amount of the flow measurement of 490nm place light absorption value is directly proportional with the quantity of viable cell in mitochondria activity and/or the culture.
In order to disclose the function of PSCA in cell, the clone through transfection endogenous expression PSCA makes PSCA reticent.
Another embodiment of the present invention is a kind ofly to synthesize the method for analyzing the cell proliferation relevant with PSCA through the DNA that measures as the propagation mark.The DNA precursor that has used mark (promptly 3The H-thymidine), it being mixed DNA carries out quantitatively.The cell fission amount that exists in the labelled precursor that mixes DNA and the cell culture is directly proportional.The another kind of method of measuring cell proliferation is to clone former test.In these tests, the cell inoculation of set amount on suitable matrix, is treated in back one section growth period formed clone's quantity to siRNA and counted.
In PSCA cancer target validation, consider with apoptosis and the test of cell cycle distribution research pair cell survival/propagation and replenish.The biochemical marker of apoptotic process is a genomic DNA fragmentization, and this is the irreversible incident that a kind of meeting causes necrocytosis.A kind of method of fragmentation DNA is to adopt immunoassay that histone compound dna fragmentation is carried out immunodetection (being that necrocytosis detects ELISA) in the observation of cell, and this method is measured the concentration (monokaryon and oligoneucleosomes) of histone compound dna fragmentation in the apoptotic cell kytoplasm.This mensuration need not pair cell and carries out preliminary making, and can detect the dna degradation (being the tumour cell of fresh separated) in external not proliferating cells.
Causing the most important effector molecule of apoptotic cell death is caspases (caspase).In case caspases is the proteolytic enzyme that can cut in the carboxyl site of an asparagicacid residue after the activation, in case this aspartic acid activation, the utmost point commitment of mediated apoptosis.All caspaseses all are as the pre-enzyme synthetic, and its activation relates to the cutting of asparagicacid residue.Particularly, as if caspases 3 plays an important role in the apoptotic cell incident causes.Through measuring the early stage incident that caspases 3 activity can detect apoptosis.RNAi has further supported the activity inducement effect to apoptosis with the existence of western blot detection of active caspases 3 or the proteolyze cleaved products in the apoptotic cell (being PARP) after handling.Because it is very complicated causing apoptotic cells mechanism, every kind of method all has its advantage and restriction.Investigate last point at other standard/end, morphocytology for example, chromatin pyknosis, cytolemma bubble, to help further sustenticular cell death be apoptosis property to apoptotic body.Because the gene target of not all adjusting cell growth all is anti-apoptosis, the dna content of changing cell has thoroughly been carried out measuring to obtain dna content distribution or cell cycle distribution.Owing to escape to tenuigenin, the nucleus of apoptotic cell contains less DNA (inferior G1 crowd).In addition, because existing DNA amount is different among G0/G1, S and the G2/M, also can adopt DNA dyeing (being iodate third ingot) to distinguish different periods cell cycle in the cell mass.In these researchs, can carry out quantitatively subgroup.
For psca gene, the understanding of RNAi research promotion the to the effect of this gene product in the cancer approach.Can these viable rnas i molecule be used for differential test and have the active MAb of antineoplaston with screening.In addition, can give the cancer patients to alleviate the malignancy of multiple cancer types (comprise in the Table I listed those) as therapeutical agent with siRNA.When PSCA works in cell survival, cell proliferation, tumour generation or apoptosis, can be with it as the target of diagnosing, predict, prevent and/or treat purpose.
Embodiment 33
Adopt IHC in cancer patients's sample, to detect PSCA albumen
Utilize antibody HA1-4.117 to detect available from the proteic expression of PSCA in cancer patients's the tumor sample.To be cut into 4 microns slabs through formaldehyde fixed, paraffin-embedded tissue, place on the glass slide.Section is dewaxed, dewaters, reclaim solution (Antigen Retrieval Citra Solution with antigen; BioGenex, 4600Norris Canyon Road, San Ramon, CA, 94583) at high temperature handle.Then under 4 ℃, section is incubated among the anti-psca antibody Ha1-4.117 of resorcinolphthalein link coupled human monoclonal 16 hours.This slide glass of washing is 3 times in damping fluid, hatches 1 hour with the anti-resorcinolphthalein of rabbit again, in damping fluid, after the washing, immerses DAKO EnVision+ TMPeroxidase-conjugated goat anti-rabbit immunoglobulin two anti-(DAKO Corporation, Carpenteria, CA) in 30 minutes.Washing slice in damping fluid develops with DAB test kit (SIGMA Chemicals) then, carries out counterstaining with phenodin, analyzes with bright-field microscope.The result show the prostate cancer tumour cell (A, B), express PSCA in bladder metastatic carcinoma tumour cell (C) and the pancreas duct carcinoma tumour cell (D).These results show that PSCA expresses in human cancer, anti-this antigenic antibody can be used as diagnostic reagent (Figure 17).
These results show that PSCA is a kind of target in diagnosis in the cancer, prediction and the therepic use.
Quote various websites data in the whole text in the application, published document, patented claim and patent (website of quoting unifies finger URL with its resource or URL representes, the address is on the World Wide Web).The disclosure of these reference is all brought among this paper as a reference by complete.
The present invention is not limited in the scope of embodiment defined described herein, and these embodiments are just in order to explain all respects of the present invention, all is included within the scope of the present invention with any revision of its function equivalence.Except described herein, those skilled in the art is according to top description and explanation, should understand the various modifications that model of the present invention and method have been done, and these modifications equally also drop within the scope of the present invention.Only otherwise depart from true scope of the present invention and just can implement this modification or other embodiments with spirit.
Form
Table I: the organ of expressing PSCA when being pernicious
Prostate gland
Pancreas
Bladder
Kidney
Colon
Lung
Ovary
Mammary gland
Table II: amino acid abbreviations
Single-letter Trigram Full name
? ? ?
F Phe Phenylalanine(Phe)
L Leu Leucine
S Ser Serine
Y Tyr Tyrosine
C Cys Halfcystine
W Trp Tryptophane
P Pro Proline(Pro)
H His Histidine
Q Gln Stimulina
R Arg L-arginine
I Ile Isoleucine
M Met Methionine(Met)
T Thr Threonine
N Asn L-asparagine
K Lys Methionin
V Val Xie Ansuan
A Ala L-Ala
D Asp Aspartic acid
E Glu L-glutamic acid
G Gly Glycocoll
[0842]Table III: aminoacid replacement matrix
Adopt from GCG software 9.0BLOSUM62 aminoacid replacement matrix (sealing substitution matrix).Numerical value possibly be substituted in relevant natural protein more greatly more.(seeing World Wide Web URLikp.unibe.ch/manual/blosum62.html)
A C D E F G H I K L M N P Q R S T V W Y .
4 0?-2?-1?-2 0?-2?-1?-1?-1?-1?-2?-1?-1?-1 1 0 0?-3?-2 A
9?-3?-4?-2?-3?-3?-1?-3?-1?-1?-3?-3?-3?-3?-1?-1?-1?-2?-2 C
6 2?-3?-1?-1?-3?-1?-4?-3 1?-1 0?-2 0?-1?-3?-4?-3 D
5?-3?-2 0?-3 1?-3?-2 0?-1 2 0 0?-1?-2?-3?-2 E
6?-3?-1 0?-3 0 0?-3?-4?-3?-3?-2?-2?-1 1 3 F
6?-2?-4?-2?-4?-3 0?-2?-2?-2 0?-2?-3?-2?-3 G
8?-3?-1?-3?-2 1?-2 0 0?-1?-2?-3?-2 2 H
4?-3 2 1?-3?-3?-3?-3?-2?-1 3?-3?-1 I
5?-2?-1 0?-1 1 2 0?-1?-2?-3?-2 K
4 2?-3?-3?-2?-2?-2?-1 1?-2?-1 L
5?-2?-2 0?-1?-1?-1 1?-1?-1 M
6?-2 0 0 1 0?-3?-4?-2 N
7?-1?-2?-1?-1?-2?-4?-3 P
5 1 0?-1?-2?-2?-1 Q
5?-1?-1?-3?-3?-2 R
4 1?-2?-3?-2 S
5 0?-2?-2 T
4?-3?-1 V
11 2 W
7 Y
Table IV:
HLA I class/II motif/hyper-base preface
Table IV (A): HLA I class hyper-base preface/motif
The hyper-base preface The position The position The position
? 2 (main anchor stations) 3 (main anchor stations) C-terminal (main anchor station)
A1 TILVMS ? FWY
A2 LIVMATQ ? IVMATL
A3 VSMATLI ? RK
A24 YFWIVLMT ? FIYWLM
B7 P ? VILFMWYA
B27 RHK ? FYLWMIVA
B44 ED ? FWYLIMVA
B58 ATS ? FWYLIVMA
B62 QLIVMP ? FWYMIVLA
? ? ? ?
Motif ? ? ?
A1 TSM ? Y
A1 ? DEAS Y
A2.1 LMVQIAT ? VLIMAT
A3 LMVISATFCGD ? KYRHFA
A11 VTMLISAGNCDF ? KRYH
A24 YFWM ? FLIW
A*3101 MVTALIS ? RK
A*3301 MVALFIST ? RK
A*6801 AVTMSLI ? RK
B*0702 P ? LMFWYAIV
B*3501 P ? LMFWYIVA
B51 P ? LIVFWYAM
B*5301 P ? IMFWYALV
B*5401 P ? ATIVLMFWY
The residue that black matrix is represented is preferred, and the residue that italic is represented is very not preferred: think that it has motif if certain peptide has main anchors in each main anchor station of motif shown in the last table or hyper-base preface.
Table IV (B): HLA II class hyper-base preface
1 6 9
? ? ?
W，F，Y，V，.I，L A，V，I，L，P，C，S，T A，V，I，L，C，S，T，M，Y
[0872]Table IV (C): HLA II class motif
Figure G05817239920061201D001471
The residue that italic is represented is very not preferred or " available reluctantly " residue
Table IV (D): HLA I class hyper-base preface
Figure G05817239920061201D001481
The residue that italic is represented is very not preferred or " available reluctantly " residue
Table IV (E): HLA I class motif
Figure G05817239920061201D001491
Figure G05817239920061201D001501
Table IV (F):
The ultra type of HLA-is summed up
Total phenotype frequency of the ultra type of HLA-among the different ethnic populations
Figure G05817239920061201D001521
Table IV (G):
B44 and A1
A2，A3，B7，A24，
B44，A1，B27，
B62, and B58
Each motif representes to confirm the residue of superspecificity.Each motif has added the residue of confirming according to the data of delivering of discerning for a plurality of allelotrope in the ultra type.Residue in the bracket also is to be expected to be the extra residue that a plurality of allelotrope tolerated in the ultra type.
Figure G05817239920061201D001531
Table VI: transcript PSCA exon border v.1
The exon number Initial Stop Length
1 10 69 60
2 70 177 108
3 178 985 808
[0897] Table VII: the MFI value that is used for each data point of avidity calculating
The MFI value
nM PSC?A PSC?A PDP3 T＝8 +4 PDP3 T＝8 +4
40 869.3 777.06 795.24 661.66
20 875.19 835.94 816.34 824.07
10 856.28 847.83 777.85 842.72
5 866.94 817.45 758.15 818.83
2.5 835.47 769.79 742.45 783.5
1.25 813.12 782.84 806.2 792.44
0.625 766.52 689.3 683.64 666.28
0.3125 588.3 541.25 549.03 499.22
0.15625 389.95 354.24 366.82 355.83
0.07813 234.85 230.37 225.82 211.77
0.03906 138.05 132.14 134.25 134.07
0.01953 84.35 78.49 77.62 80.14
0.00977 48.13 48.38 50.93 46.87
0.00488 33.49 30.58 30.71 30.04
0.00244 21.77 20.33 18.14 20.44
0.00122 14.45 13.59 13.82 13.11
0.00061 11.07 10.14 9.52 10.35
0.00031 8.61 8.3 8.57 9.09
0.00015 7.45 7.17 7.28 7.85
0.00008 6.91 6.73 7.32 7.86
0.00004 6.94 6.41 6.81 6.51
Table VIII: the avidity that adopts Graphpad Prism computed in software: S type dosage-response (variable slope) formula
The Kd value
Table I X: the avidity of total length people PSCA Mab based on FACS
Avidity based on FACS
Sample ID Kd(nM)
Ha1-4.37 0.23
Ha1-4.121 0.26
Ha1-5.99.1 0.28
Ha1-4.117 0.32
Ha1-4.120 0.41
Ha1-4.5 1.00
Ha1-1.16.1 6.91
Table X: with the antibody of monkey PSCA and/or mouse PSCA generation cross reaction.
Cross reaction mouse PSCA takes place cross reaction takes place in hybridoma ID and monkey PSCA
H1-1.10 - -
Ha1-1.16 + -
Ha1-1.78 + -
Ha1-1.41 + -
Ha1-4.5 + -
Ha1-4.37 + -
Ha1-4.117 + +
Ha1-4.120 + -
Ha1-4.121 + -
Ha1-5.99 + -
Table X I: the epi-position group that PSCA:FACS analyzes
Figure G05817239920061201D001571
Legend:
Figure G05817239920061201D001572
Sequence table
< 110>Agensys Inc. (AGENSYS, INC.)
J. Gu Dasi (Gudas, Jean)
A. Ya Geboweici (Jakobovits, Aya)
X. go into business (Xiao-Chi, Jia)
R.K. Morrison (Morrison, Robert Kendall)
K.J.M. Morrison (Morrison, Karen Jane Meyrick)
H. Shao (Shao, Hui)
P.M. Cha Lita-Ai De (Challita-Eid, Pia M.)
A.B. Lai Tanuo (Raitano, Arthur B.)
 
< 120>be incorporated into proteic antibody of PSCA and associated molecule
 
<130>51158-20088.41
 
< 140>do not authorize
< 141>in this application
 
<150>60/616,381
<151>2004-10-05
 
<150>60/617,881
<151>2004-10-12
 
<150>60/621,310
<151>2004-10-21
 
<150>60/633,077
<151>2004-12-02
 
<150>10/857,484
<151>2004-05-28
 
<150>60/475,064
<151>2003-05-30
 
<160>78
 
<170>FastSEQ?for?Windows?Version?4.0
 
<210>1
<211>990
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<220>
<221>CDS
<222>(18)...(389)
<221>misc_feature
<222>543
< 223>n=a, t, c or g
 
<400>1
agggagaggc?agtgacc?atg?aag?gct?gtg?ctg?ctt?gcc?ctg?ttg?atg?gca 50
Met?Lys?Ala?Val?Leu?Leu?Ala?Leu?Leu?Met?Ala
1 5 10
ggc?ttg?gcc?ctg?cag?cca?ggc?act?gcc?ctg?ctg?tgc?tac?tcc?tgc?aaa 98
Gly?Leu?Ala?Leu?Gln?Pro?Gly?Thr?Ala?Leu?Leu?Cys?Tyr?Ser?Cys?Lys
15 20 25
gcc?cag?gtg?agc?aac?gag?gac?tgc?ctg?cag?gtg?gag?aac?tgc?acc?cag 146
Ala?Gln?Val?Ser?Asn?Glu?Asp?Cys?Leu?Gln?Val?Glu?Asn?Cys?Thr?Gln
30 35 40
ctg?ggg?gag?cag?tgc?tgg?acc?gcg?cgc?atc?cgc?gca?gtt?ggc?ctc?ctg 194
Leu?Gly?Glu?Gln?Cys?Trp?Thr?Ala?Arg?Ile?Arg?Ala?Val?Gly?Leu?Leu
45 50 ?55
acc?gtc?atc?agc?aaa?ggc?tgc?agc?ttg?aac?tgc?gtg?gat?gac?tca?cag 242
Thr?Val?Ile?Ser?Lys?Gly?Cys?Ser?Leu?Asn?Cys?Val?Asp?Asp?Ser?Gln
60 65 70 ?75
gac?tac?tac?gtg?ggc?aag?aag?aac?atc?acg?tgc?tgt?gac?acc?gac?ttg 290
Asp?Tyr?Tyr?Val?Gly?Lys?Lys?Asn?Ile?Thr?Cys?Cys?Asp?Thr?Asp?Leu
80 85 90
tgc?aac?gcc?agc?ggg?gcc?cat?gcc?ctg?cag?ccg?gct?gcc?gcc?atc?ctt 338
Cys?Asn?Ala?Ser?Gly?Ala?His?Ala?Leu?Gln?Pro?Ala?Ala?Ala?Ile?Leu
95 100 105
gcg?ctg?ctc?cct?gca?ctc?ggc?ctg?ctg?ctc?tgg?gga?ccc?ggc?cag?cta 386
Ala?Leu?Leu?Pro?Ala?Leu?Gly?Leu?Leu?Leu?Trp?Gly?Pro?Gly?Gln?Leu
110 115 120
tag?gctctggggg?gccccgctgc?agcccacact?gggtgtggtg?ccccaggcct 439
*
ttgtgccact?cctcacagaa?cctggcccag?tgggagcctg?tcctggttcc?tgaggcacat?499
cctaacgcaa?gtttgaccat?gtatgtttgc?accccttttc?cccnaaccct?gaccttccca?559
tgggcctttt?ccaggattcc?cacccggcag?atcagtttta?gtgacacaga?tccgcctgca?619
gatggcccct?ccaacccttt?ctgttgctgt?ttccatggcc?cagcattttc?cacccttaac?679
cctgtgttca?ggcacttctt?cccccaggaa?gccttccctg?cccaccccat?ttatgaattg?739
agccaggttt?ggtccgtggt?gtcccccgca?cccagcaggg?gacaggcaat?caggagggcc?799
cagtaaaggc?tgagatgaag?tggactgagt?agaactggag?gacaagagtt?gacgtgagtt?859
cctgggagtt?tccagagatg?gggcctggag?gcctggagga?aggggccagg?cctcacattt?919
gtggggctcc?cgaatggcag?cctgagcaca?gcgtaggccc?ttaataaaca?cctgttggat?979
aagccaaaaa?a 990
 
<210>2
<211>123
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>2
Met?Lys?Ala?Val?Leu?Leu?Ala?Leu?Leu?Met?Ala?Gly?Leu?Ala?Leu?Gln
1 5 10 15
Pro?Gly?Thr?Ala?Leu?Leu?Cys?Tyr?Ser?Cys?Lys?Ala?Gln?Val?Ser?Asn
20 25 30
Glu?Asp?Cys?Leu?Gln?Val?Glu?Asn?Cys?Thr?Gln?Leu?Gly?Glu?Gln?Cys
35 40 45
Trp?Thr?Ala?Arg?Ile?Arg?Ala?Val?Gly?Leu?Leu?Thr?Val?Ile?Ser?Lys
50 55 ?60
Gly?Cys?Ser?Leu?Asn?Cys?Val?Asp?Asp?Ser?Gln?Asp?Tyr?Tyr?Val?Gly
65 70 75 80
Lys?Lys?Asn?Ile?Thr?Cys?Cys?Asp?Thr?Asp?Leu?Cys?Asn?Ala?Ser?Gly
85 90 95
Ala?His?Ala?Leu?Gln?Pro?Ala?Ala?Ala?Ile?Leu?Ala?Leu?Leu?Pro?Ala
100 105 110
Leu?Gly?Leu?Leu?Leu?Trp?Gly?Pro?Gly?Gln?Leu
115 120
 
<210>3
<211>1020
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<220>
<221>CDS
<222>(56)...(427)
 
<400>3
tttgaggcca?tataaagtca?cctgaggccc?tctccaccac?agcccaccag?tgacc?atg 58
Met
1
aag?gct?gtg?ctg?ctt?gcc?ctg?ttg?atg?gca?ggc?ttg?gcc?ctg?cag?cca 106
Lys?Ala?Val?Leu?Leu?Ala?Leu?Leu?Met?Ala?Gly?Leu?Ala?Leu?Gln?Pro
5 10 ?15
ggc?act?gcc?ctg?ctg?tgc?tac?tcc?tgc?aaa?gcc?cag?gtg?agc?aac?gag 154
Gly?Thr?Ala?Leu?Leu?Cys?Tyr?Ser?Cys?Lys?Ala?Gln?Val?Ser?Asn?Glu
20 25 ?30
gac?tgc?ctg?cag?gtg?gag?aac?tgc?acc?cag?ctg?ggg?gag?cag?tgc?tgg 202
Asp?Cys?Leu?Gln?Val?Glu?Asn?Cys?Thr?Gln?Leu?Gly?Glu?Gln?Cys?Trp
35 40 ?45
acc?gcg?cgc?atc?cgc?gca?gtt?ggc?ctc?ctg?acc?gtc?atc?agc?aaa?ggc 250
Thr?Ala?Arg?Ile?Arg?Ala?Val?Gly?Leu?Leu?Thr?Val?Ile?Ser?Lys?Gly
50 55 60 65
tgc?agc?ttg?aac?tgc?gtg?gat?gac?tca?cag?gac?tac?tac?gtg?ggc?aag 298
Cys?Ser?Leu?Asn?Cys?Val?Asp?Asp?Ser?Gln?Asp?Tyr?Tyr?Val?Gly?Lys
70 75 80
aag?aac?atc?acg?tgc?tgt?gac?acc?gac?ttg?tgc?aac?gcc?agc?ggg?gcc 346
Lys?Asn?Ile?Thr?Cys?Cys?Asp?Thr?Asp?Leu?Cys?Asn?Ala?Ser?Gly?Ala
85 90 95
cat?gcc?ctg?cag?ccg?gct?gcc?gcc?atc?ctt?gcg?ctg?ctc?cct?gca?ctc 394
His?Ala?Leu?Gln?Pro?Ala?Ala?Ala?Ile?Leu?Ala?Leu?Leu?Pro?Ala?Leu
100 105 110
ggc?ctg?ctg?ctc?tgg?gga?ccc?ggc?cag?cta?tag?gctctggggg?gccccgctgc?447
Gly?Leu?Leu?Leu?Trp?Gly?Pro?Gly?Gln?Leu *
115 120
agcccacact?gggtgtggtg?ccccaggcct?ctgtgccact?cctcacacac?ccggcccagt?507
gggagcctgt?cctggttcct?gaggcacatc?ctaacgcaag?tctgaccatg?tatgtctgcg?567
cccctgtccc?ccaccctgac?cctcccatgg?ccctctccag?gactcccacc?cggcagatcg?627
gctctattga?cacagatccg?cctgcagatg?gcccctccaa?ccctctctgc?tgctgtttcc?687
atggcccagc?attctccacc?cttaaccctg?tgctcaggca?cctcttcccc?caggaagcct?747
tccctgccca?ccccatctat?gacttgagcc?aggtctggtc?cgtggtgtcc?cccgcaccca?807
gcaggggaca?ggcactcagg?agggcccggt?aaaggctgag?atgaagtgga?ctgagtagaa?867
ctggaggaca?ggagtcgacg?tgagttcctg?ggagtctcca?gagatggggc?ctggaggcct?927
ggaggaaggg?gccaggcctc?acattcgtgg?ggctccctga?atggcagcct?cagcacagcg?987
taggccctta?ataaacacct?gttggataag?cca 1020
 
<210>4
<211>123
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>4
Met?Lys?Ala?Val?Leu?Leu?Ala?Leu?Leu?Met?Ala?Gly?Leu?Ala?Leu?Gln
1 5 10 15
Pro?Gly?Thr?Ala?Leu?Leu?Cys?Tyr?Ser?Cys?Lys?Ala?Gln?Val?Ser?Asn
20 25 30
Glu?Asp?Cys?Leu?Gln?Val?Glu?Asn?Cys?Thr?Gln?Leu?Gly?Glu?Gln?Cys
35 40 ?45
Trp?Thr?Ala?Arg?Ile?Arg?Ala?Val?Gly?Leu?Leu?Thr?Val?Ile?Ser?Lys
50 55 ?60
Gly?Cys?Ser?Leu?Asn?Cys?Val?Asp?Asp?Ser?Gln?Asp?Tyr?Tyr?Val?Gly
65 70 75 80
Lys?Lys?Asn?Ile?Thr?Cys?Cys?Asp?Thr?Asp?Leu?Cys?Asn?Ala?Ser?Gly
85 90 95
Ala?His?Ala?Leu?Gln?Pro?Ala?Ala?Ala?Ile?Leu?Ala?Leu?Leu?Pro?Ala
100 105 110
Leu?Gly?Leu?Leu?Leu?Trp?Gly?Pro?Gly?Gln?Leu
115 120
 
<210>5
<211>888
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<220>
<221>CDS
<222>(423)...(707)
 
<400>5
tttgaggcca?tataaagtca?cctgaggccc?tctccaccac?agcccaccag?tgaccatgaa 60
ggctgtgctg?cttgccctgt?tgatggcagg?cttggccctg?cagccaggca?ctgccctgct?120
gtgctactcc?tgcaaagccc?aggcgcagtt?ggcctcctga?ccgtcatcag?caaaggctgc?180
agcttgaact?gcgtggatga?ctcacaggac?tactacgtgg?gcaagaagaa?catcacgtgc?240
tgtgacaccg?acttgtgcac?tcggcctgct?gctctgggga?cccggccagc?tataggctct?300
ggggggcccc?gctgcagccc?acactgggtg?tggtgcccca?ggcctctgtg?ccactcctca?360
cacacccggc?ccagtgggag?cctgtcctgg?ttcctgaggc?acatcctaac?gcaagtctga?420
cc?atg?tat?gtc?tgc?gcc?cct?gtc?ccc?cac?cct?gac?cct?ccc?atg?gcc 467
Met?Tyr?Val?Cys?Ala?Pro?Val?Pro?His?Pro?Asp?Pro?Pro?Met?Ala
1 5 ?10 15
ctc?tcc?agg?act?ccc?acc?cgg?cag?atc?ggc?tct?att?gac?aca?gat?ccg 515
Leu?Ser?Arg?Thr?Pro?Thr?Arg?Gln?Ile?Gly?Ser?Ile?Asp?Thr?Asp?Pro
20 25 30
cct?gca?gat?ggc?ccc?tcc?aac?cct?ctc?tgc?tgc?tgt?ttc?cat?ggc?cca 563
Pro?Ala?Asp?Gly?Pro?Ser?Asn?Pro?Leu?Cys?Cys?Cys?Phe?His?Gly?Pro
35 40 45
gca?ttc?tcc?acc?ctt?aac?cct?gtg?ctc?agg?cac?ctc?ttc?ccc?cag?gaa 611
Ala?Phe?Ser?Thr?Leu?Asn?Pro?Val?Leu?Arg?His?Leu?Phe?Pro?Gln?Glu
50 55 ?60
gcc?ttc?cct?gcc?cac?ccc?atc?tat?gac?ttg?agc?cag?gtc?tgg?tcc?gtg 659
Ala?Phe?Pro?Ala?His?Pro?Ile?Tyr?Asp?Leu?Ser?Gln?Val?Trp?Ser?Val
65 70 75
gtg?tcc?ccc?gca?ccc?agc?agg?gga?cag?gca?ctc?agg?agg?gcc?cgg?taa 707
Val?Ser?Pro?Ala?Pro?Ser?Arg?Gly?Gln?Ala?Leu?Arg?Arg?Ala?Arg*
80 85 90
aggctgagat?gaagtggact?gagtagaact?ggaggacagg?agtcgacgtg?agttcctggg?767
agtctccaga?gatggggcct?ggaggcctgg?aggaaggggc?caggcctcac?attcgtgggg?827
ctccctgaat?ggcagcctca?gcacagcgta?ggcccttaat?aaacacctgt?tggataagcc?887
a 888
 
<210>6
<211>94
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>6
Met?Tyr?Val?Cys?Ala?Pro?Val?Pro?His?Pro?Asp?Pro?Pro?Met?Ala?Leu
1 5 10 15
Ser?Arg?Thr?Pro?Thr?Arg?Gln?Ile?Gly?Ser?Ile?Asp?Thr?Asp?Pro?Pro
20 25 30
Ala?Asp?Gly?Pro?Ser?Asn?Pro?Leu?Cys?Cys?Cys?Phe?His?Gly?Pro?Ala
35 40 45
Phe?Ser?Thr?Leu?Asn?Pro?Val?Leu?Arg?His?Leu?Phe?Pro?Gln?Glu?Ala
50 55 60
Phe?Pro?Ala?His?Pro?Ile?Tyr?Asp?Leu?Ser?Gln?Val?Trp?Ser?Val?Val
65 70 75 80
Ser?Pro?Ala?Pro?Ser?Arg?Gly?Gln?Ala?Leu?Arg?Arg?Ala?Arg
85 90
 
<210>7
<211>1174
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<220>
<221>CDS
<222>(424)...(993)
 
<400>7
gacagtgaac?cctgcgctga?aggcgttggg?gctcctgcag?ttctggggca?gccacaggcg 60
cccagggttt?cgtgccgatc?agcccaggac?ggtcttcccg?gtgcagtttc?tgatgcgggg?120
agggcagtgc?tgccttccgg?tcaccaggac?cagtgctcag?cccgcctgct?tgaccccctt?180
acttagctgg?ggtccaatcc?atacccaatt?tagatgattc?agacgatggg?atttgaaact?240
tttgaactgg?gtgcgactta?agcactgccc?tgctgtgcta?ctcctgcaaa?gcccaggtga?300
gcaacgagga?ctgcctgcag?gtggagaact?gcacccagct?gggggagcag?tgctggaccg?360
cgcgcatccg?cgcagttggc?ctcctgaccg?tcatcagcaa?aggctgcagc?ttgaactgcg?420
tgg?atg?act?cac?agg?act?act?acg?tgg?gca?aga?aga?aca?tca?cgt?gct 468
Met?Thr?His?Arg?Thr?Thr?Thr?Trp?Ala?Arg?Arg?Thr?Ser?Arg?Ala
1 5 10 ?15
gtg?aca?ccg?act?tgt?gca?acg?cca?gcg?ggg?ccc?atg?ccc?tgc?agc?cgg 516
Val?Thr?Pro?Thr?Cys?Ala?Thr?Pro?Ala?Gly?Pro?Met?Pro?Cys?Ser?Arg
20 25 30
ctg?ccg?cca?tcc?ttg?cgc?tgc?tcc?ctg?cac?tcg?gcc?tgc?tgc?tct?ggg 564
Leu?Pro?Pro?Ser?Leu?Arg?Cys?Ser?Leu?His?Ser?Ala?Cys?Cys?Ser?Gly
35 40 45
gac?ccg?gcc?agc?tat?agg?ctc?tgg?ggg?gcc?ccg?ctg?cag?ccc?aca?ctg 612
Asp?Pro?Ala?Ser?Tyr?Arg?Leu?Trp?Gly?Ala?Pro?Leu?Gln?Pro?Thr?Leu
50 55 ?60
ggt?gtg?gtg?ccc?cag?gcc?tct?gtg?cca?ctc?ctc?aca?cac?ccg?gcc?cag 660
Gly?Val?Val?Pro?Gln?Ala?Ser?Val?Pro?Leu?Leu?Thr?His?Pro?Ala?Gln
65 70 75
tgg?gag?cct?gtc?ctg?gtt?cct?gag?gca?cat?cct?aac?gca?agt?ctg?acc 708
Trp?Glu?Pro?Val?Leu?Val?Pro?Glu?Ala?His?Pro?Asn?Ala?Ser?Leu?Thr
80 85 90 95
atg?tat?gtc?tgc?gcc?cct?gtc?ccc?cac?cct?gac?cct?ccc?atg?gcc?ctc 756
Met?Tyr?Val?Cys?Ala?Pro?Val?Pro?His?Pro?Asp?Pro?Pro?Met?Ala?Leu
100 105 ?110
tcc?agg?act?ccc?acc?cgg?cag?atc?ggc?tct?att?gac?aca?gat?ccg?cct 804
Ser?Arg?Thr?Pro?Thr?Arg?Gln?Ile?Gly?Ser?Ile?Asp?Thr?Asp?Pro?Pro
115 120 125
gca?gat?ggc?ccc?tcc?aac?cct?ctc?tgc?tgc?tgt?ttc?cat?ggc?cca?gca 852
Ala?Asp?Gly?Pro?Ser?Asn?Pro?Leu?Cys?Cys?Cys?Phe?His?Gly?Pro?Ala
130 135 140
ttc?tcc?acc?ctt?aac?cct?gtg?ctc?agg?cac?ctc?ttc?ccc?cag?gaa?gcc 900
Phe?Ser?Thr?Leu?Asn?Pro?Val?Leu?Arg?His?Leu?Phe?Pro?Gln?Glu?Ala
145 150 155
ttc?cct?gcc?cac?ccc?atc?tat?gac?ttg?agc?cag?gtc?tgg?tcc?gtg?gtg 948
Phe?Pro?Ala?His?Pro?Ile?Tyr?Asp?Leu?Ser?Gln?Val?Trp?Ser?Val?Val
160 165 170 175
tcc?ccc?gca?ccc?agc?agg?gga?cag?gca?ctc?agg?agg?gcc?cgg?taa 993
Ser?Pro?Ala?Pro?Ser?Arg?Gly?Gln?Ala?Leu?Arg?Arg?Ala?Arg?*
180 185
aggctgagat?gaagtggact?gagtagaact?ggaggacagg?agtcgacgtg?agttcctggg?1053
agtctccaga?gatggggcct?ggaggcctgg?aggaaggggc?caggcctcac?attcgtgggg?1113
ctccctgaat?ggcagcctca?gcacagcgta?ggcccttaat?aaacacctgt?tggataagcc?1173
a 1174
 
<210>8
<211>189
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>8
Met?Thr?His?Arg?Thr?Thr?Thr?Trp?Ala?Arg?Arg?Thr?Ser?Arg?Ala?Val
1 5 10 15
Thr?Pro?Thr?Cys?Ala?Thr?Pro?Ala?Gly?Pro?Met?Pro?Cys?Ser?Arg?Leu
20 25 30
Pro?Pro?Ser?Leu?Arg?Cys?Ser?Leu?His?Ser?Ala?Cys?Cys?Ser?Gly?Asp
35 40 45
Pro?Ala?Ser?Tyr?Arg?Leu?Trp?Gly?Ala?Pro?Leu?Gln?Pro?Thr?Leu?Gly
50 55 60
Val?Val?Pro?Gln?Ala?Ser?Val?Pro?Leu?Leu?Thr?His?Pro?Ala?Gln?Trp
65 70 75 80
Glu?Pro?Val?Leu?Val?Pro?Glu?Ala?His?Pro?Asn?Ala?Ser?Leu?Thr?Met
85 90 95
Tyr?Val?Cys?Ala?Pro?Val?Pro?His?Pro?Asp?Pro?Pro?Met?Ala?Leu?Ser
100 105 110
Arg?Thr?Pro?Thr?Arg?Gln?Ile?Gly?Ser?Ile?Asp?Thr?Asp?Pro?Pro?Ala
115 120 125
Asp?Gly?Pro?Ser?Asn?Pro?Leu?Cys?Cys?Cys?Phe?His?Gly?Pro?Ala?Phe
130 135 140
Ser?Thr?Leu?Asn?Pro?Val?Leu?Arg?His?Leu?Phe?Pro?Gln?Glu?Ala?Phe
145 150 155 ?160
Pro?Ala?His?Pro?Ile?Tyr?Asp?Leu?Ser?Gln?Val?Trp?Ser?Val?Val?Ser
165 170 ?175
Pro?Ala?Pro?Ser?Arg?Gly?Gln?Ala?Leu?Arg?Arg?Ala?Arg
180 185
 
<210>9
<211>1660
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<220>
<221>CDS
<222>(910)...(1479)
 
<400>9
gacagtgaac?cctgcgctga?aggcgttggg?gctcctgcag?ttctggggca?gccacaggcg 60
cccagggttt?cgtgccgatc?agcccaggac?ggtcttcccg?gtgcagtttc?tgatgcgggg?120
agggcagtgc?tgccttccgg?tcaccaggac?cagtgctcag?cccgcctgct?tgaccccctt?180
acttagctgg?ggtccaatcc?atacccaatt?tagatgattc?agacgatggg?atttgaaact?240
tttgaactgg?gtgcgactta?agcactgccc?tgctgtgcta?ctcctgcaaa?gcccaggtga?300
gcaacgagga?ctgcctgcag?gtggagaact?gcacccagct?gggggagcag?tgctggaccg?360
cgcgcatccg?tgagtggggg?gacgacagcc?gccaggccta?ggtctctgcc?actgaactat?420
taatctttct?ggccatctgt?ccgcatctgt?gtgctgtttt?ccttccacct?gtccccgacc?480
cgtcccgcac?ctgcaccccc?aacaatcacc?cagcatctgt?ccctccagcc?atcctcctcc?540
atctgccact?cctccactca?tctgtccctc?cccatcctcc?atcttccact?cctccaccca?600
tctgtccctc?cccatccctg?agctcactta?ctcactcacc?ccatttctga?cgctcagcgg?660
gtggtccatc?tgcctcggac?atctggatag?ggctgagacc?agggccgaga?ccaggccctc?720
gcactgcttg?caatcctgag?gccagcccag?ggggactcta?gagcattagg?cagggtggga?780
caggaggagg?cctggggcag?gtcaggcagg?tgagcacaca?gggcagcccc?atccccggat?840
cccgctgctc?cccaggcgca?gttggcctcc?tgaccgtcat?cagcaaaggc?tgcagcttga?900
actgcgtgg?atg?act?cac?agg?act?act?acg?tgg?gca?aga?aga?aca?tca?cgt?951
Met?Thr?His?Arg?Thr?Thr?Thr?Trp?Ala?Arg?Arg?Thr?Ser?Arg
1 5 10
gct?gtg?aca?ccg?act?tgt?gca?acg?cca?gcg?ggg?ccc?atg?ccc?tgc?agc 999
Ala?Val?Thr?Pro?Thr?Cys?Ala?Thr?Pro?Ala?Gly?Pro?Met?Pro?Cys?Ser
15 20 25 30
cgg?ctg?ccg?cca?tcc?ttg?cgc?tgc?tcc?ctg?cac?tcg?gcc?tgc?tgc?tct 1047
Arg?Leu?Pro?Pro?Ser?Leu?Arg?Cys?Ser?Leu?His?Ser?Ala?Cys?Cys?Ser
35 40 45
ggg?gac?ccg?gcc?agc?tat?agg?ctc?tgg?ggg?gcc?ccg?ctg?cag?ccc?aca 1095
Gly?Asp?Pro?Ala?Ser?Tyr?Arg?Leu?Trp?Gly?Ala?Pro?Leu?Gln?Pro?Thr
50 55 ?60
ctg?ggt?gtg?gtg?ccc?cag?gcc?tct?gtg?cca?ctc?ctc?aca?cac?ccg?gcc 1143
Leu?Gly?Val?Val?Pro?Gln?Ala?Ser?Val?Pro?Leu?Leu?Thr?His?Pro?Ala
65 70 75
cag?tgg?gag?cct?gtc?ctg?gtt?cct?gag?gca?cat?cct?aac?gca?agt?ctg 1191
Gln?Trp?Glu?Pro?Val?Leu?Val?Pro?Glu?Ala?His?Pro?Asn?Ala?Ser?Leu
80 85 90
acc?atg?tat?gtc?tgc?gcc?cct?gtc?ccc?cac?cct?gac?cct?ccc?atg?gcc 1239
Thr?Met?Tyr?Val?Cys?Ala?Pro?Val?Pro?His?Pro?Asp?Pro?Pro?Met?Ala
95 100 105 110
ctc?tcc?agg?act?ccc?acc?cgg?cag?atc?ggc?tct?att?gac?aca?gat?ccg 1287
Leu?Ser?Arg?Thr?Pro?Thr?Arg?Gln?Ile?Gly?Ser?Ile?Asp?Thr?Asp?Pro
115 120 125
cct?gca?gat?ggc?ccc?tcc?aac?cct?ctc?tgc?tgc?tgt?ttc?cat?ggc?cca 1335
Pro?Ala?Asp?Gly?Pro?Ser?Asn?Pro?Leu?Cys?Cys?Cys?Phe?His?Gly?Pro
130 135 140
gca?ttc?tcc?acc?ctt?aac?cct?gtg?ctc?agg?cac?ctc?ttc?ccc?cag?gaa 1383
Ala?Phe?Ser?Thr?Leu?Asn?Pro?Val?Leu?Arg?His?Leu?Phe?Pro?Gln?Glu
145 150 155
gcc?ttc?cct?gcc?cac?ccc?atc?tat?gac?ttg?agc?cag?gtc?tgg?tcc?gtg 1431
Ala?Phe?Pro?Ala?His?Pro?Ile?Tyr?Asp?Leu?Ser?Gln?Val?Trp?Ser?Val
160 165 170
gtg?tcc?ccc?gca?ccc?agc?agg?gga?cag?gca?ctc?agg?agg?gcc?cgg?taa 1479
Val?Ser?Pro?Ala?Pro?Ser?Arg?Gly?Gln?Ala?Leu?Arg?Arg?Ala?Arg *
175 180 185
aggctgagat?gaagtggact?gagtagaact?ggaggacagg?agtcgacgtg?agttcctggg 1539
agtctccaga?gatggggcct?ggaggcctgg?aggaaggggc?caggcctcac?attcgtgggg 1599
ctccctgaat?ggcagcctca?gcacagcgta?ggcccttaat?aaacacctgt?tggataagcc?1659
a 1660
 
<210>10
<211>189
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>10
Met?Thr?His?Arg?Thr?Thr?Thr?Trp?Ala?Arg?Arg?Thr?Ser?Arg?Ala?Val
1 5 10 15
Thr?Pro?Thr?Cys?Ala?Thr?Pro?Ala?Gly?Pro?Met?Pro?Cys?Ser?Arg?Leu
20 25 ?30
Pro?Pro?Ser?Leu?Arg?Cys?Ser?Leu?His?Ser?Ala?Cys?Cys?Ser?Gly?Asp
35 40 45
Pro?Ala?Ser?Tyr?Arg?Leu?Trp?Gly?Ala?Pro?Leu?Gln?Pro?Thr?Leu?Gly
50 55 60
Val?Val?Pro?Gln?Ala?Ser?Val?Pro?Leu?Leu?Thr?His?Pro?Ala?Gln?Trp
65 70 75 80
Glu?Pro?Val?Leu?Val?Pro?Glu?Ala?His?Pro?Asn?Ala?Ser?Leu?Thr?Met
85 90 95
Tyr?Val?Cys?Ala?Pro?Val?Pro?His?Pro?Asp?Pro?Pro?Met?Ala?Leu?Ser
100 105 ?110
Arg?Thr?Pro?Thr?Arg?Gln?Ile?Gly?Ser?Ile?Asp?Thr?Asp?Pro?Pro?Ala
115 120 125
Asp?Gly?Pro?Ser?Asn?Pro?Leu?Cys?Cys?Cys?Phe?His?Gly?Pro?Ala?Phe
130 135 140
Ser?Thr?Leu?Asn?Pro?Val?Leu?Arg?His?Leu?Phe?Pro?Gln?Glu?Ala?Phe
145 150 155 160
Pro?Ala?His?Pro?Ile?Tyr?Asp?Leu?Ser?Gln?Val?Trp?Ser?Val?Val?Ser
165 170 ?175
Pro?Ala?Pro?Ser?Arg?Gly?Gln?Ala?Leu?Arg?Arg?Ala?Arg
180 185
 
<210>11
<211>1020
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<220>
<221>CDS
<222>(83)...(427)
 
<400>11
tttgaggcca?tataaagtca?cctgaggccc?tctccaccac?agcccaccag?tgaccatgaa 60
ggctgtgctg?cttgccctgt?tg?atg?gca?ggc?ttg?gcc?ctg?cag?cca?ggc?act 112
Met?Ala?Gly?Leu?Ala?Leu?Gln?Pro?Gly?Thr
1 5 10
gcc?ctg?ctg?tgc?tac?tcc?tgc?aaa?gcc?cag?gtg?agc?aac?gag?gac?tgc 160
Ala?Leu?Leu?Cys?Tyr?Ser?Cys?Lys?Ala?Gln?Val?Ser?Asn?Glu?Asp?Cys
15 20 25
ctg?cag?gtg?gag?aac?tgc?acc?cag?ctg?ggg?gag?cag?tgc?tgg?acc?gcg 208
Leu?Gln?Val?Glu?Asn?Cys?Thr?Gln?Leu?Gly?Glu?Gln?Cys?Trp?Thr?Ala
30 35 ?40
cgc?atc?cgc?gca?gtt?ggc?ctc?ctg?acc?gtc?atc?agc?aaa?ggc?tgc?agc 256
Arg?Ile?Arg?Ala?Val?Gly?Leu?Leu?Thr?Val?Ile?Ser?Lys?Gly?Cys?Ser
45 50 55
ttg?aac?tgc?gtg?gat?gac?tca?cag?gac?tac?tac?gtg?ggc?aag?aag?aac 304
Leu?Asn?Cys?Val?Asp?Asp?Ser?Gln?Asp?Tyr?Tyr?Val?Gly?Lys?Lys?Asn
60 65 70
atc?acg?tgc?tgt?gac?acc?gac?ttg?tgc?aac?gcc?agc?ggg?gcc?cat?gcc 352
Ile?Thr?Cys?Cys?Asp?Thr?Asp?Leu?Cys?Asn?Ala?Ser?Gly?Ala?His?Ala
75 80 85 90
ctg?cag?ccg?gct?gcc?gcc?atc?ctt?gcg?ctg?ctc?cct?gca?ctc?ggc?ctg 400
Leu?Gln?Pro?Ala?Ala?Ala?Ile?Leu?Ala?Leu?Leu?Pro?Ala?Leu?Gly?Leu
95 100 105
ctg?ctc?tgg?gga?ccc?ggc?cag?cta?tag?gctctggggg?gccccgctgc 447
Leu?Leu?Trp?Gly?Pro?Gly?Gln?Leu *
110
agcccacact?gggtgtggtg?ccccaggcct?ctgtgccact?cctcacacac?ccggcccagt?507
gggagcctgt?cctggttcct?gaggcacatc?ctaacgcaag?tctgaccatg?tatgtctgcg?567
cccctgtccc?ccaccctgac?cctcccatgg?ccctctccag?gactcccacc?cggcagatcg?627
gctctattga?cacagatccg?cctgcagatg?gcccctccaa?ccctctctgc?tgctgtttcc?687
atggcccagc?attctccacc?cttaaccctg?tgctcaggca?cctcttcccc?caggaagcct?747
tccctgccca?ccccatctat?gacttgagcc?aggtctggtc?cgtggtgtcc?cccgcaccca?807
gcaggggaca?ggcactcagg?agggcccggt?aaaggctgag?atgaagtgga?ctgagtagaa?867
ctggaggaca?ggagtcgacg?tgagttcctg?ggagtctcca?gagatggggc?ctggaggcct?927
ggaggaaggg?gccaggcctc?acattcgtgg?ggctccctga?atggcagcct?cagcacagcg?987
taggccctta?ataaacacct?gttggataag?cca 1020
 
<210>12
<211>114
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>12
Met?Ala?Gly?Leu?Ala?Leu?Gln?Pro?Gly?Thr?Ala?Leu?Leu?Cys?Tyr?Ser
1 5 10 15
Cys?Lys?Ala?Gln?Val?Ser?Asn?Glu?Asp?Cys?Leu?Gln?Val?Glu?Asn?Cys
20 25 ?30
Thr?Gln?Leu?Gly?Glu?Gln?Cys?Trp?Thr?Ala?Arg?Ile?Arg?Ala?Val?Gly
35 40 45
Leu?Leu?Thr?Val?Ile?Ser?Lys?Gly?Cys?Ser?Leu?Asn?Cys?Val?Asp?Asp
50 55 60
Ser?Gln?Asp?Tyr?Tyr?Val?Gly?Lys?Lys?Asn?Ile?Thr?Cys?Cys?Asp?Thr
65 70 75 80
Asp?Leu?Cys?Asn?Ala?Ser?Gly?Ala?His?Ala?Leu?Gln?Pro?Ala?Ala?Ala
85 90 95
Il?e?Leu?Ala?Leu?Leu?Pro?Ala?Leu?Gly?Leu?Leu?Leu?Trp?Gly?Pro?Gly
100 105 110
Gln?Leu
 
<210>13
<211>148
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>13
Val?Ala?Ala?Pro?Arg?Trp?Val?Leu?Ser?Gln?Val?Gln?Leu?Gln?Glu?Ser
1 5 10 15
Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln?Thr?Leu?Ser?Leu?Thr?Cys?Thr
20 25 30
Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Gly?Gly?Tyr?Tyr?Trp?Ile?Trp?Ile
35 40 45
Arg?Gln?His?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Tyr?Ile?Tyr?Tyr
50 55 60
Asn?Gly?Asn?Thr?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg?Val?Thr?Met
65 70 ?75 80
Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser?Ser?Val
85 90 95
Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Asp?Gly?Ile?Thr
100 105 110
Met?Ile?Arg?Gly?Tyr?Tyr?Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr
115 120 125
Thr?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Lys?Gly
130 135 140
Pro?Ser?Val?Phe
145
 
<210>14
<211>155
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>14
Gln?Leu?Thr?Gln?Ser?Pro?Ser?Ser?Val?Ser?Ala?Ser?Val?Gly?Asp?Arg
1 5 10 15
Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Arg?Gly?Ile?Ser?Ser?Trp?Leu?Ala
20 25 30
Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Thr
35 40 45
Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly
50 55 ?60
Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp
65 70 75 80
PheAla?Thr?Tyr?Tyr?Cys?Gln?Gln?Ala?Tyr?Ser?Phe?Pro?Arg?Thr?Phe
85 90 95
Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser
100 105 ?110
Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?AIa
115 120 125
Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val
130 135 140
Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly
145 150 155
 
<210>15
<211>136
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>15
Gln?Cys?Val?Ala?Ala?Pro?Arg?Trp?Val?Leu?Ser?Gln?Val?Gln?Leu?Gln
1 5 10 15
Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln?Thr?Leu?Ser?Leu?Thr
20 25 30
Cys?Thr?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Gly?Gly?Tyr?Tyr?Trp?Ser
35 40 ?45
Trp?Ile?Arg?Gln?His?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Tyr?Ile
50 55 ?60
Tyr?Tyr?Ser?Gly?Ser?Thr?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg?Val
65 70 75 80
Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser
85 90 95
Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Asp?Arg
100 105 ?110
Ile?Thr?Met?Val?Arg?Gly?Gly?Ile?Pro?Ser?Gly?Met?Asp?Val?Trp?Gly
115 120 ?125
Gln?Gly?Thr?Thr?Val?Thr?Val?Ser
130 135
 
<210>16
<211>139
<212>PRT
 
< 213>homo sapiens (Homo sapiens)
 
<400>16
Ser?Pro?Phe?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Thr?Asn?Tyr?Leu?Asn
1 5 10 15
Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Glu?Ala?Pro?Lys?Leu?Leu?Ile?His?Val
20 25 30
Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly
35 40 ?45
Ser?Gly?Arg?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp
50 55 ?60
Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?His?Ser?Ile?Pro?Arg?Thr?Phe
65 70 75 80
Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser
85 90 95
Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala
100 105 110
Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val
115 120 125
Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly
130 135
 
<210>17
<211>138
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>17
Gln?Val?His?Leu?Gln?Gln?Trp?Gly?Ala?Gly?Leu?Leu?Lys?Pro?Ser?Glu
1 5 10 15
Thr?Leu?Ser?Leu?Thr?Cys?Ala?Val?Tyr?Gly?Gly?Ser?Phe?Ser?Gly?Tyr
20 25 30
Tyr?Trp?Ser?Trp?IleArg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile
35 40 ?45
Gly?Glu?Ile?Asn?His?Ser?Gly?Ser?Thr?Ser?Tyr?Lys?Pro?Ser?Leu?Lys
50 55 60
Ser?Arg?Val?Thr?Val?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu
65 70 75 80
Lys?Leu?Ser?Tyr?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala
85 90 95
Arg?Asp?Arg?Gly?Asp?Tyr?Gly?Asp?Phe?Leu?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val
115 120 125
Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
130 135
 
<210>18
<211>141
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>18
Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Ile
1 5 10 15
Gly?Ser?Thr?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro
20 25 ?30
Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Ser?Arg?Ala?Thr?Gly?Ile?Pro?Glu
35 40 ?45
Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser
50 55 ?60
Gly?Leu?Glu?Pro?Glu?Asp?Phe?Ala?Val?Phe?Tyr?Cys?Gln?Gln?Cys?Gly
65 70 ?75 80
Ser?Ser?Pro?Pro?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
85 90 95
Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln
100 105 110
Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr
115 120 125
Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala
130 135 140
 
<210>19
<211>139
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>19
Gly?Leu?Gln?Cys?Val?Ala?Ala?Pro?Arg?Trp?Val?Leu?Ser?Gln?Val?Gln
1 5 10 15
Leu?Gln?Gln?Trp?Gly?Ala?Gly?Leu?Leu?Lys?Pro?Ser?Glu?Thr?Leu?Ser
20 25 30
Leu?Thr?Cys?Ala?Val?Tyr?Gly?Gly?Ser?Phe?Ser?Gly?Asn?Tyr?Trp?Ser
35 40 45
Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Glu?Ile
50 55 60
Asn?His?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg?Val
65 70 75 80
Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser
85 90 95
Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Gly?Gly
100 105 110
Ser?Tyr?Asn?Tyr?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val
115 120 125
Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Lys
130 135
 
<210>20
<211>236
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>20
Met?Asp?Met?Arg?Val?Pro?Ala?Gln?Leu?Leu?Gly?Leu?Leu?Leu?Leu?Trp
1 5 10 15
Leu?Ser?Gly?Ala?Arg?Cys?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser
20 25 30
Leu?Ser?Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Gln?Ala?Ser
35 40 45
Gln?Asp?Ile?Ser?Asn?Tyr?Leu?Asn?Trp?Tyr?Gln?Gln?Ser?Pro?Gly?Lys
50 55 60
Ala?Pro?Lys?Phe?Leu?Ile?Ser?Asp?Ala?Ser?Asn?Leu?Lys?Thr?Gly?Val
65 70 75 80
Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Ser?Phe?Thr
85 90 95
Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Ile?Ala?Thr?Tyr?Cys?Cys?Gln?Gln
100 105 110
Tyr?Asp?Ser?Leu?Pro?Phe?Thr?Phe?Gly?Pro?Gly?Thr?Lys?Val?Asp?Ile
115 120 125
Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp
130 135 140
Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn
145 150 155 160
Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu
165 170 175
Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp
180 185 190
Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr
195 200 205
Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser
210 215 220
Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
225 230 235
 
<210>21
<211>239
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>21
Met?Arg?Leu?Pro?Ala?Gln?Leu?Leu?Gly?Leu?Leu?Met?Leu?Trp?Val?Ser
1 5 10 15
Gly?Ser?Ser?Gly?Asp?Ile?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro
20 25 30
Val?Thr?Pro?Gly?Glu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser
35 40 45
Leu?Leu?His?Ser?Asn?Gly?Tyr?Asn?Tyr?Leu?Val?Trp?Tyr?Leu?Gln?Lys
50 55 60
Pro?Gly?Gln?Ser?Pro?Gln?Leu?Leu?Ile?Tyr?Leu?Gly?Ser?Ile?Arg?Ala
65 70 75 80
Ser?Gly?Val?Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe
85 90 95
Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr
100 105 ?110
Cys?Met?Gln?Pro?Leu?Gln?Thr?Pro?Ile?Thr?Phe?Gly?Gln?Gly?Thr?Arg
115 120 ?125
Leu?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro
130 135 140
Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu
145 150 155 160
Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp
165 170 175
Asn?Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp
180 185 190
Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys
195 200 ?205
Ala?Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln
210 215 220
Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
225 230 235
 
<210>22
<211>141
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>22
Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln
1 5 10 15
Thr?Leu?Ser?Leu?Thr?Cys?Ala?Ile?Ser?Gly?Asp?Ser?Val?Ser?Ser?Asn
20 25 30
Ser?Ala?Ala?Trp?Asn?Trp?Ile?Arg?Gln?Ser?Pro?Ser?Arg?Gly?Leu?Glu
35 40 45
Trp?Leu?Gly?Arg?Thr?Tyr?Tyr?Arg?Ser?Lys?Trp?Tyr?Asn?Gly?Tyr?Ala
50 55 60
Val?Ser?Val?Lys?Ser?Arg?Met?Thr?Ile?Asn?Pro?Asp?Thr?Ser?Lys?Asn
65 70 75 80
Gln?Phe?Ser?Leu?Gln?Leu?Asn?Ser?Val?Thr?Pro?Glu?Asp?Thr?Ala?Val
85 90 95
Tyr?Tyr?Cys?Ala?Arg?Glu?Arg?Leu?Gly?Glu?Leu?Tyr?Gly?Met?Asp?Val
100 105 110
Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly
115 120 125
Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
130 135 140
 
<210>23
<211>161
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>23
Gln?Leu?Thr?Gln?Ser?Pro?Asp?Ser?Leu?Ala?Val?Ser?Leu?Gly?Glu?Arg
1 5 10 ?15
Ala?Thr?Ile?Asn?Cys?Lys?Ser?Ser?Gln?Asn?Ile?Leu?Tyr?Arg?Ser?Ser
20 25 30
Lys?Lys?Asn?His?Leu?Val?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro
35 40 45
Lys?Leu?Leu?Ile?Tyr?Trp?Ala?Ser?Thr?Arg?Glu?Ser?Gly?Val?Pro?Ala
50 55 ?60
Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser
65 70 ?75 80
Thr?Leu?Gln?Ala?Glu?Asp?Val?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Tyr
85 90 95
Ser?Thr?Pro?Pro?Thr?Phe?Gly?Gln?Gly?Thr?Arg?Leu?Glu?Ile?Lys?Arg
100 105 110
Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln
115 120 125
Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr
130 135 140
Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser
145 150 155 160
Gly
 
<210>24
<211>137
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<220>
<221>VARIANT
<222>(0)...(0)
< 223>any amino acid of Xaa=
 
<400>24
Gly?Pro?Gly?Xaa?Xaa?Lys?Pro?Ser?Gln?Xaa?Leu?Ser?Leu?Thr?Gly?Thr
1 5 10 15
Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Gly?Gly?Tyr?Tyr?Trp?Ser?Trp?Ile
20 25 ?30
Arg?Gln?His?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Asn?Ile?Tyr?Tyr
35 40 ?45
Ser?Gly?Ser?Thr?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg?Val?Thr?Ile
50 55 ?60
Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser?Ala?Val
65 70 75 ?80
Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Asp?Asn?Ile?Thr
85 90 95
Met?Val?Arg?Gly?Val?Tyr?Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr
100 105 110
Thr?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro
115 120 125
Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Tyr
130 135
<210>25
<211>107
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>25
Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
1 5 10 15
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
20 25 ?30
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ala?His?Ser?Leu?Pro?Arg
35 40 45
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala
50 55 ?60
Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly
65 70 75 80
Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Lys?Ala
85 90 95
Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Thr?Leu?Gln
100 105
 
<210>26
<211>144
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>26
Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?Arg
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Arg?His
20 25 30
Gly?Val?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ala?Val?Ile?Trp?Tyr?Asp?Gly?Ser?Asn?Lys?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 ?60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Arg?Gly?Gly?Leu?Ile?Ala?Val?Arg?Pro?Gly?Tyr?Tyr?Tyr?Tyr?Gly
100 105 110
Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala?Ser
115 120 125
Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
130 135 140
 
<210>27
<211>157
<212>PRT
< 213>homo sapiens (Homo sapiens)
<400>27
Glu?Met?Gln?Leu?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Ser?Tyr
20 25 ?30
LeuAsn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?Tyr?Ser?Thr?Pro?Leu
85 90 95
Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala
100 105 ?110
ProSer?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly
115 120 125
Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala
130 135 140
Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly
145 150 155
 
<210>28
<211>141
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>28
Cys?Pro?Gly?Ala?Leu?Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Arg?Pro?Ser
1 5 10 15
Gln?Thr?Leu?Ser?Leu?Thr?Cys?Thr?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser
20 25 30
Gly?Gly?Thr?Tyr?Tyr?Trp?Ile?Trp?Ile?Arg?Gln?His?Pro?Gly?Lys?Gly
35 40 45
Leu?Glu?Trp?Ile?Gly?Tyr?Ile?Tyr?Tyr?Ser?Gly?Ser?Thr?Tyr?Tyr?Asn
50 55 ?60
Pro?Ser?Leu?Lys?Ser?Arg?Val?Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn
65 70 75 80
Gln?Phe?Ser?Leu?Lys?Leu?Ser?Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val
85 90 95
Tyr?Tyr?Cys?Ala?Arg?Asp?Gly?Ile?Thr?Met?Val?Arg?Gly?Ile?Ser?Gly
100 105 ?110
Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala
115 120 125
Ser?Thr?Lys?Gly?Pro?Ser?Val?Lys?Gly?Pro?Ser?Val?Phe
130 135 140
 
<210>29
<211>145
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>29
Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser
1 5 10 15
Ile?Ser?Ser?His?Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro
20 25 30
Lys?Leu?Leu?Ile?Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser
35 40 45
Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Ser?Ile?Ser
50 55 60
Ser?Leu?Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Phe?Cys?Gln?Gln?Ser?Tyr
65 70 75 80
Ser?Ile?Pro?Arg?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Thr?Arg
85 90 ?95
Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln
100 105 110
Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr
115 120 125
Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser
130 135 140
Gly
145
 
<210>30
<211>139
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>30
Gln?Leu?Gln?Gln?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln?Thr?Leu
1 5 10 15
Ser?Leu?Thr?Cys?Ala?Ile?Ser?Gly?Asp?Ser?Val?Ser?Ser?Asn?Ser?Ala
20 25 30
Ala?Trp?Asn?Trp?Ile?Arg?Gln?Ser?Pro?Ser?Arg?Gly?Leu?Glu?Trp?Leu
35 40 45
Gly?Arg?Thr?Tyr?Tyr?Arg?Ser?Lys?Trp?Tyr?Asn?Glu?Tyr?Ala?Val?Ser
50 55 60
Val?Lys?Ser?Arg?Met?Thr?Ile?Asn?Pro?Asp?Thr?Ser?Lys?Asn?Gln?Phe
65 70 75 80
Ser?Leu?Gln?Leu?Asn?Ser?Val?Thr?Pro?Glu?Asp?Thr?Ala?Val?Tyr?Tyr
85 90 95
Cys?Ala?Arg?Glu?Arg?Leu?Gly?Glu?Leu?Tyr?Gly?Met?Asp?Val?Trp?Gly
100 105 110
Gln?Gly?Thr?Met?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser
115 120 125
Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
130 135
<210>31
<211>128
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>31
Leu?Gln?Val?Gln?Pro?Glu?Cys?Leu?Tyr?Thr?Val?Ser?Asp?Lys?Asn?Asn
1 5 10 15
Phe?Leu?Cys?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro?Lys?Leu?Leu
20 25 30
Met?Tyr?Trp?Ala?Ser?Ile?Arg?Glu?Ser?Gly?Val?Pro?Asp?Arg?Phe?Ser
35 40 45
Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln
50 55 60
Ala?Glu?Asp?Val?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Tyr?Ser?Thr?Pro
65 70 75 80
Pro?Thr?Phe?Gly?Gln?Gly?Thr?Arg?Leu?Glu?Ile?Lys?Arg?Thr?Val?Ala
85 90 95
Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser
100 105 ?110
Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu
115 120 125
 
<210>32
<211>141
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>32
Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln
1 5 10 15
Thr?Leu?Ser?Leu?Thr?Cys?Ala?Ile?Ser?Gly?Asp?Ser?Val?Ser?Ser?Asn
20 25 30
Ser?Ala?Ala?Trp?Asn?Trp?Ile?Arg?Gln?Ser?Pro?Trp?Arg?Gly?Leu?Glu
35 40 45
Trp?Leu?Gly?Arg?Thr?Tyr?Tyr?Arg?Ser?Lys?Trp?Tyr?Asn?Glu?Tyr?Ala
50 55 60
Val?Ser?Val?Lys?Ser?Arg?Met?Thr?Ile?Asn?Pro?Asp?Thr?Ser?Lys?Asn
65 70 75 80
Gln?Phe?Ser?Leu?Gln?Leu?Asn?Ser?Val?Thr?Pro?Glu?Asp?Thr?Ala?Val
85 90 95
Tyr?Tyr?Cys?Ala?Arg?Glu?Arg?Leu?Gly?Glu?Leu?Tyr?Gly?Met?Asp?Val
100 105 110
Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly
115 120 125
Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
130 135 140
 
<210>33
<211>162
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>33
Met?Gln?Leu?Thr?Gln?Ser?Pro?Asp?Ser?Leu?Ala?Val?Ser?Leu?Gly?Glu
1 5 10 ?15
Arg?Ala?Thr?Ile?Asn?Cys?Lys?Ser?Ser?Gln?Asn?Val?Leu?Tyr?Arg?Ser
20 25 30
Asn?Lys?Lys?Asn?Phe?Leu?Val?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Pro
35 40 ?45
Pro?Lys?Leu?Leu?Ile?Tyr?Trp?Ala?Ser?Ile?Arg?Glu?Ser?Gly?Val?Pro
50 55 ?60
Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile
65 70 75 80
Ser?Ser?Leu?Gln?Thr?Glu?Asp?Val?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr
85 90 95
Tyr?Ser?Thr?Pro?Pro?Thr?Phe?Gly?Gln?Gly?Thr?Arg?Leu?Glu?Ile?Lys
100 105 110
Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu
115 120 125
Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe
130 135 140
Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln
145 150 155 160
Ser?Gly
 
<210>34
<211>445
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>34
gtggcagcac?ccagatgggt?cctgtcccag?gtgcagctgc?aggagtcggg?cccaggactg 60
gtgaagcctt?cacagaccct?gtccctcacc?tgcactgtct?ctggtggctc?catcagcagt?120
ggtggttact?actggatctg?gatccgccag?cacccaggga?agggcctgga?gtggattggg?180
tacatctatt?acaatgggaa?cacctactac?aacccgtccc?tcaagagtcg?agttaccatg?240
tcagtagaca?cgtctaagaa?ccagttctcc?ctgaagctga?gctctgtgac?tgccgcggac?300
acggccgtgt?attactgtgc?gagagatggt?attactatga?tacgcggcta?ctactacggt?360
atggacgtct?ggggccaagg?gaccacggtc?accgtctcct?cagcctccac?caagggccca?420
tcggtcaagg?gcccatcggt?cttca 445
 
<210>35
<211>148
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>35
Val?Ala?Ala?Pro?Arg?Trp?Val?Leu?Ser?Gln?Val?Gln?Leu?Gln?Glu?Ser
1 5 10 15
Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln?Thr?Leu?Ser?Leu?Thr?Cys?Thr
20 25 30
Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Gly?Gly?Tyr?Tyr?Trp?Ile?Trp?Ile
35 40 ?45
Arg?Gln?His?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Tyr?Ile?Tyr?Tyr
50 55 60
Asn?Gly?Asn?Thr?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg?Val?Thr?Met
65 70 75 80
Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser?Ser?Val
85 90 95
Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Asp?Gly?Ile?Thr
100 105 110
Met?Ile?Arg?Gly?Tyr?Tyr?Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr
115 120 ?125
Thr?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Lys?Gly
130 135 140
Pro?Ser?Val?Phe
145
 
<210>36
<211>466
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<220>
<221>misc_feature
<222>(0)...(0)
< 223>n=a, t, c or g
 
<400>36
cagctgacnc?agtctccatc?ttccgtgtct?gcatctgtag?gagacagagt?caccatcact 60
tgtcgggcga?gtcggggtat?tagcagctgg?ttagcctggt?atcagcagaa?accagggaaa?120
gcccctaagc?tcctgatcta?tactgcatcc?agtttgcaaa?gtggagtccc?atcaaggttc?180
agcggcagtg?gttctgggac?agatttcact?ctcaccatca?gcagcctgca?gcctgaagat?240
tttgcaactt?actattgtca?acaggcttac?agtttccctc?ggacgttcgg?ccaagggacc?300
aaggtggaaa?tcaaacgaac?tgtggctgca?ccatctgtct?tcatcttccc?gccatctgat?360
gagcagttga?aatctggaac?tgcctctgtt?gtgtgcctgc?tgaataactt?ctatcccaga?420
gaggccaaag?tacagtggaa?ggtggataac?gccctccaat?cgggta 466
 
<210>37
<211>155
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>37
Gln?Leu?Thr?Gln?Ser?Pro?Ser?Ser?Val?Ser?Ala?Ser?Val?Gly?Asp?Arg
1 5 10 15
Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Arg?Gly?Ile?Ser?Ser?Trp?Leu?Ala
20 25 30
Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Thr
35 40 45
Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly
50 55 ?60
Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp
65 70 75 80
Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ala?Tyr?Ser?Phe?Pro?Arg?Thr?Phe
85 90 95
Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser
100 105 110
Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala
115 120 125
Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val
130 135 140
Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly
145 150 155
 
<210>38
<211>409
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>38
cagtgtgtgg?cagcacccag?atgggtcctg?tcccaggtgc?agctgcagga?gtcgggccca 60
ggactggtga?agccttcaca?gaccctgtcc?ctcacctgca?ctgtctctgg?tggctccatc?120
agcagtggtg?gttactactg?gagctggatc?cgccagcacc?cagggaaggg?cctggagtgg?180
attgggtaca?tatattacag?tgggagcacc?tactacaacc?cgtccctcaa?gagtcgagtt?240
accatatcag?tagacacgtc?caagaaccag?ttctccctga?agctgagctc?tgtgactgcc?300
gcggacacgg?ccgtgtatta?ctgtgcgaga?gatcgaatta?ctatggttcg?gggaggtatt?360
cccagtggta?tggacgtctg?gggccaaggg?accacggtca?ccgtctcct 409
 
<210>39
<211>136
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>39
Gln?Cys?Val?Ala?Ala?Pro?Arg?Trp?Val?Leu?Ser?Gln?Val?Gln?Leu?Gln
1 5 10 15
Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln?Thr?Leu?Ser?Leu?Thr
20 25 30
Cys?Thr?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Gly?Gly?Tyr?Tyr?Trp?Ser
35 40 ?45
Trp?Ile?Arg?Gln?His?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Tyr?Ile
50 55 ?60
Tyr?Tyr?Ser?Gly?Ser?Thr?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg?Val
65 70 75 80
Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser
85 90 95
Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Asp?Arg
100 105 110
Ile?Thr?Met?Val?Arg?Gly?Gly?Ile?Pro?Ser?Gly?Met?Asp?Val?Trp?Gly
115 120 125
Gln?Gly?Thr?Thr?Val?Thr?Val?Ser
130 135
 
<210>40
<211>417
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>40
tcaccattca?cttgccgggc?aagtcagagc?attaccaact?atttaaattg?gtatcagcag 60
aaaccagggg?aagcccctaa?gctcctgatc?catgttgcat?ccagtttgca?aagtggggtc?120
ccatcaaggt?tcagtggcag?tggatctggg?agagatttca?ctctcaccat?cagcagtctg?180
caacctgaag?attttgcaac?ttactactgt?caacagagtc?acagtatccc?tcggacgttc?240
ggccaaggga?ccaaggtgga?aatcaaacga?actgtggctg?caccatctgt?cttcatcttc?300
ccgccatctg?atgagcagtt?gaaatctgga?actgcctctg?ttgtgtgcct?gctgaataac?360
ttctatccca?gagaggccaa?agtacagtgg?aaggtggata?acgccctcca?atcgggt 417
 
<210>41
<211>139
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>41
Ser?Pro?Phe?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Thr?Asn?Tyr?Leu?Asn
1 5 10 ?15
Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Glu?Ala?Pro?Lys?Leu?Leu?Ile?His?Val
20 25 30
Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly
35 40 45
Ser?Gly?Arg?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp
50 55 60
Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?His?Ser?Ile?Pro?Arg?Thr?Phe
65 70 75 80
Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser
85 90 95
Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala
100 105 ?110
Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val
115 120 125
Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly
130 135
 
<210>42
<211>416
<212>DNA
< 213>homo sapiens (Homo sapiens)
<400>42
caggtgcatc?tacagcagtg?gggcgcagga?ctgttgaagc?cttcggagac?cctgtccctc 60
acctgcgctg?tctatggtgg?gtccttcagt?ggttactact?ggagctggat?ccgccagccc?120
ccggggaagg?gactggagtg?gattggggaa?atcaatcata?gtggaagcac?cagctacaag?180
ccgtccctca?agagtcgagt?caccgtatca?gtggacacgt?ccaagaacca?gttctccctg?240
aagctgagct?atgtgaccgc?cgcggacacg?gctgtgtatt?actgtgcgag?agataggggt?300
gactacggtg?acttcctctt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcctca?360
gcctccacca?agggcccatc?ggtcttcccc?ctggcaccct?cctccaagag?caccta 416
 
<210>43
<211>138
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>43
Gln?Val?His?Leu?Gln?Gln?Trp?Gly?Ala?Gly?Leu?Leu?Lys?Pro?Ser?Glu
1 5 10 15
Thr?Leu?Ser?Leu?Thr?Cys?Ala?Val?Tyr?Gly?Gly?Ser?Phe?Ser?Gly?Tyr
20 25 30
Tyr?Trp?Ser?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile
35 40 45
Gly?Glu?Ile?Asn?His?Ser?Gly?Ser?Thr?Ser?Tyr?Lys?Pro?Ser?Leu?Lys
50 55 60
Ser?Arg?Val?Thr?Val?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu
65 70 75 80
Lys?Leu?Ser?Tyr?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala
85 90 95
Arg?Asp?Arg?Gly?Asp?Tyr?Gly?Asp?Phe?Leu?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val
115 120 125
Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
130 135
 
<210>44
<211>425
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>44
tctccagggg?aaagagccac?cctctcctgc?agggccagtc?agagtattgg?cagcacctac 60
ttagcctggt?accagcagaa?acctggccag?gctcccaggc?tcctcatcta?tggtgcatcc?120
agcagggcca?ctggcatccc?agaaaggttc?agtggcagtg?ggtctgggac?agacttcact?180
ctcaccatca?gcggactgga?gcctgaagat?tttgcagtgt?tttactgtca?acagtgtggt?240
agctcacctc?cgacgttcgg?ccaagggacc?aaggtggaaa?tcaaacgaac?tgtggctgca?300
ccatctgtct?tcatcttccc?gccatctgat?gagcagttga?aatctggaac?tgcctctgtt?360
gtgtgcctgc?tgaataactt?ctatcccaga?gaggccaaag?tacagtggaa?ggtggataac?420
gccct 425
 
<210>45
<211>141
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>45
Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Ile
1 5 10 15
Gly?Ser?Thr?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro
20 25 ?30
Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Ser?Arg?Ala?Thr?Gly?Ile?Pro?Glu
35 40 45
Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser
50 55 60
Gly?Leu?Glu?Pro?Glu?Asp?Phe?Ala?Val?Phe?Tyr?Cys?Gln?Gln?Cys?Gly
65 70 75 80
Ser?Ser?Pro?Pro?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
85 90 95
Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln
100 105 ?110
Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr
115 120 125
Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala
130 135 140
 
<210>46
<211>417
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>46
ggtctgcagt?gtgtggcagc?acccagatgg?gtcctgtccc?aggtgcagct?acagcagtgg 60
ggcgcaggac?tgttgaagcc?ttcggagacc?ctgtccctca?cctgcgctgt?ctatggtggg?120
tccttcagtg?gtaactactg?gagctggatc?cgccagcccc?cagggaaggg?gctggagtgg?180
attggggaaa?tcaatcatag?tggaagcacc?aactacaacc?cgtccctcaa?gagtcgagtc?240
accatatcag?tagacacgtc?caagaaccag?ttctccctga?agctgagctc?tgtgaccgcc?300
gcggacacgg?ctgtgtatta?ctgtgcgaga?ggggggagct?acaactactt?tgactactgg?360
ggccagggaa?ccctggtcac?cgtctcctca?gcctccacca?agggcccatc?ggtcaag 417
 
<210>47
<211>139
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>47
Gly?Leu?Gln?Cys?Val?Ala?Ala?Pro?Arg?Trp?Val?Leu?Ser?Gln?Val?Gln
1 5 10 15
Leu?Gln?Gln?Trp?Gly?Ala?Gly?Leu?Leu?Lys?Pro?Ser?Glu?Thr?Leu?Ser
20 25 30
Leu?Thr?Cys?Ala?Val?Tyr?Gly?Gly?Ser?Phe?Ser?Gly?Asn?Tyr?Trp?Ser
35 40 45
Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Glu?Ile
50 55 60
Asn?His?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg?Val
65 70 75 80
Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser
85 90 95
Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Gly?Gly
100 105 110
Ser?Tyr?Asn?Tyr?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val
115 120 125
Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Lys
130 135
 
<210>48
<211>933
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>48
aaaaaagtca?gtcccaacca?ggacacagca?tggacatgag?ggtccctgct?cagctcctgg 60
ggctcctgct?gctctggctc?tcaggtgcca?gatgtgacat?ccagatgact?cagtctccat?120
cctccctgtc?tgcatctgta?ggagacagag?tcaccatcac?ttgccaggcg?agtcaggaca?180
ttagcaacta?tttgaattgg?tatcagcaga?gtcccgggaa?agcccctaag?ttcctgatct?240
ccgatgcatc?caatttaaaa?acaggggtcc?catcaaggtt?cagtggaagt?ggatctggga?300
cagatttttc?tttcaccatc?agcagcctac?agcctgaaga?tattgcgacc?tattgctgtc?360
aacagtatga?tagtctccca?ttcactttcg?gccctgggac?caaagtggat?atcaaacgaa?420
ctgtggctgc?accatctgtc?ttcatcttcc?cgccatctga?tgagcagttg?aaatctggaa?480
ctgcctctgt?tgtgtgcctg?ctgaataact?tctatcccag?agaggccaaa?gtacagtgga?540
aggtggataa?cgccctccaa?tcgggtaact?cccaggagag?tgtcacagag?caggacagca?600
aggacagcac?ctacagcctc?agcagcaccc?tgacgctgag?caaagcagac?tacgagaaac?660
acaaagtcta?cgcctgcgaa?gtcacccatc?agggcctgag?ctcgcccgtc?acaaagagct?720
tcaacagggg?agagtgttag?agggagaagt?gcccccacct?gctcctcagt?tccagcctga?780
ccccctccca?tcctttggcc?tctgaccctt?tttccacagg?ggacctaccc?ctattgcggt?840
cctccagctc?atctttcacc?tcacccccct?cctcctcctt?ggctttaatt?atgctaatgt?900
tggaggagaa?tgaataaata?aagtgaatct?ttg 933
 
<210>49
<211>236
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>49
Met?Asp?Met?Arg?Val?Pro?Ala?Gln?Leu?Leu?Gly?Leu?Leu?Leu?Leu?Trp
1 5 10 ?15
Leu?Ser?Gly?Ala?Arg?Cys?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser
20 25 ?30
Leu?Ser?Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Gln?Ala?Ser
35 40 45
Gln?Asp?Ile?Ser?Asn?Tyr?Leu?Asn?Trp?Tyr?Gln?Gln?Ser?Pro?Gly?Lys
50 55 60
Ala?Pro?Lys?Phe?Leu?Ile?Ser?Asp?Ala?Ser?Asn?Leu?Lys?Thr?Gly?Val
65 70 ?75 80
Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Ser?Phe?Thr
85 90 95
Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Ile?Ala?Thr?Tyr?Cys?Cys?Gln?Gln
100 105 ?110
Tyr?Asp?Ser?Leu?Pro?Phe?Thr?Phe?Gly?Pro?Gly?Thr?Lys?Val?Asp?Ile
115 120 125
Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp
130 135 140
Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn
145 150 155 160
Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu
165 170 175
Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp
180 185 ?190
Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr
195 200 205
Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser
210 215 220
Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
225 230 235
 
<210>50
<211>968
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>50
aaagatcagg?actcctcagt?tcaccttctc?acaatgaggc?tccctgctca?gctcctgggg 60
ctgctaatgc?tctgggtctc?tggatccagt?ggggatattg?tgatgactca?gtctccactc?120
tccctgcccg?tcacccctgg?agagccggcc?tccatctcct?gcaggtctag?tcagagcctc?180
ctacatagta?atggatacaa?ctatttggtt?tggtacctgc?agaagccagg?acagtctcca?240
cagctcctga?tctatttggg?ttctattcgg?gcctccgggg?tccctgacag?gttcagtggc?300
agtggatcag?gcacagattt?tacactgaaa?atcagcagag?tggaggctga?ggatgttggg?360
gtttattact?gcatgcaacc?tctacaaact?ccgatcacct?tcggccaagg?gacacgactg?420
gagattaaac?gaactgtggc?tgcaccatct?gtcttcatct?tcccgccatc?tgatgagcag?480
ttgaaatctg?gaactgcctc?tgttgtgtgc?ctgctgaata?acttctatcc?cagagaggcc?540
aaagtacagt?ggaaggtgga?taacgccctc?caatcgggta?actcccagga?gagtgtcaca?600
gagcaggaca?gcaaggacag?cacctacagc?ctcagcagca?ccctgacgct?gagcaaagca?660
gactacgaga?aacacaaagt?ctacgcctgc?gaagtcaccc?atcagggcct?gagctcgccc?720
gtcacaaaga?gcttcaacag?gggagagtgt?tagagggaga?agtgccccca?cctgctcctc?780
agttccagcc?tgaccccctc?ccatcctttg?gcctctgacc?ctttttccac?aggggaccta?840
cccctattgc?ggtcctccag?ctcatctttc?acctcacccc?cctcctcctc?cttggcttta?900
attatgctaa?tgttggagga?gaatgaataa?ataaagtgaa?tctttgcaaa?aaaaaaaaaa?960
aaaaaaaa 968
 
<210>51
<211>239
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>51
Met?Arg?Leu?Pro?Ala?Gln?Leu?Leu?Gly?Leu?Leu?Met?Leu?Trp?Val?Ser
1 5 10 15
Gly?Ser?Ser?Gly?Asp?Ile?Val?Met?Thr?Gln?Ser?Pro?Leu?Ser?Leu?Pro
20 25 30
Val?Thr?Pro?Gly?Glu?Pro?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Gln?Ser
35 40 ?45
Leu?Leu?His?Ser?Asn?Gly?Tyr?Asn?Tyr?Leu?Val?Trp?Tyr?Leu?Gln?Lys
50 55 60
Pro?Gly?Gln?Ser?Pro?Gln?Leu?Leu?Ile?Tyr?Leu?Gly?Ser?Ile?Arg?Ala
65 70 75 80
Ser?Gly?Val?Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe
85 90 95
Thr?Leu?Lys?Ile?Ser?Arg?Val?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr
100 105 110
Cys?Met?Gln?Pro?Leu?Gln?Thr?Pro?Ile?Thr?Phe?Gly?Gln?Gly?Thr?Arg
115 120 125
Leu?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro
130 135 140
Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu
145 150 155 160
Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp
165 170 175
Asn?Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp
180 185 190
Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys
195 200 205
Ala?Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln
210 215 220
Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
225 230 235
 
<210>52
<211>425
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>52
caggtacagc?tgcagcagtc?aggtccagga?ctggtgaagc?cctcgcagac?cctctcactc 60
acctgtgcca?tctccgggga?cagtgtctct?agcaacagtg?ctgcttggaa?ctggatcagg?120
cagtccccat?cgagaggcct?tgagtggctg?ggaaggacat?actacaggtc?caagtggtat?180
aatggttatg?cagtatctgt?gaaaagtcga?atgaccatca?acccagacac?atccaagaac?240
cagttctccc?tgcagctgaa?ctctgtgact?cccgaggaca?cggctgtgta?ttactgtgca?300
agagagaggt?taggggagtt?atacggtatg?gacgtctggg?gccaagggac?cacggtcacc?360
gtctcctcag?cctccaccaa?gggcccatcg?gtcttccccc?tggcaccctc?ctccaagagc?420
accta 425
 
<210>53
<211>141
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>53
Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln
1 5 10 15
Thr?Leu?Ser?Leu?Thr?Cys?Ala?Ile?Ser?Gly?Asp?Ser?Val?Ser?Ser?Asn
20 25 30
Ser?Ala?Ala?Trp?Asn?Trp?Ile?Arg?Gln?Ser?Pro?Ser?Arg?Gly?Leu?Glu
35 40 45
Trp?Leu?Gly?Arg?Thr?Tyr?Tyr?Arg?Ser?Lys?Trp?Tyr?Asn?Gly?Tyr?Ala
50 55 ?60
Val?Ser?Val?Lys?Ser?Arg?Met?Thr?Ile?Asn?Pro?Asp?Thr?Ser?Lys?Asn
65 70 75 80
Gln?Phe?Ser?Leu?Gln?Leu?Asn?Ser?Val?Thr?Pro?Glu?Asp?Thr?Ala?Val
85 90 95
Tyr?Tyr?Cys?Ala?Arg?Glu?Arg?Leu?Gly?Glu?Leu?Tyr?Gly?Met?Asp?Val
100 105 110
Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly
115 120 125
Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
130 135 140
 
<210>54
<211>484
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>54
cagctgacgc?agtctccaga?ctccctggct?gtgtctctgg?gcgagagggc?caccatcaac 60
tgcaagtcca?gccagaatat?tttatacagg?tccagcaaga?agaaccactt?agtttggtac?120
cagcagaaac?caggacagcc?tcctaagctg?ctcatttact?gggcatctac?ccgggaatcc?180
ggggtccctg?cccgattcag?tggcagcggg?tctgggacag?atttcactct?caccatcagc?240
accctgcagg?ctgaagatgt?ggcagtttat?tactgtcagc?aatattatag?tactcctccc?300
accttcggcc?aagggacacg?actggagatt?aaacgaactg?tggctgcacc?atctgtcttc?360
atcttcccgc?catctgatga?gcagttgaaa?tctggaactg?cctctgttgt?gtgcctgctg?420
aataacttct?atcccagaga?ggccaaagta?cagtggaagg?tggataacgc?cctccaatcg?480
ggta 484
 
<210>55
<211>161
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>55
Gln?Leu?Thr?Gln?Ser?Pro?Asp?Ser?Leu?Ala?Val?Ser?Leu?Gly?Glu?Arg
1 5 10 15
Ala?Thr?Ile?Asn?Cys?Lys?Ser?Ser?Gln?Asn?Ile?Leu?Tyr?Arg?Ser?Ser
20 25 30
Lys?Lys?Asn?His?Leu?Val?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro
35 40 45
Lys?Leu?Leu?Ile?Tyr?Trp?Ala?Ser?Thr?Arg?Glu?Ser?Gly?Val?Pro?Ala
50 55 ?60
Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser
65 70 75 80
Thr?Leu?Gln?Ala?Glu?Asp?Val?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Tyr
85 90 95
Ser?Thr?Pro?Pro?Thr?Phe?Gly?Gln?Gly?Thr?Arg?Leu?Glu?Ile?Lys?Arg
100 105 110
Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln
115 120 125
Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr
130 135 140
Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser
145 150 155 160
Gly
 
<210>56
<211>409
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<220>
<221>misc_feature
<222>(0)...(0)
< 223>n=a, t, c or g
 
<400>56
ggcccaggac?nggngaagcc?ttcacagacc?tgtccctcac?cggcactgtc?tctggtggcc 60
catcagcagt?ggtggttatt?actggagctg?gatccgccag?cacccaggga?agggcctgga?120
gtggattggg?aacatctatt?acagtgggag?cacctactac?aacccgtccc?tcaagagtcg?180
agttaccata?tcagtagaca?cgtctaagaa?ccagttctcc?ctgaagctga?gcgctgtgac?240
tgccgcggac?acggccgtgt?attactgtgc?gagagataat?attactatgg?ttcggggagt?300
ctactacggt?atggacgtct?ggggccaagg?gaccacggtc?accgtctcct?cagcctccac?360
caagggccca?tcggtcttcc?ccctggcacc?ctcctccaag?agcacctat 409
 
<210>57
<211>136
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<220>
<221>VARIANT
<222>(0)...(0)
< 223>any amino acid of Xaa=
 
<400>57
Ala?Gln?Asp?Xaa?Xaa?Ser?Leu?His?Arg?Pro?Val?Pro?His?Arg?His?Cys
1 5 10 15
Leu?Trp?Trp?Pro?Ile?Ser?Ser?Gly?Gly?Tyr?Tyr?Trp?Ser?Trp?Ile?Arg
20 25 ?30
Gln?His?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Asn?Ile?Tyr?Tyr?Ser
35 40 45
Gly?Ser?Thr?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg?Val?Thr?Ile?Ser
50 55 60
Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser?Ala?Val?Thr
65 70 75 80
Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Asp?Asn?Ile?Thr?Met
85 90 95
Val?Arg?Gly?Val?Tyr?Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr
100 105 110
Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu
115 120 125
Ala?Pro?Ser?Ser?Lys?Ser?Thr?Tyr
130 135
 
<210>58
<211>321
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>58
tatgcagcat?ccagtttgca?aagtggggtc?ccatcaaggt?tcagcggcag?tggatctgga 60
acagacttca?ctctcaccat?cagcagcctg?cagcctgaag?attttgcaac?ttactattgt?120
caacaggctc?acagtctccc?tcggacgttc?ggccaaggga?ccaaggtgga?aatcaaacga?180
actgtggctg?caccatctgt?cttcatcttc?ccgccatctg?atgagcagtt?gaaatctgga?240
actgcctctg?ttgtgtgcct?gctgaataac?ttctatccca?gaaaggccaa?agtacagtgg?300
aaggtggata?acaccctcca?a 321
 
<210>59
<211>107
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>59
Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
1 5 10 15
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
20 25 30
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ala?His?Ser?Leu?Pro?Arg
35 40 45
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala
50 55 60
Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly
65 70 75 80
Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Lys?Ala
85 90 95
Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Thr?Leu?Gln
100 105
 
<210>60
<211>433
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>60
caggtgcagc?tggtggagtc?tgggggaggc?gtggtccagc?ctgggaggtc?cctgagactc 60
tcctgtgcag?cgtccggatt?caccttcagt?cgccatggcg?tgcactgggt?ccgccaggct?120
ccaggcaagg?ggctggagtg?ggtggcagtt?atatggtatg?atggaagtaa?taaatactat?180
gcagactccg?tgaagggccg?attcaccatc?tccagagaca?attccaagaa?cacgctgtat?240
ctgcaaatga?acagcctgag?agccgaggac?acggctgtgt?attactgtgc?gagaggaggc?300
cttatagcag?ttcgtccggg?gtactactac?tacggtatgg?acgtctgggg?ccaagggacc?360
acggtcaccg?tctcctcagc?ctccaccaag?ggcccatcgg?tcttccccct?ggcaccctcc?420
tccaagagca?cct 433
 
<210>61
<211>144
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>61
Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?Arg
1 5 10 ?15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Arg?His
20 25 30
Gly?Val?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ala?Val?Ile?Trp?Tyr?Asp?Gly?Ser?Asn?Lys?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 ?60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Arg?Gly?Gly?Leu?Ile?Ala?Val?Arg?Pro?Gly?Tyr?Tyr?Tyr?Tyr?Gly
100 105 ?110
Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala?Ser
115 120 125
Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
130 135 140
 
<210>62
<211>471
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>62
gaaatgcagc?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?cagagtcacc 60
atcacttgcc?gggcaagtca?gagcattagc?agctatttaa?attggtatca?gcagaaacca?120
gggaaagccc?ctaagctcct?gatctatgct?gcatccagtt?tgcaaagtgg?ggtcccatca?180
aggttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct?240
gaagattttg?caacttacta?ctgtcaacag?agttacagta?ccccgctcac?tttcggcgga?300
gggaccaagg?tggagatcaa?acgaactgtg?gctgcaccat?ctgtcttcat?cttcccgcca?360
tctgatgagc?agttgaaatc?tggaactgcc?tctgttgtgt?gcctgctgaa?taacttctat?420
cccagagagg?ccaaagtaca?gtggaaggtg?gataacgccc?tccaatcggg?t 471
 
<210>63
<211>157
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>63
Glu?Met?Gln?Leu?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 ?15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Ser?Tyr
20 25 30
Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 ?60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?Tyr?Ser?Thr?Pro?Leu
85 90 95
Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala
100 105 ?110
Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly
115 120 125
Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala
130 135 140
Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly
145 150 155
 
<210>64
<211>424
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>64
tgtccaggtg?cactgcagga?gtcgggccca?ggactggtga?ggccttcaca?gaccctgtcc 60
ctcacctgca?ctgtctctgg?tggctccatc?agcagtggtg?gtacttacta?ctggatctgg?120
atccgccagc?acccagggaa?gggcctggag?tggattgggt?acatctatta?cagtgggagc?180
acctactaca?acccgtccct?caagagtcga?gttaccatat?cagtagacac?gtctaagaac?240
cagttctccc?tgaagctgag?ctctgtgact?gccgcggaca?cggccgtgta?ttactgtgcg?300
agagatggaa?ttactatggt?tcggggaatt?agcgggggca?tggacgtctg?gggccaaggg?360
accacggtca?ccgtctcctc?agcctccacc?aagggcccat?cggtcaaggg?cccatcggtc?420
ttca 424
<210>65
<211>141
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>65
Cys?Pro?Gly?Ala?Leu?Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Arg?Pro?Ser
1 5 10 15
Gln?Thr?Leu?Ser?Leu?Thr?Cys?Thr?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser
20 25 30
Gly?Gly?Thr?Tyr?Tyr?Trp?Ile?Trp?Ile?Arg?Gln?His?Pro?Gly?Lys?Gly
35 40 45
Leu?Glu?Trp?Ile?Gly?Tyr?Ile?Tyr?Tyr?Ser?Gly?Ser?Thr?Tyr?Tyr?Asn
50 55 60
Pro?Ser?Leu?Lys?Ser?Arg?Val?Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn
65 70 75 80
Gln?Phe?Ser?Leu?Lys?Leu?Ser?Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val
85 90 95
Tyr?Tyr?Cys?Ala?Arg?Asp?Gly?Ile?Thr?Met?Val?Arg?Gly?Ile?Ser?Gly
100 105 ?110
Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala
115 120 125
Ser?Thr?Lys?Gly?Pro?Ser?Val?Lys?Gly?Pro?Ser?Val?Phe
130 135 140
 
<210>66
<211>436
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>66
gcatctgtag?gagacagagt?caccatcact?tgccgggcaa?gtcagagcat?tagtagtcat 60
ttaaattggt?atcagcagaa?accagggaaa?gcccctaagc?tcctgatcta?tgctgcttcc?120
agtttgcaaa?gtggggtccc?atcaaggttc?agtggcagtg?gatctgggac?agatttcact?180
ctctccatca?gcagtctgca?acctgaagat?tttgcaactt?acttctgtca?acagagttac?240
agtatccctc?ggacgttcgg?ccaagggacc?aaggtggaaa?tcacacgaac?tgtggctgca?300
ccatctgtct?tcatcttccc?gccatctgat?gagcagttga?aatctggaac?tgcctctgtt?360
gtgtgcctgc?tgaataactt?ctatcccaga?gaggccaaag?tacagtggaa?ggtggataac?420
gccctccaat?cgggta 436
 
<210>67
<211>145
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>67
Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser
1 5 10 15
Ile?Ser?Ser?His?Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro
20 25 30
Lys?Leu?Leu?Ile?Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser
35 40 45
Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Ser?Ile?Ser
50 55 60
Ser?Leu?Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Phe?Cys?Gln?Gln?Ser?Tyr
65 70 75 80
Ser?Ile?Pro?Arg?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Thr?Arg
85 90 95
Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln
100 105 ?110
Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr
115 120 125
Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser
130 135 140
Gly
145
 
<210>68
<211>417
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>68
cagctgcagc?agtcaggtcc?aggactggtg?aagccctcgc?agaccctctc?actcacctgt 60
gccatctccg?gggacagtgt?ctctagcaac?agtgctgctt?ggaactggat?caggcagtcc?120
ccatcgagag?gccttgagtg?gctgggaagg?acatactaca?ggtccaagtg?gtataatgaa?180
tatgcagtat?ctgtgaaaag?tcgaatgacc?atcaacccag?acacatccaa?gaaccagttc?240
tccctgcagc?tgaactctgt?gactcccgag?gacacggctg?tgtattactg?tgcaagagag?300
aggttagggg?agttatacgg?tatggacgtc?tggggccaag?ggaccatggt?caccgtctcc?360
tcagcctcca?ccaagggccc?atcggtcttc?cccctggcac?cctcctccaa?gagcacc 417
 
<210>69
<211>139
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>69
Gln?Leu?Gln?Gln?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln?Thr?Leu
1 5 10 ?15
Ser?Leu?Thr?Cys?Ala?Ile?Ser?Gly?Asp?Ser?Val?Ser?Ser?Asn?Ser?Ala
20 25 30
Ala?Trp?Asn?Trp?Ile?Arg?Gln?Ser?Pro?Ser?Arg?Gly?Leu?Glu?Trp?Leu
35 40 45
Gly?Arg?Thr?Tyr?Tyr?Arg?Ser?Lys?Trp?Tyr?Asn?Glu?Tyr?Ala?Val?Ser
50 55 60
Val?Lys?Ser?Arg?Met?Thr?Ile?Asn?Pro?Asp?Thr?Ser?Lys?Asn?Gln?Phe
65 70 75 80
Ser?Leu?Gln?Leu?Asn?Ser?Val?Thr?Pro?Glu?Asp?Thr?Ala?Val?Tyr?Tyr
85 90 95
Cys?Ala?Arg?Glu?Arg?Leu?Gly?Glu?Leu?Tyr?Gly?Met?Asp?Val?Trp?Gly
100 105 110
Gln?Gly?Thr?Met?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser
115 120 125
Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
130 135
 
<210>70
<211>384
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>70
ctgcaagtcc?agccagagtg?tctttataca?gtgtccgaca?agaacaactt?cttatgttgg 60
taccagcaga?aaccaggaca?gcctcctaaa?ctgctcatgt?actgggcatc?tatccgggaa?120
tccggggtcc?ctgaccgatt?cagtggcagc?gggtctggga?cagatttcac?tctcaccatc?180
agcagcctgc?aggctgaaga?tgtggcagtt?tattactgtc?agcaatatta?tagtactcct?240
cccaccttcg?gccaagggac?acgactggag?attaaacgaa?ctgtggctgc?accatctgtc?300
ttcatcttcc?cgccatctga?tgagcagttg?aaatctggaa?ctgcctctgt?tgtgtgcctg?360
ctgaataact?tctatcccag?agag 384
 
<210>71
<211>128
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>71
Leu?Gln?Val?Gln?Pro?Glu?Cys?Leu?Tyr?Thr?Val?Ser?Asp?Lys?Asn?Asn
1 5 10 15
Phe?Leu?Cys?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro?Lys?Leu?Leu
20 25 30
Met?Tyr?Trp?Ala?Ser?Ile?Arg?Glu?Ser?Gly?Val?Pro?Asp?Arg?Phe?Ser
35 40 45
Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln
50 55 60
Ala?Glu?Asp?Val?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Tyr?Ser?Thr?Pro
65 70 ?75 80
Pro?Thr?Phe?Gly?Gln?Gly?Thr?Arg?Leu?Glu?Ile?Lys?Arg?Thr?Val?Ala
85 90 95
Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser
100 105 110
Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu
115 120 ?125
 
<210>72
<211>425
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<400>72
caggtacagc?tgcagcagtc?aggtccagga?ctggtgaagc?cctcgcagac?cctctcactc 60
acctgtgcca?tctccgggga?cagtgtctct?agcaacagtg?ctgcttggaa?ctggatcagg?120
cagtccccat?ggagaggcct?tgagtggctg?ggaaggacat?actacaggtc?caagtggtat?180
aatgaatatg?cagtatctgt?gaaaagtcga?atgaccatca?acccagacac?atccaagaac?240
cagttctccc?tgcagctgaa?ctctgtgact?cccgaggaca?cggctgtgta?ttactgtgca?300
agagagaggt?taggggagtt?atacggtatg?gacgtctggg?gccaagggac?cacggtcacc?360
gtctcctcag?cctccaccaa?gggcccatcg?gtcttccccc?tggcaccctc?ctccaagagc?420
accta 425
 
<210>73
<211>141
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>73
Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln
1 5 10 15
Thr?Leu?Ser?Leu?Thr?Cys?Ala?Ile?Ser?Gly?Asp?Ser?Val?Ser?Ser?Asn
20 25 30
Ser?Ala?Ala?Trp?Asn?Trp?Ile?Arg?Gln?Ser?Pro?Trp?Arg?Gly?Leu?Glu
35 40 45
Trp?Leu?Gly?Arg?Thr?Tyr?Tyr?Arg?Ser?Lys?Trp?Tyr?Asn?Glu?Tyr?Ala
50 55 60
Val?Ser?Val?Lys?Ser?Arg?Met?Thr?Ile?Asn?Pro?Asp?Thr?Ser?Lys?Asn
65 70 75 80
Gln?Phe?Ser?Leu?Gln?Leu?Asn?Ser?Val?Thr?Pro?Glu?Asp?Thr?Ala?Val
85 90 95
Tyr?Tyr?Cys?Ala?Arg?Glu?Arg?Leu?Gly?Glu?Leu?Tyr?Gly?Met?Asp?Val
100 105 ?110
Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly
115 120 125
Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
130 135 140
 
<210>74
<211>487
<212>DNA
< 213>homo sapiens (Homo sapiens)
 
<220>
<221>misc_feature
<222>(0)...(0)
< 223>n=a, t, c or g
 
<400>74
atgcagctga?cncagtctcc?agactccctg?gctgtgtctc?tgggcgagag?ggccaccatc 60
aactgcaagt?ccagccagaa?tgttttatac?aggtccaaca?agaagaactt?cttagtttgg?120
taccagcaga?aaccaggaca?gcctcctaag?ctgctcattt?actgggcatc?tatccgggaa?180
tccggggtcc?ctgaccgatt?cagtggcagc?gggtctggga?cagatttcac?tctcaccatc?240
agcagcctgc?agactgaaga?tgtggcagtt?tattactgtc?agcaatatta?tagtactcct?300
cccaccttcg?gccaagggac?acgactggag?attaaacgaa?ctgtggctgc?accatctgtc?360
ttcatcttcc?cgccatctga?tgagcagttg?aaatctggaa?ctgcctctgt?tgtgtgcctg?420
ctgaataact?tctatcccag?agaggccaaa?gtacagtgga?aggtggataa?cgccctccaa?480
tcgggta 487
 
<210>75
<211>162
<212>PRT
< 213>homo sapiens (Homo sapiens)
 
<400>75
Met?Gln?Leu?Thr?Gln?Ser?Pro?Asp?Ser?Leu?Ala?Val?Ser?Leu?Gly?Glu
1 5 10 15
Arg?Ala?Thr?Ile?Asn?Cys?Lys?Ser?Ser?Gln?Asn?Val?Leu?Tyr?Arg?Ser
20 25 30
Asn?Lys?Lys?Asn?Phe?Leu?Val?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Pro
35 40 45
Pro?Lys?Leu?Leu?Ile?Tyr?Trp?Ala?Ser?Ile?Arg?Glu?Ser?Gly?Val?Pro
50 55 60
Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile
65 70 ?75 80
Ser?Ser?Leu?Gln?Thr?Glu?Asp?Val?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr
85 90 95
Tyr?Ser?Thr?Pro?Pro?Thr?Phe?Gly?Gln?Gly?Thr?Arg?Leu?Glu?Ile?Lys
100 105 110
Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu
115 120 125
Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe
130 135 140
Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln
145 150 155 160
Ser?Gly
 
<210>76
<211>24
<212>DNA
< 213>artificial sequence
 
<220>
<223>Flag?Tag
 
<400>76
gattacaagg?atgacgacga?taag 24
 
<210>77
<211>24
<212>DNA
< 213>artificial sequence
 
<220>
< 223>control sequence
 
<400>77
aagggacgaa?gacgaacacu?uctt 24
 
<210>78
<211>23
<212>DNA
 
< 213>artificial sequence
 
<220>
< 223>control sequence
 
<400>78
aactgaagac?ctgaagacaa?taa 23

Claims (31)

1. a monoclonal antibody or its Fab, it comprises that specificity is incorporated into the proteic antigen binding site of PSCA, and the proteic aminoacid sequence of said PSCA is shown in SEQ ID NO:2, and wherein said monoclonal antibody comprises the V shown in the SEQ ID NO:47 HV shown in region amino acid sequence and the SEQ ID NO:51 LRegion amino acid sequence.
2. antibody as claimed in claim 1 or fragment is characterized in that, said fragment is Fab, F (ab ') 2, Fv or sFv fragment.
3. a monoclonal antibody or its Fab; It comprises that specificity is incorporated into the proteic antigen binding site of PSCA; The proteic aminoacid sequence of said PSCA is shown in SEQ ID NO:2, and it is the V of the monoclonal antibody that hybridoma produced of PTA-6701 that wherein said monoclonal antibody comprises by the A.T.C.C preserving number HDistrict and V LRegion amino acid sequence.
4. antibody as claimed in claim 3 or fragment is characterized in that, said fragment is Fab, F (ab ') 2, Fv or sFv fragment.
5. like each described antibody or fragment among the claim 1-4, it is characterized in that said antibody or fragment are and the detectable label link coupled.
6. like each described antibody or fragment among the claim 1-4, it is characterized in that said antibody or fragment are with toxin conjugated.
7. like each described antibody or fragment among the claim 1-4, it is characterized in that said antibody or fragment are and the therapeutical agent link coupled.
8. like each described antibody or fragment among the claim 1-4, it is characterized in that said antibody or fragment are and the chemotherapeutics link coupled.
9. a hybridoma that produces claim 1 or 3 described monoclonal antibodies is characterized in that, the A.T.C.C preserving number of said hybridoma is PTA-6701.
10. polynucleotide, its encoded packets contains the sequence that right requires the variable region of light chain and the variable region of heavy chain of 1 described antibody.
11. a carrier, it comprises the described polynucleotide of claim 10.
12. one kind with the described carrier cells transfected of claim 11.
13. a method that produces antibody or its Fab, said method comprises:
Culturing cell, wherein
(a) said cell contains the carrier transfection that right requires the polynucleotide of the sequence of the variable region of heavy chain of antibody described in 1 with comprising the carrier that contains the polynucleotide of the sequence of the variable region of light chain of antibody described in the right requirement 1 of encoding with comprising to encode; Or
(b) said cell has been used the described carrier transfection of claim 11.
14. a pharmaceutical composition that is used for prostate gland or treatment of pancreatic cancer, said pharmaceutical composition comprise each described antibody or its Fab, F (ab ') 2, Fv or sFv fragment among the claim 1-8.
15. each described antibody is used for the purposes in the bonded test kit of PSCA albumen and said antibody in proteic existence of detection of biological sample PSCA and the test sample in preparation among the claim 1-4, the said PSCA albumen in the wherein said sample comprises the aminoacid sequence shown in the SEQ ID NO:2.
16. a carcinostatic agent that reduces tumor growth is characterized in that, said carcinostatic agent comprises the combination of each described antibody among the claim 1-4 or its Fab and chemotherapeutics, and wherein said tumour is prostate gland or pancreas tumor.
17. carcinostatic agent as claimed in claim 16; It is characterized in that; Said antibody or its fragment and detectable label coupling, said detectable label are ri, metal chelator, enzyme, fluorescent chemicals, bioluminescent compound or chemiluminescence compound.
18. carcinostatic agent as claimed in claim 17 is characterized in that, said ri is: 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi or 32P.
19. carcinostatic agent as claimed in claim 16; It is characterized in that said chemotherapeutics is: the 40kD fragment of ricin, ricin A-chain, Zorubicin, daunorubicin, maytansinol, taxol, ethidium bromide, MTC, etoposide, VM-26, vincristine(VCR), vinealeucoblastine(VLB), NST-757, dihydroxyl anthracin diketone, NSC-3053, diphtheria toxin, ETA, ETA, abrin, abrin A chain, modeccin A chain, α-sarcina, gelonin, mitogillin, restrictocin, phenomycin, enomycin, Curie's rhzomorph, crotin, calicheamycin, Sapaonaria officinalis suppressor factor, glucocorticosteroid, Jin Tating, duomycin, yttrium, bismuth, Kang Letading, a Ka-7038, many Luo Tating, rachelmycin or cis-platinum.
20. each described antibody or its Fab are used for suppressing the purposes in the medicine of method of object growth of cancer cells in preparation among the claim 1-4; Wherein said cancer cells is expressed the PSCA albumen that comprises aminoacid sequence shown in the SEQ ID NO:2, and wherein said cancer cells is prostate cancer cell or pancreatic cancer cell.
21. purposes as claimed in claim 20 is characterized in that, said antibody or its Fab and cytotoxic agent or detectable label coupling.
22. purposes as claimed in claim 21 is characterized in that, with said antibody or fragment link coupled detectable label be ri, metal chelator, enzyme, fluorescent chemicals, bioluminescent compound or chemiluminescence compound.
23. purposes as claimed in claim 22 is characterized in that, said ri is: 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi or 32P.
24. purposes as claimed in claim 21; It is characterized in that said cytotoxic agent is: the 40kD fragment of ricin, ricin A-chain, Zorubicin, daunorubicin, maytansinol, taxol, ethidium bromide, MTC, etoposide, VM-26, vincristine(VCR), vinealeucoblastine(VLB), NST-757, dihydroxyl anthracin diketone, NSC-3053, diphtheria toxin, ETA, ETA, abrin, abrin A chain, modeccin A chain, α-sarcina, gelonin, mitogillin, restrictocin, phenomycin, enomycin, Curie's rhzomorph, crotin, calicheamycin, Sapaonaria officinalis suppressor factor, glucocorticosteroid, Jin Tating, duomycin, yttrium, bismuth, Kang Letading, a Ka-7038, many Luo Tating, rachelmycin or cis-platinum.
25. be used in patient treatment method for cancer like claim 1-8 in each described antibody or its Fab, wherein said cancer is prostate cancer or carcinoma of the pancreas.
26. antibody or its Fab that is used for cancer treatment method as claimed in claim 25 is characterized in that said antibody or its Fab and chemotherapeutics, radiotherapy or both couplings.
27. antibody as claimed in claim 26 or its Fab is characterized in that, said monoclonal antibody before beginning to give chemotherapeutics, in or give afterwards.
28. antibody as claimed in claim 26 or its Fab is characterized in that, said monoclonal antibody the beginning radiotherapy before, in or give afterwards.
29., it is characterized in that said monoclonal antibody gives between 1-60 days like claim 27 or 28 described antibody or its Fab before beginning radiotherapy and/or chemotherapy.
30. antibody as claimed in claim 26 or its Fab; It is characterized in that; Said chemotherapeutics is selected from: cis-platinum, dicarbazine, dactinomycin, mechlorethamine, streptozocin, endoxan,, carmustine, lomustine, Dx, daunorubicin, Procarbazine, MTC, cytosine arabinoside, VP, Rheumatrex, 5 FU 5 fluorouracil, vinealeucoblastine(VLB), vincristine(VCR), bleomycin, taxol, docetaxel, rIL-2, asparaginase, busulfan, carboplatin, CldAdo, dicarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprorelin, megestrol, melphalan, mercaptopurine, Plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, Plicamycin, streptozocin, Tamoxifen, teniposide, testolactone, Tioguanine, plug be for group, uracil mustard, vinorelbine, TV, taxol or their combination.
31. antibody as claimed in claim 26 or its Fab is characterized in that, also comprise giving additional chemotherapeutic agents.
CN200580017239.9A 2004-05-28 2005-05-17 Antibodies and related molecules that bind to PSCA proteins Active CN1997751B (en)

Applications Claiming Priority (15)

Application Number Priority Date Filing Date Title
US10/857,484 US7622564B2 (en) 2003-05-30 2004-05-28 Prostate stem cell antigen (PSCA) variants and subsequences thereof
USPCT/US2004/017231 2004-05-28
US10/857,484 2004-05-28
PCT/US2004/017231 WO2005014780A2 (en) 2003-05-30 2004-05-28 Prostate stem cell antigen (psca) variants and subsequences thereof
US61638104P 2004-10-05 2004-10-05
US60/616,381 2004-10-05
US61788104P 2004-10-12 2004-10-12
US60/617,881 2004-10-12
US62131004P 2004-10-21 2004-10-21
US60/621,310 2004-10-21
US63307704P 2004-12-02 2004-12-02
US60/633,077 2004-12-02
US67200005P 2005-04-14 2005-04-14
US60/672,000 2005-04-14
PCT/US2005/017412 WO2005118864A2 (en) 2004-05-28 2005-05-17 Antibodies and related molecules that bind to psca proteins

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CN1997751B true CN1997751B (en) 2012-08-29

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CN116514959A (en) * 2018-03-24 2023-08-01 瑞泽恩制药公司 Genetically modified non-human animals for producing therapeutic antibodies against peptide-MHC complexes, methods of making and uses thereof
CN112820355B (en) * 2020-12-16 2024-03-22 浙江工业大学 Molecular virtual screening method based on protein sequence comparison
CN113287569B (en) * 2021-05-27 2023-04-07 四川康城生物科技有限公司 Construction method of animal model with low immunity
CN113604505A (en) * 2021-08-11 2021-11-05 华农（肇庆）生物产业技术研究院有限公司 pSFV-p32 virus-like particle and preparation method and application thereof
CN117736295B (en) * 2024-02-21 2024-05-07 中国科学院烟台海岸带研究所 Lectin and application thereof

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WO2003022879A2 (en) * 2001-09-07 2003-03-20 Polymun Scientific Immunbiologische Forschung Gmbh Peptides mimicking a cryptic epitope of gp41 hiv-1 and antibodies directed against them

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2003022879A2 (en) * 2001-09-07 2003-03-20 Polymun Scientific Immunbiologische Forschung Gmbh Peptides mimicking a cryptic epitope of gp41 hiv-1 and antibodies directed against them

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